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From cells to tissues
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Better ways to give medicines
Cancer is one of the major causes of death in our society. The anti-cancer drugs currently used in chemotherapy produce several side effects and, although fatal for almost all cells in a tumor, a small percentage of cells appear to resist the treatment. It is therefore urgent to design new therapies that specifically target these cells while causing no harm to healthy tissue. The resistance to multiple drugs is linked to the presence of specific molecules at the cellular plasma membrane, that actively pump out chemical drugs (efflux pumps). Additionally, some cancer cells have the ability to self-renew, thereby initiating secondary tumor growth. Multi-drug resistant cells and cancer stem cells are suggested to be the main cause of treatment failure and relapse.
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Structure of Biomimetic Materials
A large number of natural and synthetic hydrogels are currently used for tissue engineering and regenerative medicine. Over the last decade, there has been an increasing awareness of the role of material properties of the substrates in guiding cellular behaviour. This has inspired chemists to create a new generation of materials with mechanical properties closed to that of natural occurring biopolymer networks. Recently, the groups of Prof. Alan Rowan (Queens University, Australia) and Prof. Paul Kouwer (Radboud University of Nijmegen, The Netherlands) were able to develop a fully synthetic material that mimics in all aspects the gels prepared from cellular filaments. These synthetics gels are prepared from polyisocyanopeptides (PICs) grafted with oligo(ethylene glycol) chains and share structural features of biopolymers: their helical structure renders the polymer molecules relatively stiff while the interaction between the side chains enable the formation of bundles or fibrils of defined dimensions. The triethylene glycol side chains attached to the polymer backbone render the material thermo-responsive (it will gel upon heating beyond 20 °C and become liquid again upon cooling). Despite being characterized extensively in bulk, the fundamental dynamics and the relation between the macroscopic properties and the microscopic structure at cellular length scales of PIC-based hydrogels remains obscure.
Classically, structural characterization of materials is performed with electron microscopy or scanning probe microscopy. Despite the high spatial resolution achievable with these techniques, they are unable to measure dynamics ‘in situ’ and sample preparation can be a laborious process. In contrast, optical microscopy has the potential to unravel the dynamics in complex heterogeneous systems but has been limited to a spatial resolution of ca. 200 nm. In the past 10 years fluorescence imaging has been revolutionized by the successful development of sub-diffraction (super-resolution) microscopy modalities which can achieve resolutions down to tens of nanometers (see Molecular Organization at the Nanoscale).The various possibilities of fluorescence microscopy to probe dynamics and heterogeneities, with molecular resolution, for a wide range of time scales makes it an ideal tool to address many topics of polymer science. In this project we are using STED to image the polymer network at the nanometer scale.
For more information on PIC-based hydrogels:
- Kouwer P.H.J., et al. (2013) Responsive biomimetic networks from polyisocyanopeptide hydrogels, Nature, 493, pages 651–655 (article can be found here)
- Jasper M., et al. (2014) Ultra-responsive soft matter from strain-stiffening hydrogels, Nature Communications, 5, 5808 (article can be found here)
- Jasper M., et al. (2016) Bundle Formation in Biomimetic Hydrogels, Macromolecules, 17(8), pages 2642–2649 (article can be found here)
Polymer reptation in 3D
Our current theoretical understanding of entangled polymer chain dynamics is based on the reptation model. First proposed by Doi and Edwards, and further expanded by de Gennes, the reptation model assumes that a polymer chain is confined by the surrounding matrix and is therefore forced to move inside an imaginary tube defined by the transient network of entangled neighboring chains. Intuitively this motion resembles that of a snake or worm. The reptation model predicts five dynamical regimes for segment diffusion, summarized in the figure below. These regimes are as follows: (0) sub-segmental processes (“glassy dynamics”) at very short times (microseconds), (I) small motion subject only to chain connectivity, (II) “local reptation”: short-distance motion within the constraints imposed by the surrounding chains (“tube”), (III) “reptation”: diffusive motion along the curvilinear tube over distances larger than the polymer size, and (IV) free diffusion.
Rheology at the micrometer scale
Due to the crucial role of physical cues in regulating cell behaviour, the mechanical properties of hydrogels are a key design parameter in tissue engineering applications. The shear elastic properties of viscoelastic materials are commonly measured by mechanical rheometers. Storage and loss moduli of a material can be measured by application of strain while measuring stress or vice versa. In contrast, recently developed optical micro-rheology techniques use nanometer- or micrometer-sized particles embedded in the material to obtain the viscoelastic response parameters. Thermal or passive micro-rheology for viscoelastic materials is based on an extension of the concepts of Brownian motion of particles in simple liquids. The movement of the embedded particles can be monitored using particle tracking. Initially developed to investigate the rheological properties of uniform complex fluids, particle tracking micro-rheology (PTM) is becoming a popular technique to analyze polymer blends and gels, as well as the deformability and elasticity within cells. However, if the beads locally modify the structure of the gel or are contained in a pore in an inhomogeneous matrix, the bulk rheological properties will not be retrieved. A solution is to use the cross-correlated thermal fluctuation of pairs of tracer particles, ‘two-point micro-rheology’. This method provides a better agreement between micro and macro-rheology, even in complex micro-structured fluids. However, technical constrains limit the wide application of this technique. One of the major limitations of two-point micro-rheology is the reduced number of trajectories that can be used for analysis. During particle tracking micro-rheology, the length of the calculated trajectories is limited by the time spent by the tracers in the field of view (x,y) and depth of focus (z). Consequently, mechanical characterization of complex polymer matrixes at the micrometer scale would benefit greatly of a new method for (fast) tracking in 3D. We are developing a new method for fast tracking of (fluorescent) beads in 3D using a multi-plane wide field microscope. This will allow a better mechanical characterization of soft materials, at the microscale.
Cellular adhesion in 3D matrices
Cells sense physical forces and the mechanical properties of the microenvironment via several distinct mechanisms and cellular components. The first step of cellular adhesion to the ECM occurs via transmembrane heterodimers of the integrin family. Once integrin molecules adhere to the ECM, they are activated and form clusters. As the number of bound molecules increases, some of the focal complexes evolve from small (0.5-1µm in diameter) transient ‘dot-like’ contacts to elongated structures (3-10µm) which couple with actin and associated proteins. The mechanical coupling between the ECM and the cell cytoskeleton is controlled by the dynamics of the focal adhesion complexes (assembly, disassembly and turnover).
Protein-Protein Interactions
Protein-protein interactions (PPIs) are intrinsic to all cellular processes, driving both metabolic and regulatory pathways. Despite the numerous techniques available, detection of transient short-lived PPIs remains challenging4. The main fluorescence microscopic techniques developed for visualizing PPIs in a cellular context are based on Föster resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC)5. Both techniques detect the interaction between a pair of labeled molecules. Although highly informative, they require fine positioning of the labels and in the majority of the applications the spatial resolution achieved is limited by the diffraction of light to about 200 nm. More information concerning the use of FRET to detect PPIs can be found at Cellular Signalling).
We have use a single molecule localization based super-resolution technique to detect and map PPIs at the cell membrane. This new variant of PAINT that enables mapping of short-lived transient interactions between cytosolic and membrane-bound proteins inside living mammalian cells, at the nanometer scale. In this method the protein of interest is labeled with a light-controllable fluorescent protein and imaged under TIRF illumination, which leads to the selective activation and subsequent detection of molecules in close proximity with the plasma membrane. Interacting molecules are discriminated using a stringent fitting of the fluorescence signal recorded for every single molecule.
Organoids: models for cell communication
Nowadays, human organoids are becoming a highly promising tool to model organ development, function and especially human diseases in vitro. In general, organoids are miniature, simplified organs that can easily propagate in vitro originating from one or a few cells, typically stem cells.
Single cell manipulation by endoscopy
Nanowire-based endoscopy has attracted interest due to its ability to manipulate cells at the single-cell level with minimal cellular perturbation. High-density, vertically aligned nanowire arrays have been used as an efficient gene delivery system. Despite the high transfection rates, culturing the cells on nanowire arrays might have other influences on the cellular behaviour. For example, stem cells cultured on silicon nanowires show significantly different adhesion, proliferation and differentiation, compared with flat silicon or other control substrates. Furthermore, such arrays are not location-specific and require optimization of the nanowire density and dimension for the different the cell types. In collaboration with the group of Prof. Hiroshi Uji-i we are developing a method to delivery genetic material using a single nanowire. In contrast to the existing methods, this approach can be applied to any cell type and is extremely specific: it can target a single cell and it can deliver the genetic material exactly at the desired position, such as inside of the nucleus, with no damage to the cell. Since gene editing is a stochastic event occurring in only a fraction of the cells, the transfer of genetic material (or proteins) is of crucial importance in genome editing methods, where the nucleases must be efficiently delivered. The duration and magnitude of the nuclease expression are critical parameters for the level of both on-target and off-target nuclease activity. Additionally, the dose of donor template DNA is important to ensure efficient homologous recombination. The proposed method offers the possibility to deliver different molecules at different times, in synchronization with the cell cycle. The lab of Prof. Uji-i is one of the first (and few) groups worldwide to have developed and optimized a novel nanoscopic technique using 1D nanowires, with a diameter of less than 100 nm, for SERS endoscopic studies. It has been already proven by us that the thin diameter and 1D structure of the NW greatly reduces the damage induced to a live cell during probe insertion. Although designed for a different purpose, this nanoprobe is ideal as a starting point to develop a new NW-based gene delivery system.
New Drug delivery systems
In this decade, the pharmacology field has been intensively exploring different approaches to deliver multiple drugs with a single drug nano-carrier, such as liposomes, polymer nanoparticles, and inorganic nanoparticles. The advantage of nanoparticle based drug delivery is the ability to unify pharmacokinetics by simultaneous delivery of multiple drugs to specific target cells.
Ever since first reported in 2001, mesoporous silica nanoparticles (MSNPs) have manifested themselves as highly potential candidates for targeted drug delivery. They owe their popularity to their high drug load capacity, chemical stability, biocompatibility and easy functionalization. Since the diameter of the nanoparticles (100 to 200 nm) is tunable, one can obtain a size suitable for passive targeting through the hyperpermeable tumor vasculature, thereby promoting accumulation of the nanoparticles in tumor tissue due to the enhanced permeability and retention effect (EPR). Additionally, functionalization of the nanoparticles with ligands which have a high affinity for tumor cell specific surface receptors promotes more specific internalization in cancer cells. For example, hyaluronic acid (HA) has been extensively used as a targeting ligand due to its affinity for CD44, a transmembrane glycoprotein receptor that plays a critical role in malignant cell activities and, most importantly, it is overexpressed in many solid tumor cells, in metastasis and cancer stem cells.
Correlative AFM and Fluorescence Microscopy
Biological processes are often carried out in the context of macromolecular assemblies. In addition, arrangements of these complexes can be dynamic, resulting in a heterogeneous ensemble. Single molecule techniques can resolve distinct populations in heterogeneous systems, in contrast to bulk experiments where heterogeneity is averaged out. In turn, mechanistic details of bio-macromolecular interactions can be uncovered. Atomic force microscopy (AFM) is a technique that can generate 3D reconstructions of individual biomolecules and complexes thereof in a label-free fashion, and with ~ nm resolution. To this end a very sharp tip, mounted on a flexible cantilever, scans a sample surface in a raster pattern using a piezo-scanner, while keeping the interaction force between sample and tip constant. In every pixel (x,y) of the scanned area, the z-position is recorded. Consequently, a 3D representation of the surface topography can be reconstructed. An alternative way to study single molecules is by fluorescence microscopy. The molecule of interest is labeled with a fluorescent tag providing high contrast. Emission of the tag after excitation, is detected through an optical system. Due to the wave character of light, the emitted light is spread out on the detector described by the point spread function (PSF) of the optical system. This effect limits the resolution achieved with optical microscopy, referred to as the diffraction limit. However, when the signal of a single molecule is detected, the position of this molecule can be determined by fitting of the recorded fluorescence signal with a mathematical approximation of the PSF such as a two-dimensional Gaussian function. This principle underlies single molecule localization microscopy (SMLM). AFM and SMLM are highly complementary technologies: AFM can provide insight in topographic features at a nanometer resolution while SMLM is sensitive towards specifically labelled molecules in complex samples. Integrated setups combining both technologies can therefore provide orthogonal information at the single-molecule level.
Cell signalling: probes and methods
Cell signaling involves the sensing of an extracellular signal by a cell surface receptor, which then transduces this signal to an intracellular response. Despite the numerous studies performed on signaling pathways and mechanisms, little is known about the initial steps occurring at the plasma membrane: receptor pre-assembly at the molecular level and potential reorganization after ligand activation. Traditionally crystallography is used to investigate receptor multimerization. However, the crystallized state might not represent the biochemically active form due to the harsh preparation conditions and the absence of the cellular environment. Other approaches include macroscopic biochemical or biophysical methods, such as chemical cross-linking, ion-channel gating, immunoprecipitation or binding assays. Nowadays, established fluorescence imaging and spectroscopic techniques offer a versatile toolbox to study membrane receptor organization in (living) cells.
In the lab we are using fluorescence fluctuation spectroscopy to quantify physicochemical processes (mobility, binding affinity, stoichiometry, absolute concentration) occurring on a micro-to-millisecond time scale. Fluorescence experiments down to picoseconds are also commonly possible with methods such as time-correlated single photon counting (TCSPC), that allow, e.g., measuring fluorescence lifetimes and molecular tumbling. Additionally, spatially resolved microscopy with high temporal resolution also has clear benefits. For example, combined with confocal laser scanning microscopy (LSM), TCSPC allows protein-protein interactions (PPIs) to be imaged via Förster resonance energy transfer (FRET) based fluorescence lifetime imaging microscopy (FLIM). Imaging based FCS methods such as raster (RICS), number and brightness analysis (N&B) or (spatio-) temporal image correlation spectroscopy [(S)TICS] combine the quantitative analytical power of fluctuation methods with spatial information to map, among many other things, mobility and stoichiometry inside living systems. Simultaneous dual-color fluorescence imaging is possible when fast alternating excitation (alias pulsed interleaved excitation, PIE) is employed. PIE renders analysis of dual-color point FCS experiments considerably more straightforward. The combination of PIE with fluctuation imaging (PIE-FI) allows extracting the maximum amount of molecular information (mobility, stoichiometry, interactions…) from each species present in dual-color LSM images.
For more information on these methods:
- Hendrix J., Lamb D.C. (2014) Implementation and Application of Pulsed Interleaved Excitation for Dual-Color FCS and RICS. In: Engelborghs Y., Visser A. (eds) Fluorescence Spectroscopy and Microscopy. Methods in Molecular Biology (Methods and Protocols), vol 1076. Humana Press, Totowa, NJ (chapter can be found here)
- Hendrix J., Schrimpf W., Höller M., Lamb D.C. (2013) Pulsed Interleaved Excitation Fluctuation Imaging, Biophysical Journal, 105(4), 848-861 (article can be found here)
Imaging single HIV virions
Viruses are simple agents exhibiting complex reproductive mechanisms. Decades of research have provided crucial basic insights, antiviral medication and moderately successful gene therapy trials. The most infectious viral particle is, however, not always the most abundant one in a population, questioning the utility of classic ensemble-averaging virology. Indeed, viral replication is often not particularly efficient, prone to errors or containing parallel routes. In collaboration with Prof. Zeger Debeyser (KU Leuven) and Prof Hendrix (UHasselt) we have applied different single-molecule sensitive fluorescence methods to investigate viruses, one-by-one. While this collaboration is still ongoing, there is already several publications that show-case the potential of imaging single virions.
publications
Encoding and decoding spatio-temporal information for super-resolution microscopy
Abstract
The challenge of increasing the spatial resolution of an optical microscope beyond the diffraction limit can be reduced to a spectroscopy task by proper manipulation of the molecular states. The nanoscale spatial distribution of the molecules inside the detection volume of a scanning microscope can be encoded within the fluorescence dynamics and decoded by resolving the signal into its dynamics components. Here we present a robust and general method to decode this information using phasor analysis. As an example of the application of this method, we optically generate spatially controlled gradients in the fluorescence lifetime by stimulated emission. Spatial resolution can be increased indefinitely by increasing the number of resolved dynamics components up to a maximum determined by the amount of noise. We demonstrate that the proposed method provides nanoscale imaging of subcellular structures, opening new routes in super-resolution microscopy based on the encoding/decoding of spatial information through manipulation of molecular dynamics.
Published in Nat. Comm., 2015
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STED super-resolved microscopy
Abstract
Stimulated emission depletion (STED) microscopy provides subdiffraction resolution while preserving useful aspects of fluorescence microscopy, such as optical sectioning, and molecular specificity and sensitivity. However, sophisticated microscopy architectures and high illumination intensities have limited STED microscopy's widespread use in the past. Here we summarize the progress that is mitigating these problems and giving substantial momentum to STED microscopy applications. We discuss the future of this method in regard to spatiotemporal limits, live-cell imaging and combination with spectroscopy. Advances in these areas may elevate STED microscopy to a standard method for imaging in the life sciences.
Published in Nat. Methods, 2018
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A robust and versatile platform for image scanning microscopy enabling super-resolution FLIM
Abstract
Image scanning microscopy (ISM) can improve the effective spatial resolution of confocal microscopy to its theoretical limit. However, current implementations are not robust or versatile, and are incompatible with fluorescence lifetime imaging (FLIM). We describe an implementation of ISM based on a single-photon detector array that enables super-resolution FLIM and improves multicolor, live-cell and in-depth imaging, thereby paving the way for a massive transition from confocal microscopy to ISM.
Published in Nat. Methods, 2019
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Abstract
Published in , 2019
Easy Two-Photon Image-Scanning Microscopy With Spad Array And Blind Image Reconstruction
Abstract
Two-photon excitation (2PE) laser scanning microscopy is the imaging modality of choice when one desires to work with thick biological samples. However, its spatial resolution is poor, below confocal laser scanning microscopy. Here, we propose a straightforward implementation of 2PE image scanning microscopy (2PE-ISM) that, by leveraging our recently introduced ISM platform – based on a new single-photon avalanche diode (SPAD) array detector – coupled with a novel blind image reconstruction method, is shown to improve the effective resolution, as well as the overall image quality of 2PE microscopy. Indeed, in stark contrast to conventional single-point detectors, SPAD array detectors give access to the images of any excited scanning region, from which it is possible to decode information about the aberrations/distortions – occurring during imaging – able to substantially improve the reconstruction. Most importantly, our 2PE-ISM implementation requires no calibration or other input from the user; it works like any familiar two-photon system, but produces higher resolution images deep into thick samples. In our view, this novel implementation is the key for making 2PE-ISM mainstream.
Published in bioRxiv, 2019
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Fourier ring correlation simplifies image restoration in fluorescence microscopy
Abstract
Fourier ring correlation (FRC) has recently gained popularity among fluorescence microscopists as a straightforward and objective method to measure the effective image resolution. While the knowledge of the numeric resolution value is helpful in e.g., interpreting imaging results, much more practical use can be made of FRC analysis—in this article we propose blind image restoration methods enabled by it. We apply FRC to perform image de-noising by frequency domain filtering. We propose novel blind linear and non-linear image deconvolution methods that use FRC to estimate the effective point-spread-function, directly from the images. We show how FRC can be used as a powerful metric to observe the progress of iterative deconvolution. We also address two important limitations in FRC that may be of more general interest: how to make FRC work with single images (within certain practical limits) and with three-dimensional images with highly anisotropic resolution.
Published in Nat. Comm., 2019
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Synergic Combination of Stimulated Emission Depletion Microscopy with Image Scanning Microscopy to Reduce Light Dosage
Abstract
Stimulated emission depletion (STED) microscopy is one of the most influential nanoscopy techniques; by increasing the STED beam intensity, it theoretically improves the spatial resolution to any desired value. However, the higher is the dose of stimulating photons, the stronger are the photo-bleaching and photo-toxicity effects, which potentially compromise live-cell and long-term imaging. For this reason the scientific community is looking for strategies to reduce the STED beam intensity needed to achieve a target resolution. Here, we show how the combination of STED microscopy with image scanning microscopy (ISM) meets this request. In particular, we introduce a new STED-ISM architecture – based on our recent single-photon-avalanche-diode (SPAD) detector array – which allows covering the near-diffraction limit resolution range with reduced STED beam intensity. We demonstrate this ability both with simulated data and in live-cell experiments. Because of (i) the minimal changes in the optical architecture of the typical point-scanning STED microscope; (ii) the parameter-free, robust and real-time pixel-reassignment method to obtain the STED-ISM image; (iii) the compatibility with all the recent progresses in STED microscopy, we envisage a natural and rapid upgrade of any STED microscope to the proposed STED-ISM architecture.
Published in bioRxiv, 2019
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Photon-separation to enhance the spatial resolution of pulsed STED microscopy
Abstract
Stimulated emission depletion microscopy (STED) is one of the pivotal super-resolution techniques. It overcomes the spatial resolution limit imposed by the diffraction by using an additional laser beam, the STED beam, intensity of which is directly related to the achievable resolution. Despite reaching nanometer resolution, much effort in recent years has been devoted to reducing the STED beam intensity because it may lead to photo-damaging effects. Accessing the spatial information encoded in the temporal dynamics of the detected fluorescent photons has been proved to be a powerful strategy and has contributed to the separation by lifetime tuning (SPLIT) technique. The SPLIT method uses the phasor analysis to efficiently distinguish photons emitted from the center and the periphery of the excitation spot. It thus improves the resolution without increasing the STED beam intensity. This method was proposed for architectures based on the STED beam running in continuous waves (CW-STED microscopy). Here, we extend it to pulsed STED beam implementations (pSTED microscopy). We show, through simulated and experimental data, that the pSTED-SPLIT method reduces the detection volume of the pSTED microscope without significantly decreasing the signal-to-noise ratio of the final image, thus effectively improving the resolution without increasing the STED beam intensity.
Published in Nanoscale, 2019
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Efficient Multiphoton STED Nanoscope Exploiting Spatio-Temporal Information
Abstract
Stimulated emission depletion (STED) microscopy is a powerful bioimaging technique that theoretically provides molecular spatial resolution while preserving the most important assets of fluorescence microscopy. When combined with two-photon excitation (2PE) microscopy (2PE-STED), subdiffraction resolution may be achieved for thick biological samples. The most straightforward implementation of 2PE-STED microscopy entails introduction of an STED beam operating in continuous wave (CW) into a conventional Ti:sapphire-based 2PE microscope (2PE CW-STED). In this implementation, resolution enhancement is typically achieved using time-gated detection schemes, often resulting in drastic signal-to-noise/-background ratio (SNR/SBR) reductions. Herein, we employ a pixel-by-pixel phasor approach to discard fluorescence photons lacking super-resolution information to enhance image SNR/SBR in 2PE CW-STED microscopy. We compare this separation of photons by lifetime tuning approach against other postprocessing algorithms and combine it with image deconvolution to further optimize image quality.
Published in Neurophotonics, 2019
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Pixel reassignment in image scanning microscopy: a re-evaluation
Abstract
Image scanning microscopy is a technique based on confocal microscopy, in which the confocal pinhole is replaced by a detector array, and the resulting image is reconstructed, usually by the process of pixel reassignment. The detector array collects most of the fluorescent light, so the signal-to-noise ratio is much improved compared with confocal microscopy with a small pinhole, while the resolution is improved compared with conventional (wide-field) microscopy. In previous studies, it has usually been assumed that pixels should be reassigned by a constant factor, to a point midway between the illumination and detection spots. Here it is shown that the peak intensity of the effective point spread function (PSF) can be further increased by 4% by a new choice of the pixel reassignment factor. For an array of two Airy units, the peak of the effective PSF is 1.90 times that of a conventional microscope, and the transverse resolution is 1.53 times better. It is confirmed that image scanning microscopy gives optical sectioning strength identical to that of a confocal microscope with a pinhole equal to the size of the detector array. However, it is shown that image scanning microscopy exhibits axial resolution superior to a confocal microscope with a pinhole the same size as the detector array. For a two-Airy-unit array, the axial resolution is 1.34 times better than in a conventional microscope for the standard reassignment factor, and 1.28 times better for the new reassignment factor. The axial resolution of a confocal microscope with a two-Airy-unit pinhole is only 1.04 times better than conventional microscopy. We also examine the signal-to-noise ratio of a point object in a uniform background (called the detectability), and show that it is 1.6 times higher than in a confocal microscope.
Published in J. Opt. Soc. Am. A, 2019
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SPAD-based asynchronous-readout array detectors for image-scanning microscopy
Abstract
Fluorescence microscopy and derived techniques are continuously looking for photodetectors able to guarantee increased sensitivity, high spatial and temporal resolution and ease of integration into modern microscopy architectures. Recent advances in single photon avalanche diodes (SPADs) fabricated with industry-standard microelectronic processes allow the development of new detection systems tailored to address the requirements of advanced imaging techniques (such as image-scanning microscopy). To this aim, we present the complete design and characterization of two bidimensional SPAD arrays composed of 25 fully independent and asynchronously-operated pixels, both having fill-factor of about 50% and specifically designed for being integrated into existing laser scanning microscopes. We used two different microelectronics technologies to fabricate our detectors: the first technology exhibiting very low noise (roughly 200 dark counts per second at room temperature), and the second one showing enhanced detection efficiency (more than 60% at a wavelength of 500 nm). Starting from the silicon-level device structures and moving towards the in pixel and readout electronics description, we present performance assessments and comparisons between the two detectors. Images of a biological sample acquired after their integration into our custom image-scanning microscope finally demonstrate their exquisite on-field performance in terms of spatial resolution and contrast enhancement. We envisage that this work can trigger the development of a new class of SPAD-based detector arrays able to substitute the typical single-element sensor used in fluorescence laser scanning microscopy.
Published in arXiv, 2020
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Improving SPLIT-STED Super-Resolution Imaging with Tunable Depletion and Excitation Power
Abstract
The SPLIT approach is a simple and efficient way to improve the spatial resolution of a super-resolved STED multi-dimensional image, i.e. a STED image in which an additional dimension encodes spatial information. Recently, we have demonstrated that the SPLIT can be applied to multidimensional STED images obtained with tunable depletion power. In this SPLIT-STED implementation, the additional dimension is represented by the depletion power, a parameter that can be easily tuned on any STED microscope. In this work, we introduce a modified implementation in which we tune also the excitation power. The tuning of the excitation power is used to modulate the number of photons collected for each STED image. We show that the modified SPLIT-STED method produces an improvement of spatial resolution for very different tuning patterns of the excitation intensity, improving the versatility of the SPLIT-STED approach. Interestingly, we find that the extent of photobleaching can be modulated by the excitation pattern, as it depends on the simultaneous impact of high STED and excitation powers. Thus, the tuning of the excitation power can improve applicability of the method to live cell imaging, potentially minimizing the photobleaching of the fluorophores and the phototoxicity on the biological samples during a SPLIT-STED acquisition. We apply the modified SPLIT-STED method to super-resolution imaging of nuclear periphery, in both fixed and live cells.
Published in J. Phys. D, 2020
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Two-photon image-scanning microscopy with SPAD array and blind image reconstruction
Abstract
Two-photon excitation (2PE) laser scanning microscopy is the imaging modality of choice when one desires to work with thick biological samples. However, its spatial resolution is poor, below confocal laser scanning microscopy. Here, we propose a straightforward implementation of 2PE image scanning microscopy (2PE-ISM) that, by leveraging our recently introduced single-photon avalanche diode (SPAD) array detector and a novel blind image reconstruction method, is shown to enhance the effective resolution, as well as the overall image quality of 2PE microscopy. With our adaptive pixel reassignment procedure ∼1.6 times resolution increase is maintained deep into thick semi-transparent samples. The integration of Fourier ring correlation based semi-blind deconvolution is shown to further enhance the effective resolution by a factor of ∼2 – and automatic background correction is shown to boost the image quality especially in noisy images. Most importantly, our 2PE-ISM implementation requires no calibration measurements or other input from the user, which is an important aspect in terms of day-to-day usability of the technique.
Published in Biomed. Opt. Express, 2020
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SPAD-based asynchronous-readout array detectors for image-scanning microscopy
Abstract
Fluorescence microscopy and derived techniques are continuously looking for photodetectors able to guarantee increased sensitivity, high spatial and temporal resolution, and ease of integration into modern microscopy architectures. Recent advances in single-photon avalanche diodes (SPADs) fabricated with industry-standard microelectronic processes allow the development of new detection systems tailored to address the requirements of advanced imaging techniques (such as image-scanning microscopy). To this aim, we present the complete design and characterization of two bidimensional SPAD arrays composed of 25 fully independent and asynchronously operated pixels, both having fill factor of about 50% and specifically designed for being integrated into existing laser scanning microscopes. We used two different microelectronics technologies to fabricate our detectors: the first technology exhibiting very low noise (roughly 200 dark counts per second at room temperature) and the second one showing enhanced detection efficiency (more than 60% at a wavelength of 500 nm). Starting from the silicon-level device structures and moving towards the in-pixel and readout electronics description, we present performance assessments and comparisons between the two detectors. Images of a biological sample acquired after their integration into our custom image-scanning microscope finally demonstrate their exquisite on-field performance in terms of spatial resolution and contrast enhancement. We envisage that this work can trigger the development of a new class of SPAD-based detector arrays able to substitute the typical single-element sensor used in fluorescence laser scanning microscopy.
Published in Optica, 2020
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Image scanning microscopy with multiphoton excitation or Bessel beam illumination
Abstract
Image scanning microscopy is a technique of confocal microscopy in which the confocal pinhole is replaced by a detector array, and the image is reconstructed most straightforwardly by pixel reassignment. In the fluorescence mode, the detector array collects most of the fluorescent light, so the signal-to-noise ratio is much improved compared with confocal microscopy with a small pinhole, while the resolution is improved compared with conventional fluorescence microscopy. Here we consider two cases in which the illumination and detection point spread functions are dissimilar: illumination with a Bessel beam and multiphoton microscopy. It has been shown previously that for Bessel beam illumination in image scanning microscopy with a large array, the imaging performance is degraded. On the other hand, it is also known that the resolution of confocal microscopy is improved by Bessel beam illumination. Here we analyze image scanning microscopy with Bessel beam illumination together with a small array and show that an improvement in transverse resolution (width of the point spread function) by a factor of 1.78 compared with a conventional fluorescence microscope can be obtained. We also examine the behavior of image scanning microscopy in two- or three-photon fluorescence and for two-photon excitation also with Bessel beam illumination. The combination of the optical sectioning effect of image scanning microscopy with multiphoton microscopy reduces background from the sample surface, which can increase penetration depth. For a detector array size of two Airy units, the resolution of two-photon image scanning microscopy is a factor 1.85 better and the peak of the point spread function 2.84 times higher than in nonconfocal two-photon fluorescence. The resolution of three-photon image scanning microscopy is a factor 2.10 better, and the peak of the point spread function is 3.77 times higher than in nonconfocal three-photon fluorescence. The resolution of two-photon image scanning microscopy with Bessel beam illumination is a factor 2.13 better than in standard two-photon fluorescence. Axial resolution and optical sectioning in two-photon or three-photon fluorescence are also improved by using the image scanning modality.
Published in J. Opt. Soc. Am. A, 2020
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Pixel reassignment in image scanning microscopy with a doughnut beam: example of maximum likelihood restoration
Abstract
In image scanning microscopy, the pinhole of a confocal microscope is replaced by a detector array. The point spread function for each detector element can be interpreted as the probability density function of the signal, the peak giving the most likely origin. This thus allows a form of maximum likelihood restoration, and compensation for aberrations, with similarities to adaptive optics. As an example of an aberration, we investigate theoretically and experimentally illumination with a vortex doughnut beam. After reassignment and summation over the detector array, the point spread function is compact, and the resolution and signal level higher than in a conventional microscope.
Published in J. Opt. Soc. Am. A, 2021
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Confocal-based fluorescence fluctuation spectroscopy with a SPAD array detector
Abstract
The combination of confocal laser-scanning microscopy (CLSM) and fluorescence fluctuation spectroscopy (FFS) is a powerful tool in studying fast, sub-resolution biomolecular processes in living cells. A detector array can further enhance CLSM-based FFS techniques, as it allows the simultaneous acquisition of several samples–essentially images—of the CLSM detection volume. However, the detector arrays that have previously been proposed for this purpose require tedious data corrections and preclude the combination of FFS with single-photon techniques, such as fluorescence lifetime imaging. Here, we solve these limitations by integrating a novel single-photon-avalanche-diode (SPAD) array detector in a CLSM system. We validate this new implementation on a series of FFS analyses: spot-variation fluorescence correlation spectroscopy, pair-correlation function analysis, and image-derived mean squared displacement analysis. We predict that the unique combination of spatial and temporal information provided by our detector will make the proposed architecture the method of choice for CLSM-based FFS.
Published in Light Sci. Appl., 2021
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Chromatin investigation in the nucleus using a phasor approach to structured illumination microscopy
Abstract
Chromatin in the nucleus is organized in functional sites at variable level of compaction. Structured illumination microscopy (SIM) can be used to generate three-dimensional super-resolution (SR) imaging of chromatin by changing in phase and in orientation a periodic line illumination pattern. The spatial frequency domain is the natural choice to process SIM raw data and to reconstruct an SR image. Using an alternative approach, we demonstrate that the additional spatial information encoded in the knowledge of the position of the illumination pattern can be efficiently decoded using a generalized version of separation of photon by lifetime tuning (SPLIT) that does not require lifetime measurements. In the resulting SPLIT-SIM, the SR image is obtained by isolating a fraction of the intensity corresponding to the center of the diffraction-limited point spread function. This extends the use of the SPLIT approach from stimulated emission depletion microscopy to SIM. The SPLIT-SIM algorithm is based only on phasor analysis and does not require deconvolution. We show that SPLIT-SIM can be used to generate SR images of chromatin organizational motifs with tunable resolution and can be a valuable tool for the imaging of functional sites in the nucleus.
Published in Biophys. J., 2021
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PRRT2 modulates presynaptic Ca2+ influx by interacting with P/Q-type channels
Abstract
Loss-of-function mutations in proline-rich transmembrane protein-2 (PRRT2) cause paroxysmal disorders associated with defective Ca2+ dependence of glutamatergic transmission. We find that either acute or constitutive PRRT2 deletion induces a significant decrease in the amplitude of evoked excitatory postsynaptic currents (eEPSCs) that is insensitive to extracellular Ca2+ and associated with a reduced contribution of P/Q-type Ca2+ channels to the EPSC amplitude. This synaptic phenotype parallels a decrease in somatic P/Q-type Ca2+ currents due to a decreased membrane targeting of the channel with unchanged total expression levels. Co-immunoprecipitation, pull-down assays, and proteomics reveal a specific and direct interaction of PRRT2 with P/Q-type Ca2+ channels. At presynaptic terminals lacking PRRT2, P/Q-type Ca2+ channels reduce their clustering at the active zone, with a corresponding decrease in the P/Q-dependent presynaptic Ca2+ signal. The data highlight the central role of PRRT2 in ensuring the physiological Ca2+ sensitivity of the release machinery at glutamatergic synapses.
Published in Cell Rep., 2021
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Evaluation of STED super-resolution image quality by image correlation spectroscopy (QuICS)
Abstract
Stimulated emission depletion (STED) microscopy is one of the most influential nanoscopy techniques; by increasing the STED beam intensity, it theoretically improves the spatial resolution to any desired value. However, the higher is the dose of stimulating photons, the stronger are the photo-bleaching and photo-toxicity effects, which potentially compromise live-cell and long-term imaging. For this reason the scientific community is looking for strategies to reduce the STED beam intensity needed to achieve a target resolution. Here, we show how the combination of STED microscopy with image scanning microscopy (ISM) meets this request. In particular, we introduce a new STED-ISM architecture – based on our recent single-photon-avalanche-diode (SPAD) detector array – which allows covering the near-diffraction limit resolution range with reduced STED beam intensity. We demonstrate this ability both with simulated data and in live-cell experiments. Because of (i) the minimal changes in the optical architecture of the typical point-scanning STED microscope; (ii) the parameter-free, robust and real-time pixel-reassignment method to obtain the STED-ISM image; (iii) the compatibility with all the recent progresses in STED microscopy, we envisage a natural and rapid upgrade of any STED microscope to the proposed STED-ISM architecture.
Published in Sci. Rep., 2021
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Evaluation of STED super-resolution image quality by image correlation spectroscopy (QuICS)
Abstract
Stimulated emission depletion (STED) microscopy is one of the most influential nanoscopy techniques; by increasing the STED beam intensity, it theoretically improves the spatial resolution to any desired value. However, the higher is the dose of stimulating photons, the stronger are the photo-bleaching and photo-toxicity effects, which potentially compromise live-cell and long-term imaging. For this reason the scientific community is looking for strategies to reduce the STED beam intensity needed to achieve a target resolution. Here, we show how the combination of STED microscopy with image scanning microscopy (ISM) meets this request. In particular, we introduce a new STED-ISM architecture – based on our recent single-photon-avalanche-diode (SPAD) detector array – which allows covering the near-diffraction limit resolution range with reduced STED beam intensity. We demonstrate this ability both with simulated data and in live-cell experiments. Because of (i) the minimal changes in the optical architecture of the typical point-scanning STED microscope; (ii) the parameter-free, robust and real-time pixel-reassignment method to obtain the STED-ISM image; (iii) the compatibility with all the recent progresses in STED microscopy, we envisage a natural and rapid upgrade of any STED microscope to the proposed STED-ISM architecture.
Published in bioRxiv, 2021
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Cooled SPAD array detector for low light-dose fluorescence laser scanning microscopy
Abstract
The single-photon timing and sensitivity performance and the imaging ability of asynchronous-readout single-photon avalanche diode (SPAD) array detectors have opened up enormous perspectives in fluorescence (lifetime) laser scanning microscopy (FLSM), such as super-resolution image scanning microscopy and high-information content fluorescence fluctuation spectroscopy (FFS). However, the strengths of these FLSM techniques depend on the many different characteristics of the detector, such as dark-noise, photon-detection efficiency, after-pulsing probability, and optical-cross talk, whose overall optimization is typically a trade-off between these characteristics. To mitigate this trade-off, we present a novel SPAD array detector with an active cooling system, which substantially reduces the dark-noise without significantly deteriorating any other detector characteristics. In particular, we show that lowering the temperature of the sensor to −15°C significantly improves the signal-to-noise ratio due to a 10-fold decrease in the dark-count rate compared to room temperature. As a result, for imaging, the laser power can be decreased by more than a factor of three, which is particularly beneficial for live-cell super-resolution imaging, as demonstrated in fixed and living cells expressing GFP-tagged proteins. For FFS, together with the benefit of the reduced laser power, we show that cooling the detector is necessary to remove artifacts in the correlation function, such as spurious negative correlations observed in the hot elements of the detector, i.e., elements whose dark-noise is substantially higher than the median value. Overall, this detector represents a further step towards the integration of SPAD array detectors in any FLSM system.
Published in bioRxiv, 2021
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Cooled SPAD array detector for low light-dose fluorescence laser scanning microscopy
Abstract
The single-photon timing and sensitivity performance and the imaging ability of asynchronous-readout single-photon avalanche diode (SPAD) array detectors have opened up enormous perspectives in fluorescence (lifetime) laser scanning microscopy (FLSM), such as super-resolution image scanning microscopy and high-information content fluorescence fluctuation spectroscopy. However, the strengths of these FLSM techniques depend on the many different characteristics of the detector, such as dark noise, photon-detection efficiency, after-pulsing probability, and optical cross talk, whose overall optimization is typically a trade-off between these characteristics. To mitigate this trade-off, we present, to our knowledge, a novel SPAD array detector with an active cooling system that substantially reduces the dark noise without significantly deteriorating any other detector characteristics. In particular, we show that lowering the temperature of the sensor to −15°C significantly improves the signal/noise ratio due to a 10-fold decrease in the dark count rate compared with room temperature. As a result, for imaging, the laser power can be decreased by more than a factor of three, which is particularly beneficial for live-cell super-resolution imaging, as demonstrated in fixed and living cells expressing green-fluorescent-protein-tagged proteins. For fluorescence fluctuation spectroscopy, together with the benefit of the reduced laser power, we show that cooling the detector is necessary to remove artifacts in the correlation function, such as spurious negative correlations observed in the hot elements of the detector, i.e., elements for which dark noise is substantially higher than the median value. Overall, this detector represents a further step toward the integration of SPAD array detectors in any FLSM system.
Published in Biophys. Rep., 2021
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The BrightEyes-TTM: an Open-Source Time-Tagging Module for Single-Photon Microscopy
Abstract
Fluorescence laser-scanning microscopy (LSM) is experiencing a revolution thanks to the introduction of new asynchronous read-out single-photon (SP) array detectors. These detectors give access to an entirely new set of single-photon information typically lost in conventional fluorescence LSM, thus triggering a new imaging/spectroscopy paradigm – the so-called singlephoton LSM (SP-LSM). The revolution’s outcomes are, from one side, the blooming of new SP-LSM techniques and tailored SP array detectors; from the other side, the need for data-acquisition (DAQ) systems effectively supporting such innovations. In particular, there is a growing need for DAQ systems capable of handling the high throughput and high temporal resolution information generated by the single-photon detectors. To fill this gap, we developed an open-source multi-channel timetagging module (TTM) based on a field-programmable-gatearray (FPGA), that can temporally tag single-photon events – with 30 ps precision – and synchronisation events – with 4 ns precision. Furthermore, being an open-access project, the TTM can be upgraded, modified, and customized by the microscopy-makers. We connected the TTM to a fluorescence LSM equipped with a single-photon avalanche diode (SPAD) bi-dimensional array detector, and we implemented fluorescence lifetime image scanning microscopy (FLISM) and, for the first time, fluorescence lifetime fluctuation spectroscopy (FLFS). We expect that our BrigthEyes-TTM will support the microscopy community to spread SP-LSM in many life science labs.
Published in bioRxiv, 2021
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ISM-FLUX: single-step MINFLUX with an array detector
Abstract
Single-molecule localization based on the concept of MINFLUX allows for molecular resolution imaging and tracking. However, MINFLUX has a limited field-of-view and therefore requires a precise and photon-costly pre-localization step. We propose ISM-FLUX, a localization technique that combines structured illumination with structured detection. We show through simulations that replacing the point-detector with a small (e.g. 5 × 5 pixels) single-photon camera and sequentially exciting the sample with four spatially separated doughnut-shaped beams, a localization precision between 1 and 15 nm can be obtained over a field-of-view (FOV) of more than 800 nm with 100 photons. The large FOV, induced by the camera, makes the pre-localization step obsolete. We calculate the effect of different parameters, such as the position of the doughnut beams and the number of pixels, on the localization precision. We predict that the combination of a good localization precision and the experimental simplicity of ISM-FLUX will help the wide adoption of MINFLUX based microscopy.
Published in bioRxiv, 2022
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Focus-ISM for Sharp and Gentle Super-Resolved Microscopy
Abstract
Super-resolution microscopy is routinely used for fixed and thin samples, while its feasibility for imaging live and thick samples is still limited. In the case of stimulated emission depletion (STED) microscopy, the high-intensity illumination required to achieve effective sub-diffraction resolution can introduce photo-damage, thus reducing the compatibility of the technique with live-cell imaging. Moreover, the out-of-focus fluorescence background may overcome the often faint signal stemming from the focal point, thus constraining imaging to thin samples. Here, we combined STED microscopy with image-scanning microscopy (ISM) to mitigate these limitations without any practical disadvantages. We first enhanced a laser scanning microscope (LSM) by introducing a detector array, hence providing access to a set of additional spatial information that is not available with a typical single-element detector. Then, we exploited this extended dataset to implement focus-ISM, a novel method that relaxes the high-intensity requirement of STED microscopy and removes the out-of-focus background. Additionally, we generalized the focus-ISM method to conventional LSM, namely without a STED beam. The proposed approach requires minimal architectural changes compared with conventional STED microscopes but provides substantial advantages for live and thick sample imaging while maintaining all compatibility with all recent advances in STED and confocal microscopy. As such, focus-ISM represents an essential step towards a universal super-resolved LSM technique for subcellular imaging.
Published in bioRxiv, 2022
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Reconstructing the Image Scanning Microscopy Dataset: an Inverse Problem
Abstract
Confocal laser-scanning microscopy (CLSM) is one of the most popular optical architectures for fluorescence imaging. In CLSM, a focused laser beam excites the fluorescence emission from a specific specimen position. Some actuators scan the probed region across the sample and a photodetector collects a single intensity value for each scan point, building a two-dimensional image pixel-by-pixel. Recently, new fast single-photon array detectors have allowed the recording of a full bi-dimensional image of the probed region for each scan point, transforming CLSM into image scanning microscopy (ISM). This latter offers significant improvements over traditional imaging but requires an optimal processing tool to extract a super-resolved image from the four-dimensional dataset. Here we describe the image formation process in ISM from a statistical point of view, and we use the Bayesian framework to formulate a multi-image deconvolution problem. Notably, the single-photon detector suffers exclusively from the photon shot noise, enabling the development of an effective likelihood model. We derive an iterative likelihood maximization algorithm and test it on experimental and simulated data. Furthermore, we demonstrate that the ISM dataset is redundant, enabling the possibility of obtaining reconstruction sampled at twice the scanning step. Our results prove that in ISM, under appropriate conditions, the Nyquist-Shannon sampling criterium is effectively relaxed. This finding can be exploited to speed up the acquisition process by a factor of four, further improving the versatility of ISM systems.
Published in arXiv, 2022
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The BrightEyes-TTM as an open-source time-tagging module for democratising single-photon microscopy
Abstract
Fluorescence laser-scanning microscopy (LSM) is experiencing a revolution thanks to new single-photon (SP) array detectors, which give access to an entirely new set of single-photon information. Together with the blooming of new SP LSM techniques and the development of tailored SP array detectors, there is a growing need for (i) DAQ systems capable of handling the high-throughput and high-resolution photon information generated by these detectors, and (ii) incorporating these DAQ protocols in existing fluorescence LSMs. We developed an open-source, low-cost, multi-channel time-tagging module (TTM) based on a field-programmable gate array that can tag in parallel multiple single-photon events, with 30 ps precision, and multiple synchronisation events, with 4 ns precision. We use the TTM to demonstrate live-cell super-resolved fluorescence lifetime image scanning microscopy and fluorescence lifetime fluctuation spectroscopy. We expect that our BrightEyes-TTM will support the microscopy community in spreading SP-LSM in many life science laboratories.
Published in Nat. Comm., 2022
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Focus image scanning microscopy for sharp and gentle super-resolved microscopy
Abstract
To date, the feasibility of super-resolution microscopy for imaging live and thick samples is still limited. Stimulated emission depletion (STED) microscopy requires high-intensity illumination to achieve sub-diffraction resolution, potentially introducing photodamage to live specimens. Moreover, the out-of-focus background may degrade the signal stemming from the focal plane. Here, we propose a new method to mitigate these limitations without drawbacks. First, we enhance a STED microscope with a detector array, enabling image scanning microscopy (ISM). Therefore, we implement STED-ISM, a method that exploits the working principle of ISM to reduce the depletion intensity and achieve a target resolution. Later, we develop Focus-ISM, a strategy to improve the optical sectioning and remove the background of any ISM-based imaging technique, with or without a STED beam. The proposed approach requires minimal architectural changes to a conventional microscope but provides substantial advantages for live and thick sample imaging.
Published in Nat. Comm., 2022
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Signal strength and integrated intensity in confocal and image scanning microscopy
Abstract
The properties of signal strength and integrated intensity in a scanned imaging system are reviewed. These properties are especially applied to confocal imaging systems, including image scanning microscopy. The integrated intensity, equal to the image of a uniform planar (sheet) object, rather than the peak of the point spread function, is a measure of the flux in an image. Analytic expressions are presented for the intensity in the detector plane for a uniform volume object, and for the resulting background. The variation in the integrated intensity with defocus for an offset point detector is presented. This axial fingerprint is independent of any pixel reassignment. The intensity in the detector plane is shown to contain the defocus information, and simple processing of the recorded data can improve optical sectioning and background rejection.
Published in J. Opt. Soc. Am. A, 2022
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SPLIT-PIN software enabling confocal and super-resolution imaging with a virtually closed pinhole
Abstract
In point-scanning microscopy, optical sectioning is achieved using a small aperture placed in front of the detector, i.e. the detection pinhole, which rejects the out-of-focus background. The maximum level of optical sectioning is theoretically obtained for the minimum size of the pinhole aperture, but this is normally prevented by the dramatic reduction of the detected signal when the pinhole is closed, leading to a compromise between axial resolution and signal-to-noise ratio. We have recently demonstrated that, instead of closing the pinhole, one can reach a similar level of optical sectioning by tuning the pinhole size in a confocal microscope and by analyzing the resulting image series. The method, consisting in the application of the separation of photons by lifetime tuning (SPLIT) algorithm to series of images acquired with tunable pinhole size, is called SPLIT-pinhole (SPLIT-PIN). Here, we share and describe a SPLIT-PIN software for the processing of series of images acquired at tunable pinhole size, which generates images with reduced out-of-focus background. The software can be used on series of at least two images acquired on available commercial microscopes equipped with a tunable pinhole, including confocal and stimulated emission depletion (STED) microscopes. We demonstrate applicability on different types of imaging modalities: (1) confocal imaging of DNA in a non-adherent cell line; (2) removal of out-of-focus background in super-resolved STED microscopy; (3) imaging of live intestinal organoids stained with a membrane dye.
Published in Sci. Rep., 2023
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ISM-FLUX: MINFLUX with an array detector
Abstract
Single-molecule localization based on the concept of MINFLUX allows for molecular resolution imaging and tracking with a laser-scanning microscopy architecture. In MINFLUX, a doughnut-shaped structured illumination beam sequentially excites the sample, thereby minimizing the localization uncertainty for a given number of photons. However, MINFLUX has a limited field of view (FOV) and therefore requires a precise prelocalization step. We mitigate this problem by introducing the single-molecule image scanning microscopy (smISM) concept. In smISM, the point detector of the laser-scanning microscope is replaced with a small single-photon detector array, thereby leveraging the benefits of both structured illumination and structured detection. We show via simulations that by combining smISM and MINFLUX (i.e., ISM-FLUX), we obtained a localization uncertainty between 1 and 15 nm over a FOV of more than 800 nm with 100 photons. In addition, smISM allows the localization of multiple molecules simultaneously. We calculate the effect of different parameters, such as the relative position of the doughnut beams, the number of detector pixels, the number of photons, and the signal-to-background ratio, on the localization uncertainty. We predict that the combination of a good localization precision and the enhanced robustness of ISM-FLUX will help the wide adoption of MINFLUX.
Published in Phys. Rev. Research, 2023
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Reconstructing the Image Scanning Microscopy Dataset: an Inverse Problem
Abstract
Confocal laser-scanning microscopy (CLSM) is one of the most popular optical architectures for fluorescence imaging. In CLSM, a focused laser beam excites the fluorescence emission from a specific specimen position. Some actuators scan the probed region across the sample and a photodetector collects a single intensity value for each scan point, building a two-dimensional image pixel-by-pixel. Recently, new fast single-photon array detectors have allowed the recording of a full bi-dimensional image of the probed region for each scan point, transforming CLSM into image scanning microscopy (ISM). This latter offers significant improvements over traditional imaging but requires an optimal processing tool to extract a super-resolved image from the four-dimensional dataset. Here we describe the image formation process in ISM from a statistical point of view, and we use the Bayesian framework to formulate a multi-image deconvolution problem. Notably, the single-photon detector suffers exclusively from the photon shot noise, enabling the development of an effective likelihood model. We derive an iterative likelihood maximization algorithm and test it on experimental and simulated data. Furthermore, we demonstrate that the ISM dataset is redundant, enabling the possibility of obtaining reconstruction sampled at twice the scanning step. Our results prove that in ISM, under appropriate conditions, the Nyquist-Shannon sampling criterium is effectively relaxed. This finding can be exploited to speed up the acquisition process by a factor of four, further improving the versatility of ISM systems.
Published in Inverse Probl., 2023
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Open-source tools enable accessible and advanced image scanning microscopy data analysis
Published in Nat. Photon., 2023
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Background Rejection in Two-Photon Fluorescence Image Scanning Microscopy
Abstract
We discuss the properties of signal strength and integrated intensity in two-photon excitation confocal microscopy and image scanning microscopy. The resolution, optical sectioning and background rejection are all improved over nonconfocal two-photon microscopy. Replacing the pinhole of confocal two-photon microscopy with a detector array increases the peak intensity of the point spread function. The outer pixels of a detector array give signals from defocused regions, and thus the processing of these, such as through subtraction, can further improve optical sectioning and background rejection.
Published in Photonics, 2023
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Content-enriched fluorescence lifetime fluctuation spectroscopy to study bio-molecular condensate formation
Abstract
Quantitative fluorescence microscopy is experiencing an important revolution thanks to single-photon array detectors. These detectors provide users with so far inaccessible specimen information: The distribution of the specimen’s fluorescence emission at single-photon level and high spatiotemporal sampling. In laser-scanning microscopy, this photon-resolved measurement has enabled robust fluorescence lifetime imaging at sub-diffraction spatial resolution, thus opening new perspectives for structural and functional imaging. Despite these significant advances in imaging, studying the time evolution of biological processes remains a considerable challenge. Here we present a com-prehensive framework of live-cell spectroscopy methodologies – compatible with imaging – to investigate bio-molecular processes at various spatiotemporal scales. We use photon-resolved spatial and temporal measurements granted by a single-photon array detector to boost the information content of a unified fluorescence fluctuation spectroscopy and fluorescence lifetime experiment. To demonstrate the potential of this approach, we investigate the phase transition of liquid-like condensates during oxidative stress inside living cells. These condensates are generally found in several cellular processes and exhibit substantial variations in molecular composition, size, and kinetics, posing a significant challenge for quantifying their underlying molecular dynamics. This study demonstrates how the pro-posed approach reveals the mutual dynamics of different RNA-binding proteins involved in the stress granules formation – inaccessible to imaging alone. We observe condensate formation by performing time-lapse super-resolved imaging of the cellular macro-environment while simultaneously monitoring the molecular mobility, the sub-diffraction environment organization, interactions, and nano-environment properties through fluorescence lifetime fluctuation spectroscopy. We are confident that our framework offers a versatile toolkit for investigating a broad range of bio-molecular processes – not limited to liquid-liquid phase transition – and we anticipate their widespread application in future life-science research.
Published in bioRxiv, 2023
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Compact and effective photon-resolved image scanning microscope
Abstract
Fluorescence confocal laser-scanning microscopy (LSM) is one of the most popular tools for life science research. This popularity is expected to grow thanks to single-photon array detectors tailored for LSM. These detectors offer unique single-photon spatiotemporal information, opening new perspectives for gentle and quantitative super-resolution imaging. However, a flawless recording of this information poses significant challenges for the microscope data-acquisition system. Here, we present a data-acquisition module based on the digital frequency domain principle, able to record photons’ essential spatial and temporal features. We use this module to extend the capabilities of established imaging techniques based on single-photon avalanche diode (SPAD) array detectors, such as fluorescence lifetime image scanning microscopy. Furthermore, we use the module to introduce a robust multi-species approach encoding the fluorophore’s excitation spectra in the time domain. Lastly, we combine time-resolved stimulated emission depletion microscopy with image scanning microscopy, boosting spatial resolution. Our results demonstrate how a conventional fluorescence laser scanning microscope can transform into a simple, information-rich, super-resolved imaging system with the simple addition of a SPAD array detector with a tailored data acquisition system. We expected a blooming of advanced single-photon imaging techniques which effectively harness all the sample information encoded in each photon.
Published in bioRxiv, 2023
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Image scanning microscopy with a doughnut beam: signal strength and integrated intensity
Abstract
We discuss the effects of image scanning microscopy using doughnut beam illumination on the properties of signal strength and integrated intensity. Doughnut beam illumination can give better optical sectioning and background rejection than Airy disk illumination. The outer pixels of a detector array give a signal from defocused regions, so digital processing of these (e.g., by simple subtraction) can further improve optical sectioning and background rejection from a single in-focus scan.
Published in J. Opt. Soc. Am. A, 2023
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4D Single-Particle Tracking with Asynchronous Read-Out SPAD-Array Detector
Abstract
Single-particle tracking (SPT) techniques are essential for investigating the com-plex functions and interactions of individual, specifically labelled particles in biological environments. Many SPT techniques exist, each optimised towards a different balance between spatiotemporal resolution and range, technical com-plexity, and information content. This bargain is exemplified by the contrast between wide-field camera-based and real-time SPT approaches, with the latter being generally more advanced but at the cost of high complexity. Further-more, the fluorescence lifetime, a powerful tool for investigating the particle’s interactions and nano-environment, has yet to be measured consistently. To overcome these limitations, we propose a novel real-time three-dimensional SPT technique based on a hybrid approach. In our implementation, we equip a confocal laser-scanning microscope with an asynchronous read-out single-photon avalanche diode (SPAD) array detector and few other optics. Each sensitive detector element acts as a confocal pinhole, and the recorded intensity distribu-tion reflects the particle’s position in three dimensions relative to the excitation volume. This localization is used in a real-time feedback system to keep the par-ticle in the centre of the excitation volume. Importantly, as each pixel is an independent single-photon detector, SPT is combined with fluorescence lifetime measurement. Our system achieves a localization precision of up to 30 nm with 100 photons and microsecond time resolution, while also performing fluorescence lifetime mea-surements. First, we validated the technique by tracking fluorescent particles in artificial environments. Secondly, as further validation, we investigated the move-ment of lysosomes in living SK-N-BE cells and measured the fluorescence lifetime of the GFP marker expressed on a membrane protein. We observed an unprece-dented correlation between the changes in fluorescence lifetime and the motion state of the lysosomes. Thanks to its simplicity and the great momentum of confocal microscopy based on SPAD array detector, we expect that this implementation will open to many information-rich correlative imaging and tracking studies.
Published in bioRxiv, 2023
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M6A reduction relieves FUS-associated ALS granules
Abstract
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease due to gradual motorneurons (MN) degeneration. Among the processes associated to ALS pathogenesis, there is the formation of cytoplasmic inclusions produced by mutant protein aggregation, among which the RNA binding protein FUS. In this work we show that such inclusions are significantly reduced in number and dissolve faster when the RNA m6A content is diminished as a consequence of the m6A writer METTL3 knock-down. These effects were observed both in neuronal cell lines and in iPSC-derived human motor neurons expressing mutant FUS. Importantly, stress granules formed in ALS condition showed a distinctive transcriptome with respect to control cells; interestingly, after METTL3 downregulation, it reverted to similar to control. Finally, we show that FUS inclusions are reduced also in patient-derived fibroblasts treated with STM-2457, a well characterized inhibitor of METTL3 activity, paving the way for its possible use for counteracting aggregate formation in ALS.
Published in bioRxiv, 2023
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Single-photon microscopy to study biomolecular condensates
Abstract
Biomolecular condensates serve as membrane-less compartments within cells, concentrating proteins and nucleic acids to facilitate precise spatial and temporal orchestration of various biological processes. The diversity of these processes and the substantial variability in condensate characteristics present a formidable challenge for quantifying their molecular dynamics, surpassing the capabilities of conventional microscopy. Here, we show that our single-photon microscope provides a comprehensive live-cell spectroscopy and imaging framework for investigating biomolecular condensation. Leveraging a single-photon detector array, single-photon microscopy enhances the potential of quantitative confocal microscopy by providing access to fluorescence signals at the single-photon level. Our platform incorporates photon spatiotemporal tagging, which allowed us to perform time-lapse super-resolved imaging for molecular sub-diffraction environment organization with simultaneous monitoring of molecular mobility, interactions, and nano-environment properties through fluorescence lifetime fluctuation spectroscopy. This integrated correlative study reveals the dynamics and interactions of RNA-binding proteins involved in forming stress granules, a specific type of biomolecular condensates, across a wide range of spatial and temporal scales. Our versatile framework opens up avenues for exploring a broad spectrum of biomolecular processes beyond the formation of membrane-less organelles.
Published in Nat. Comm., 2023
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Compact and effective photon-resolved image scanning microscope
Abstract
Fluorescence confocal laser-scanning microscopy (LSM) is one of the most popular tools for life science research. This popularity is expected to grow thanks to single-photon array detectors tailored for LSM. These detectors offer unique single-photon spatiotemporal information, opening new perspectives for gentle and quantitative superresolution imaging. However, a flawless recording of this information poses significant challenges for the microscope data acquisition (DAQ) system. We present a DAQ module based on the digital frequency domain principle, able to record essential spatial and temporal features of photons. We use this module to extend the capabilities of established imaging techniques based on single-photon avalanche diode (SPAD) array detectors, such as fluorescence lifetime image scanning microscopy. Furthermore, we use the module to introduce a robust multispecies approach encoding the fluorophore excitation spectra in the time domain. Finally, we combine time-resolved stimulated emission depletion microscopy with image scanning microscopy, boosting spatial resolution. Our results demonstrate how a conventional fluorescence laser scanning microscope can transform into a simple, information-rich, superresolved imaging system with the simple addition of a SPAD array detector with a tailored data acquisition system. We expected a blooming of advanced single-photon imaging techniques, which effectively harness all the sample information encoded in each photon.
Published in Adv. Phot., 2024
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ALS-associated FUS mutation reshapes the RNA and protein composition of Stress Granules
Abstract
Stress Granules (SG) formation is a cellular protection mechanism, constituting a storage for untranslated mRNAs and RNA-binding proteins (RBPs); however, these condensates can turn into pathological aggregates, related to the onset of neurodegenerative diseases like Amyotrophic Lateral Sclerosis (ALS). This transition towards cytotoxic inclusions is triggered by ALS-causative mutations in the RBP FUS, which lead to its cytoplasmic mis-localization and accumulation in SG. Here, we describe the SG transcriptome in a neural context and describe several features for RNA recruitment in SG. We demonstrate that SG dynamics and RNA content are strongly modified by the incorporation of mutant FUS, switching to a more unstructured, AU-rich SG transcriptome. Moreover, we show that mutant FUS, together with its protein interactors and their target RNAs, are responsible for the reshaping of the mutant SG transcriptome with alterations that can be linked to neurodegeneration. Therefore, our data give a comprehensive view of the molecular differences between physiological and pathological SG in ALS conditions, showing how FUS mutations impact the RNA and protein population of these condensates.
Published in bioRxiv, 2024
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research
Image Processing and Analysis
Team members:
Giacomo Garrè
Francesco Del Bufalo
Marco Castello
Giorgio Tortarolo
Sami Koho
Publications:
 | Open-source tools enable accessible and advanced image scanning microscopy data analysis *Alessandro Zunino*, *Eli Slenders*, *Francesco Fersini*, *Andrea Bucci*, *Mattia Donato*, *Giuseppe Vicidomini* Published in Nat. Photon., May 2023 (see publication ) - Paper, Corresponding author |
 | Reconstructing the Image Scanning Microscopy Dataset: an Inverse Problem *Alessandro Zunino*, *Marco Castello*, *Giuseppe Vicidomini* Published in Inverse Probl., April 2023 (see publication ) - Paper, Corresponding author |
 | SPLIT-PIN software enabling confocal and super-resolution imaging with a virtually closed pinhole Elisabetta Di Franco, Angelita Costantino, Elena Cerutti, Morgana D’Amico, Anna P Privitera, Paolo Bianchini, *Giuseppe Vicidomini*, Massimo Gulisano, Alberto Diaspro, Luca Lanzanò Published in Sci. Rep., March 2023 (see publication ) - Paper |
 | Reconstructing the Image Scanning Microscopy Dataset: an Inverse Problem *Alessandro Zunino*, *Marco Castello*, *Giuseppe Vicidomini* Published in arXiv, November 2022 (see publication ) - Preprint, Corresponding author |
 | Evaluation of STED super-resolution image quality by image correlation spectroscopy (QuICS) Elena Cerutti, Morgana D'Amico, Isotta Cainero, Gaetano Ivan Dellino, Mario Faretta, *Giuseppe Vicidomini*, Pier Giuseppe Pelicci, Paolo Bianchini, Alberto Diaspro Published in bioRxiv, August 2021 (see publication ) - Paper |
| | Evaluation of STED super-resolution image quality by image correlation spectroscopy (QuICS) Elena Cerutti, Morgana D'Amico, Isotta Cainero, Gaetano Ivan Dellino, Mario Faretta, *Giuseppe Vicidomini*, Pier Giuseppe Pelicci, Paolo Bianchini, Alberto Diaspro Published in Sci. Rep., August 2021 (see publication ) - Paper |
 | Chromatin investigation in the nucleus using a phasor approach to structured illumination microscopy Isotta Cainero, Elena Cerutti, Mario Faretta, Gaetano Ivan Dellino, Pier Giuseppe Pelicci, Paolo Bianchini, *Giuseppe Vicidomini*, Alberto Diaspro, Luca Lanzanò Published in Biophys. J., May 2021 (see publication ) - Paper |
 | Two-photon image-scanning microscopy with SPAD array and blind image reconstruction *Sami V. Koho*, *Eli Slenders*, *Giorgio Tortarolo*, *Marco Castello*, Mauro Buttafava, Federica Villa, Elena Tcarenkova, Marcel Ameloot, Paolo Bianchini, Colin J.R. Sheppard, Albeto Diaspro, Alberto Tosi, *Giuseppe Vicidomini* Published in Biomed. Opt. Express, May 2020 (see publication ) - Paper, Corresponding author |
 | Fourier ring correlation simplifies image restoration in fluorescence microscopy *Sami V. Koho*, *Giorgio Tortarolo*, *Marco Castello*, Talahiro Deguchi, Albeto Diaspro, *Giuseppe Vicidomini* Published in Nat. Comm., July 2019 (see publication ) - Paper, Corresponding author |
| | Easy Two-Photon Image-Scanning Microscopy With Spad Array And Blind Image Reconstruction *Sami V. Koho*, *Eli Slenders*, *Giorgio Tortarolo*, *Marco Castello*, Mauro Buttafava, Federica Villa, Elena Tcarenkova, Marcel Ameloot, Paolo Bianchini, Colin J.R. Sheppard, Albeto Diaspro, Alberto Tosi, *Giuseppe Vicidomini* Published in bioRxiv, April 2019 (see publication ) - Preprint, Corresponding author |
Published:
Single-Photon Laser-Scanning Microscopy with SPAD Array Detector
Laser-scanning microscopy (LSM) is one of the most popular optical microscopy architectures in Life Sciences. This popularity is due to the possibility to combine LSM with the fluorescence mechanism and with many advanced microscopy techniques: confocal microscopy, which provides three-dimensional imaging; two-photon excitation (TPE) microscopy, which offers deep imaging; fluorescence lifetime, which offers functional imaging; fluorescence fluctuation spectroscopy (FFS), which allows deciphering molecular dynamics, to mention a few. This synergy results in a microscope system with a unique combination of spatial and temporal characteristics and a strong ability to provide a vast pool of information about the sample investigated.
However, the performance of current fluorescence LSM can be tremendously improved by changing the image recording process. In conventional fluorescence LSM, the so-called detection/probing volume is raster scanned across the specimen. For each specimen position, a single-element detector (e.g., a photo-multiplier tube (PMT)) samples the fluorescence signal in time, but spatial integrates across its sensitive area. Successively, the data-acquisition system temporally integrates the signal along the pixel-dwell time (in the range of microseconds) and produces a single intensity value per position/pixel. Finally, a computer build-up the digital image. In short, for each sample position (i.e., detection volume), the induced fluorescence photons are integrated regardless of their spatial and temporal distribution; thus, the information potentially encoded in the dynamic and image of each probing volume are lost. Similar information loss occurs in a single-point fluorescence correlation spectroscopy (FCS) experiment, where, since the detection volume is stationary, the photons are not integrated along the pixel-dwell time, but along the bin of the time-trace (in the range of microseconds).
To solve this limitation, we have introduced a series of asynchronous read-out single-photon avalanche diode (SPAD) array detectors able to fully preserve the spatial and temporal information encoded in the probing volume of the fluorescence laser-scanning microscope. In particular, the SPAD array detectors are composed of a small number (e.g., 5 by 5) of micro-sized element/pixel able to independently deliver a digital signal, with a precision of a few hundred picoseconds, every time that a photon is registered. Thanks to the micron size of the elements, a zoom-lens system allows it to quickly obtain a sub-Nyquist sampling of the detection volume image. Simultaneously, a time-resolved data-acquisition system, synchronized with a pulse laser beam, allows recording the fluorescence signal with a temporal resolution compatible with most of the photo-physical phenomena relevant to Life science experiments, such as the excited-state fluorescence lifetime. As a mother of fact, these SPAD array detectors allow implementing an LSM architecture where the image (more in general data) recording process does not introduce any information lost described above.
However, the performance of current fluorescence LSM can be tremendously improved by changing the image recording process. In conventional fluorescence LSM, the so-called detection/probing volume is raster scanned across the specimen. For each specimen position, a single-element detector (e.g., a photo-multiplier tube (PMT)) samples the fluorescence signal in time, but spatial integrates across its sensitive area. Successively, the data-acquisition system temporally integrates the signal along the pixel-dwell time (in the range of microseconds) and produces a single intensity value per position/pixel. Finally, a computer build-up the digital image. In short, for each sample position (i.e., detection volume), the induced fluorescence photons are integrated regardless of their spatial and temporal distribution; thus, the information potentially encoded in the dynamic and image of each probing volume are lost. Similar information loss occurs in a single-point fluorescence correlation spectroscopy (FCS) experiment, where, since the detection volume is stationary, the photons are not integrated along the pixel-dwell time, but along the bin of the time-trace (in the range of microseconds).
To solve this limitation, we have introduced a series of asynchronous read-out single-photon avalanche diode (SPAD) array detectors able to fully preserve the spatial and temporal information encoded in the probing volume of the fluorescence laser-scanning microscope. In particular, the SPAD array detectors are composed of a small number (e.g., 5 by 5) of micro-sized element/pixel able to independently deliver a digital signal, with a precision of a few hundred picoseconds, every time that a photon is registered. Thanks to the micron size of the elements, a zoom-lens system allows it to quickly obtain a sub-Nyquist sampling of the detection volume image. Simultaneously, a time-resolved data-acquisition system, synchronized with a pulse laser beam, allows recording the fluorescence signal with a temporal resolution compatible with most of the photo-physical phenomena relevant to Life science experiments, such as the excited-state fluorescence lifetime. As a mother of fact, these SPAD array detectors allow implementing an LSM architecture where the image (more in general data) recording process does not introduce any information lost described above.
Published:
Time-Resolved STED Microscopy
Diffraction does not allow light to be focused to a volume smaller than roughly one-half of the light wavelength along the lateral directions (x,y) and three times larger along the optical axis (z). Stimulated emission depletion (STED) microscopy (Hell et al., Opt. Letters, 19(11):780-782, 1994, Vicidomini et al., Nat. Methods, 8:571–573, 2018) overcomes this diffraction limit by reversibly silencing (depleting) fluorophores at predefined positions of the diffraction-limited excitation volumes. Only the non-silenced fluorophores in the complementary regions emit light, allowing features closer than the diffraction limit to be separated. In the most typical STED microscopy implementation, the fluorescent confinement is obtained by coaligning the Gaussian excitation beam of a scanning microscope with a second beam, called the STED beam, which (i) is tuned in wavelength to de-excite fluorophores via stimulated emission (SE) and (ii) is engineered to create a doughnut-shaped focal intensity distribution with a ‘zero’-intensity point in the center. Although the STED beam focal intensity distribution is diffraction limited, high intensities saturate the SE transition and keep virtually all the fluorophores in the ground state, except those located in a region around the ‘zero’-intensity point, whose size reaches sub-diffraction values and decreases with increasing STED beam intensity. Thus, scanning the coaligned beams together across a specimen leads to an image where the (sub-diffraction) spatial resolution is given by the size of the effective fluorescent volume around the ‘zero’.
Theoretically, STED microscopy resolution can reach the molecule’s size (the ultimate limit of a fluorescent microscope). In practice, it is limited by different factors, and in particular by the photo-damages effects. To efficiently deplete a fluorophore, SE has to win the competition with spontaneous emission, which typically occurs within a few nanoseconds after the excitation event (fluorophore’s excited-state lifetime). This short temporal window and the small cross-section of SE demand a high flux of stimulating photons. For example, to quench by half the fluorescence of a fluorophore with 4 ns excited-state fluorescence lifetime and 25 cm2/J stimulated emission cross-section requires 10 MW/cm2 intensity (saturation intensity). Because a complete quenching of a fluorophore requires much higher intensities, and because the intensity reduces quadratically from the doughnut-crest to the ‘zero’-intensity point, effective resolution enhancements require > 1 GW/cm2 intensity (at the doughnut-crest). At first, this request was achieved using expensive and complex mode-locked pulsed laser architectures (pulsed-STED microscopy), which, together with the photo-damage problem, initially slowed the growth and dissemination of STED microscopy.
A class of methods which mitigates both problems, i.e., which reduce the (peak) intensity -- to achieve a certain spatial resolution, and the system complexity, base on the analysis of the fluorescence dynamics (time-resolved STED microscopy). Since the SE process opens a new de-excitation pathway for an excited fluorophore, the fluorophores illuminated by the STED beam show a shorter effective excited-state fluorescence lifetime than the fluorophores not illuminated. In particular, the higher is the insensity of the STED beam the shorter is the effective lifetime. Because the doughnut-shaped distribution of the SE intensity, the fluorophores located at the doughnut-crest show the shortest lifetime, which increases up to the natural lifetime moving toward to the 'zero'-intensity point (Vicidomini et al., PLoS One, 8(1):e54421, 2013). This spatial lifetime signature of the fluorophore inside the excitation volume can be used to isolate the fluorescecne signal generated by the longer-lived fluorophores located at the 'zero'-intensity point from the signal generated from the fluorophores in the pheriphey. The final result is a detection volume, thus an (optical) spatial resolution, which reduces also without a complete depletion of the pheripheral fluorophores (incomplete depletion), and without increasing the STED beam intensity.
The first implementation using the time-resolved STED principle is the so-called gated-STED microscope (Vicidomini et al., Nat. Methods, 8:571–573, 2011, Moffit et al., Opt. Express, 19(5):4242-4254, 2011). The fluorescence signal of a STED microscope is registered in a time-correlated-single-photon-counting (TCSPC) mode and the image is formed solely with the photons registered after a certain time from the excitation events, i.e., time-gated detection. Thanks to this scheme, the photons contributing to the image are likely to be emitted by the long-lived fluorophores located in the inner part of the STED effective fluorescence volume. Initially, gated-STED microscopy used STED beams running in continuous-wave (CW), thus obtaining not only a sensitive reduction of the STED bams (peak) intensity to achieve effective sub-diffraction resolution, but also a reduction in complexity and costs. On the contrary, the time-gating benefits for the early architectures based on mode-locked pulsed lasers were negligible. However, while STED beams running in CW reduce the peak intensity, they also result in useless illumination: typically, excitation uses 80 MHz (or lower) pulsed beams and the fluorescence lifetime is only a few nanoseconds. Thus the duty-cycle is small. In light of this consideration and with the time-resolved STED principle in mind, the mode-locked lasers of the pulsed-STED implementations were replaced by (sub)nanosecond pulsed laser. In this gated pulsed-STED implementation the STED beam illumination is optimized and time-gating reduces the (peak) intensity (Castello et al., Microsc. Res. Tech., 79(9):785-791, 2016).
Notably, the analysis of the fluorescence dynamics to improve the resolution of conventional microscopy (Enderlein, Appl. Phys. Lett., 87:094105, 2005), the combination of TCSPC with STED microscopy (Auksorius et al., Opt. Lett., 33(2): 113-115, 2008), and the effects of SE on the fluorophore lifetime (Marsh et al., Chem. Phys. Lett., 366(3):398-405, 2002), were all topics already investigated. However, to the best of our knowledge, the combination of all these aspects to reduce the (peak) intensity in STED microscopy was never explored up to the introduction of gated-STED microscopy. Thereby, the gated-STED story can be well described by the famous sentence of Sir Isaac Newton: "If I have seen further, it is by standing upon the shoulders of giants."
The major limitation of gated-STED microscopy is that reducing the detection volume, i.e., the improvement of the (optical) resolution, is obtained at the cost of a signal reduction. Thus, for low fluorescent photon flux or high background, reducing the signal to noise/background ratio can cancel-out the effective resolution enhancement. Indeed, time-gated detection removes completely the fluorescent photons originating from the periphery of the effective fluorescent volume, but partially also the photons from the center. Two different approaches partially solve this limitation. The first approach uses image deconvolution (Castello et al., Appl. Phys. Lett., 105:234106, 2014): The TCSPC imaging modality provides a three-dimensional STED image (x,y,t, where t is the lag-time of the photon-arrival histogram), which is deconvolved with an effective temporal point-spread-function describing the fluorescence dynamics both spatially and temporally. The second method, called separation-by-lifetime-tuning (SPLIT), uses the phasor plot representation (Lanzanò et al., Nat. Commun., 6:6701, 2015): Each pixel of the TCSPC image is represented in the phasor domain. The phasor's linear property allows separating the photons emitted from the long-lived fluorophores in the center of the effective fluorescent volume from the photons emitted by the short-lived fluorophore at the periphery.
The aim of this project is to develop new algorithms which combine deconvolution and phasor plot representation to further ehnance the effective resolution of time-resolved STED microscopy. Within the same goal, the project will take also advantages from new photon detectors, lasers, optical strategies, and probes.
Theoretically, STED microscopy resolution can reach the molecule’s size (the ultimate limit of a fluorescent microscope). In practice, it is limited by different factors, and in particular by the photo-damages effects. To efficiently deplete a fluorophore, SE has to win the competition with spontaneous emission, which typically occurs within a few nanoseconds after the excitation event (fluorophore’s excited-state lifetime). This short temporal window and the small cross-section of SE demand a high flux of stimulating photons. For example, to quench by half the fluorescence of a fluorophore with 4 ns excited-state fluorescence lifetime and 25 cm2/J stimulated emission cross-section requires 10 MW/cm2 intensity (saturation intensity). Because a complete quenching of a fluorophore requires much higher intensities, and because the intensity reduces quadratically from the doughnut-crest to the ‘zero’-intensity point, effective resolution enhancements require > 1 GW/cm2 intensity (at the doughnut-crest). At first, this request was achieved using expensive and complex mode-locked pulsed laser architectures (pulsed-STED microscopy), which, together with the photo-damage problem, initially slowed the growth and dissemination of STED microscopy.
A class of methods which mitigates both problems, i.e., which reduce the (peak) intensity -- to achieve a certain spatial resolution, and the system complexity, base on the analysis of the fluorescence dynamics (time-resolved STED microscopy). Since the SE process opens a new de-excitation pathway for an excited fluorophore, the fluorophores illuminated by the STED beam show a shorter effective excited-state fluorescence lifetime than the fluorophores not illuminated. In particular, the higher is the insensity of the STED beam the shorter is the effective lifetime. Because the doughnut-shaped distribution of the SE intensity, the fluorophores located at the doughnut-crest show the shortest lifetime, which increases up to the natural lifetime moving toward to the 'zero'-intensity point (Vicidomini et al., PLoS One, 8(1):e54421, 2013). This spatial lifetime signature of the fluorophore inside the excitation volume can be used to isolate the fluorescecne signal generated by the longer-lived fluorophores located at the 'zero'-intensity point from the signal generated from the fluorophores in the pheriphey. The final result is a detection volume, thus an (optical) spatial resolution, which reduces also without a complete depletion of the pheripheral fluorophores (incomplete depletion), and without increasing the STED beam intensity.
The first implementation using the time-resolved STED principle is the so-called gated-STED microscope (Vicidomini et al., Nat. Methods, 8:571–573, 2011, Moffit et al., Opt. Express, 19(5):4242-4254, 2011). The fluorescence signal of a STED microscope is registered in a time-correlated-single-photon-counting (TCSPC) mode and the image is formed solely with the photons registered after a certain time from the excitation events, i.e., time-gated detection. Thanks to this scheme, the photons contributing to the image are likely to be emitted by the long-lived fluorophores located in the inner part of the STED effective fluorescence volume. Initially, gated-STED microscopy used STED beams running in continuous-wave (CW), thus obtaining not only a sensitive reduction of the STED bams (peak) intensity to achieve effective sub-diffraction resolution, but also a reduction in complexity and costs. On the contrary, the time-gating benefits for the early architectures based on mode-locked pulsed lasers were negligible. However, while STED beams running in CW reduce the peak intensity, they also result in useless illumination: typically, excitation uses 80 MHz (or lower) pulsed beams and the fluorescence lifetime is only a few nanoseconds. Thus the duty-cycle is small. In light of this consideration and with the time-resolved STED principle in mind, the mode-locked lasers of the pulsed-STED implementations were replaced by (sub)nanosecond pulsed laser. In this gated pulsed-STED implementation the STED beam illumination is optimized and time-gating reduces the (peak) intensity (Castello et al., Microsc. Res. Tech., 79(9):785-791, 2016).
Notably, the analysis of the fluorescence dynamics to improve the resolution of conventional microscopy (Enderlein, Appl. Phys. Lett., 87:094105, 2005), the combination of TCSPC with STED microscopy (Auksorius et al., Opt. Lett., 33(2): 113-115, 2008), and the effects of SE on the fluorophore lifetime (Marsh et al., Chem. Phys. Lett., 366(3):398-405, 2002), were all topics already investigated. However, to the best of our knowledge, the combination of all these aspects to reduce the (peak) intensity in STED microscopy was never explored up to the introduction of gated-STED microscopy. Thereby, the gated-STED story can be well described by the famous sentence of Sir Isaac Newton: "If I have seen further, it is by standing upon the shoulders of giants."
The major limitation of gated-STED microscopy is that reducing the detection volume, i.e., the improvement of the (optical) resolution, is obtained at the cost of a signal reduction. Thus, for low fluorescent photon flux or high background, reducing the signal to noise/background ratio can cancel-out the effective resolution enhancement. Indeed, time-gated detection removes completely the fluorescent photons originating from the periphery of the effective fluorescent volume, but partially also the photons from the center. Two different approaches partially solve this limitation. The first approach uses image deconvolution (Castello et al., Appl. Phys. Lett., 105:234106, 2014): The TCSPC imaging modality provides a three-dimensional STED image (x,y,t, where t is the lag-time of the photon-arrival histogram), which is deconvolved with an effective temporal point-spread-function describing the fluorescence dynamics both spatially and temporally. The second method, called separation-by-lifetime-tuning (SPLIT), uses the phasor plot representation (Lanzanò et al., Nat. Commun., 6:6701, 2015): Each pixel of the TCSPC image is represented in the phasor domain. The phasor's linear property allows separating the photons emitted from the long-lived fluorophores in the center of the effective fluorescent volume from the photons emitted by the short-lived fluorophore at the periphery.
The aim of this project is to develop new algorithms which combine deconvolution and phasor plot representation to further ehnance the effective resolution of time-resolved STED microscopy. Within the same goal, the project will take also advantages from new photon detectors, lasers, optical strategies, and probes.
Published:
Fluorescence Fluctuation Spectroscopy with SPAD Array Detector
Over the last decades, fluorescence correlation spectroscopy (FCS) has been successfully applied to many biological systems to study molecular transports, dynamic behavior of macromolecules, membrane structures, and organization of chromatin. By measuring and analyzing the fluorescence intensity fluctuations generated by biomolecules moving in-and-out of the microscope detection volume(s), parameters such as diffusion coefficients, molecular concentrations, and the fluorescence brightness can be calculated. The (sub)microsecond temporal resolution and the low background typically needed for FCS are usually obtained by employing a point-detector - such as a photomultiplier tube or an avalanche photodiode - in a confocal microscope system. The detector integrates all photons in the detection volume, thus ignoring all information encoded in the spatial distribution of the photons. Consequently, FCS has a limited value when studying more complex situations, e.g. FCS is not able to measure the direction of active transport in a sample undergoing anomalous diffusion. Other fluorescence fluctuation spectroscopy techniques that do register spatial information with a camera, such as spatiotemporal image scanning correlation spectroscopy, exist, but the spatial information comes at the cost of a very poor temporal resolution.
Published:
Investigating the dynamics of bio-molecular condensates and organelles by spectroscopy tools and super-resolution imaging
A critical aspect of all biological processes is their temporal organization at all biological levels, ranging from embryo development, cell/tissue homeostasis, and, in general, all biomolecular functions. Despite the massive improvements in optical resolution, studying temporal processes and biomolecular dynamics is still challenging. We are working towards shining new light into the complex behaviors of single biomolecules and organelles by applying a comprehensive class of novel microscopy methods based on the high throughput of single-photon avalanche diode (SPAD) array detectors.
Published:
talks
Talk 1 on Relevant Topic in Your Field
This is a description of your talk, which is a markdown files that can be all markdown-ified like any other post. Yay markdown!
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Conference Proceeding talk 3 on Relevant Topic in Your Field
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teaching
Optics for Microscopy and Spectroscopy
Ph.D. student course, DIBRIS, University of Genoa in collaboration with Istituto Italiano di Tecnologia, 2025
Principle of Image Scanning Microscopy
Download - Presentation Slides
Lecture, online, 2025
team
Giorgio Tortarolo
Short Bio
Giorgio Tortarolo studied engineering at the Dipartimento di Informatica, Bioingegneria, Robotica e Ingegneria dei Sistemi (DIBRIS) of the University of Genoa. In 2015, he obtained the M.Sc. degree (110/110 cum laude, right of publication) for the thesis titled “Modular integration of a STED imaging system into a custom confocal microscope”, later awarded from Società Italiana di Ottica e Fotonica (SIOF). After the graduation, Giorgio joined IIT as a fellow student, under the supervisions of Dr. Giuseppe Vicidomini and Prof. Alberto Diaspro. During the six-months fellowship period, he contributed to develop the Fourier Ring Correlation Analysis, a quantitative criterium to assess the effective spatial resolution of any point-scanning microscopy image. At the end of the fellowship, Giorgio spent two months in Illinois, USA, collaborating with a well established microscopy company, ISS, to introduce in their product line a novel STED microscope.
Published:
Giuseppe Vicidomini
Short Bio
Published:
Alessandro Rossetta
Note: Alessandro Rossetta has a joint position at Nanoscopy (NIC@IIT) and MMS laboratories.
Published:
Eli Slenders
Short Bio
Eli Slenders has a bachelor’s and master’s degree in physics from Hasselt University (Hasselt, Belgium) and KU Leuven (Leuven, Belgium), respectively. He graduated in 2013. From 2014 to 2018, he worked as a PhD student at Hasselt University in the Biophysics group of prof. M. Ameloot. His PhD thesis was entitled “Resolution in coherent and incoherent optical imaging with two-photon excitation microscopy”. From 2019 to 2021, Eli worked as a post-doctoral researcher under the supervision of Giuseppe Vicidomini in the Molecular Microscopy and Spectroscopy research line at the Italian Institute of Technology (IIT, Genoa, Italy) where he developed SPAD-FFS, an optical tool to study biomolecular dynamics. Since March 2021, Eli is a Marie Skłodowska-Curie Actions research fellow in dr. Vicidomini’s group, working on time-resolved low light-dose single molecule imaging with SPAD array detectors.
Published:
Andrea Bucci
Short Bio
“Not a Physicist. Not an Engineer. All he knows is what he’s not. If only he could see what he is.”
Andrea Bucci studied Engineering Physics at Politecnico of Milan (Milano, Italy) and subsequently at KTH (Stockholm, Sweden), where he got his M.Sc. in 2019 with a thesis entitled “Extended Parallelization of the Illumination in Optical Nanoscopy”. After his graduation, he worked for 6 months as a student intern at KAUST (Thuwal, Saudi Arabia).
Since 2020, Andrea is a PhD student under the supervision of Giuseppe Vicidomini in the Molecular Microscopy and Spectroscopy group at the Italian Institute of Technology (Genoa, Italy), working in the research line of the “BrightEyes” ERC project.
His research interests include the design, realization and characterization of optical bioimaging methods with a special focus on smart data analysis and automation. He also has a crush for simulations and analytical models.
Published:
Francesco Fersini
Short Bio
Francesco Fersini studied Electronics Engineering - Photonics at University of Pavia. He got his M.Sc. in 2019 with a research thesis developed at SciLifeLab (TestaLab group, Stockholm). He worked on a super-resolution microscope RESOLFT set-up (REversible Saturable Optical Linear Fluorescence Transitions), combining with Fluorescence Lifetime Imaging Microscopy technique (FLIM) to study the kinetics of super fluorescence proteins decay. After the graduation, he joined to Pharmaceutical Company Bracco Imaging in Colleretto Giacosa (Turin, Italy) in the R&D group as Junior Researcher. The work was orientated to design a FLIM time-gated wilde-field microscope and a GUI. The final goal was to provide lifetime information and spatial variations by parametric maps. In November 2020, Francesco obtained a Ph.D. scholarship in "Bioengineering and Robotics" at the University of Genoa, to join the Molecular Microscopy and Spectroscopy group at the Italian Institute of Technology (IIT, Genoa, Italy) under the supervision of Dr. Giuseppe Vicidomini. His research interests include the design of super-resolution optical microscopy and characterization of fluorescence lifetime analysis in time and frequency domain.
Published:
Eleonora Perego
Short Bio
Eleonora Perego studied Physics at the University of Milano-Bicocca (Italy). She got a M. Sc. in Physics in 2015, after a period at the AMOLF Institute(Amsterdam, the Netherlands), working in the group of “Developmental Biology” of Dr. Jeroen van Zon. In 2020 she obtained a PhD in Biophysics, titled “Studying molecular interaction under flow with fluorescence fluctuation spectroscopy”, working in the group of “Cellular Biophysics” of Prof. Dr. Sarah Koester (Göttingen, Germany), where she studied with fluorescence spectroscopy methods combined with microfluidic approaches protein-protein interactions.
Published:
Alessandro Zunino
Short Bio
Published:
Giacomo Garrè
Short Bio
Published:
Francesco Del Bufalo
Short Bio
Francesco Del Bufalo pursued a Bachelor’s degree in Electronics Engineering and a Master’s degree in Biomedical Engineering from Università degli Studi Roma Tre. In November 2023 he joined, as a PhD Student, the Molecular Microscopy and Spectroscopy Laboratory at Istituto Italiano di Tecnologia in Genoa. His research interests involve the development of innovative data analysis tools for fluorescence lifetime imaging
Published:
Ivan Coto Hernandez
ShortBio
Published:
Marco Castello
Note: Dr. Marco Castello is primarly involved in the realization of the Genoa Instruments Startup.
Published:
Luca Bega
ShortBio
Publications with our group
| 4D Single-Particle Tracking with Asynchronous Read-Out SPAD-Array Detector Andrea Bucci, Giorgio Tortarolo, Marcus Oliver Held, Luca Bega, Eleonora Perego, Francesco Castagnetti, Irene Bozzoni, Eli Slenders, Giuseppe Vicidomini Published in bioRxiv, August 2023 (see publication ) |
Published:
Mattia Donato
Short Bio
Mattia Donato studied at the Department of Physics, University of Genoa (Italy) and he obtained the physics bachelor degree (2010) with a thesis about the tests and the characterization of a prototype of compact gamma-camera for Single Photon Emission Computed Tomography (SPECT).
Published:
Marcus Oliver Held
Short Bio and Projects Description
As physicist we tend to seek the optimal result possible within the given laws of physics and I am also enthusiastic about it. My area of interest is photonic, the field I did my studies, before I specialised in my PhD on fluorescence microscopy to measure single fluorescent molecules. In my thesis, I could show that using complex but optimal illumination conditions the localization precision of a single molecule reached previously unattained values for a low number of detected photons.
Published:
Manuela Salvatori
Note: Manuela Salvatory jointly does assistance between the laboratory of Prof. Alberto Diaspro (Nanoscopy and NIC@IIT, IIT) and my own.
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Marco Scotto d’Abbusco
Note: Marco Scotto jointly does research between the laboratory of Prof. Alberto Diaspro (Nanoscopy and NIC@IIT, IIT) and my own.
Published:
Simonluca Piazza
Note: Dr. Simonluca Piazza is primarly involved in the realization of the Genoa Instruments Startup.
Short Bio
Published:
Sami Koho
Short Bio
Sami Koho studied Information Technology in Tampere University of Technology, with dual Majors in Medical Electronics and Embedded Systems, and a minor in Industrial Management. He graduated in 2009, after which he embarked on an academic career. He held different teaching staff positions at University of Turku from 2009 to 2019. In spring 2011, he also decided to do a PhD in super-resolution optical microscopy; the Laboratory of Biophysics, lead by Prof. Pekka Hänninen has a long history in microcopy research. The subject of his thesis, completed in 2016 is “Bioimage informatics in STED super-resolution microscopy”. Since 2017 Sami has been with the Molecular Microscopy and Spectorscopy lab at IIT, first as a Post-doctoral researcher and currently as a Marie Curie EF-IF research fellow. His research interests include the design of super-resolution optical microscopy methods, bioimage informatics (image reconstruction, restoration, analysis) and adaptive optics.
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Sabrina Zappone
Short Bio
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Sanket Patil
Short Bio
Sanket Patil has a bachelor’s and master’s degree in Biotechnology from the Indian Institute of Technology (IIT), Kanpur (India). He got his master’s degree in 2022 with a thesis entitled “Automating Single Particle Tracking in Optical Microscopy with a Novel Deep Learning Architecture to Probe Intracellular Trafficking.” During his master’s degree, he also built a custom super-resolution STORM setup. After graduation, he joined the Molecular Microscopy and Spectroscopy group under the supervision of Giuseppe Vicidomini at the Istituto Italiano di Tecnologia (Genoa, Italy) to pursue his PhD studies.
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