scholarly journals Image scanning microscopy (ISM) with a single photon avalanche diode (SPAD) array detector

2018 ◽  
Author(s):  
Colin J. R. Sheppard ◽  
Alberto Tosi ◽  
Marco Castello ◽  
Giorgio Tortarolo ◽  
Giuseppe Vicidomini ◽  
...  
Keyword(s):  
Single Photon ◽  
Scanning Microscopy ◽  
Array Detector ◽  
Image Scanning ◽  
Avalanche Diode

scholarly journals EASY TWO-PHOTON IMAGE-SCANNING MICROSCOPY WITH SPAD ARRAY AND BLIND IMAGE RECONSTRUCTION

2019 ◽  
Author(s):  
S. V. Koho ◽  
E. Slenders ◽  
G. Tortarolo ◽  
M. Castello ◽  
M. Buttafava ◽  
...  
Keyword(s):  
Image Reconstruction ◽  
Single Photon ◽  
Imaging Modality ◽  
Scanning Microscopy ◽  
Array Detector ◽  
Reconstruction Method ◽  
Image Scanning ◽  
Two Photon ◽  
Blind Image

ABSTRACTTwo-photon excitation (2PE) microscopy is the imaging modality of choice, when one desires to work with thick biological samples, possibly in-vivo. However, the resolution in two-photon microscopy is poor, below confocal microscopy, and the lack of an optical pinhole becomes apparent in complex samples as reduced quality of optical sectioning. 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 array detector – coupled with a novel blind image reconstruction method, is shown to improve the optical resolution, as well as the overall image quality in various test samples. Most importantly, our 2PE-ISM implementation requires no calibration or other input from the user – it works like any old and familiar two-photon system, but simply produces higher resolution images (in real-time). Making the complexity disappear, in our view, is the biggest novelty here, and the key for making 2PE-ISM mainstream.

scholarly journals Synergic Combination of Stimulated Emission Depletion Microscopy with Image Scanning Microscopy to Reduce Light Dosage

2019 ◽  
Author(s):  
Giorgio Tortarolo ◽  
Marco Castello ◽  
Sami Koho ◽  
Giuseppe Vicidomini
Keyword(s):  
Single Photon ◽  
Simulated Data ◽  
Live Cell ◽  
Stimulated Emission ◽  
Beam Intensity ◽  
Scanning Microscopy ◽  
Image Scanning ◽  
Sted Microscopy ◽  
Stimulated Emission Depletion

AbstractStimulated 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.

scholarly journals Cooled SPAD array detector for low light-dose fluorescence laser scanning microscopy

2021 ◽  
Author(s):  
Eli Slenders ◽  
Eleonora Perego ◽  
Mauro Buttafava ◽  
Giorgio Tortarolo ◽  
Enrico Conca ◽  
...  
Keyword(s):  
Laser Power ◽  
Laser Scanning ◽  
Single Photon ◽  
Super Resolution ◽  
Laser Scanning Microscopy ◽  
Scanning Microscopy ◽  
Array Detector ◽  
Trade Off ◽  
Dark Noise ◽  
Array Detectors

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.

scholarly journals The BrightEyes-TTM: an Open-Source Time-Tagging Module for Single-Photon Microscopy

2021 ◽  
Author(s):  
Alessandro Rossetta ◽  
Eli Slenders ◽  
Mattia Donato ◽  
Eleonora Perego ◽  
Francesco Diotalevi ◽  
...  
Keyword(s):  
Open Source ◽  
Fluorescence Lifetime ◽  
Laser Scanning ◽  
Single Photon ◽  
Imaging Spectroscopy ◽  
Laser Scanning Microscopy ◽  
Scanning Microscopy ◽  
Array Detector ◽  
Array Detectors ◽  
Science Labs

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 single-photon 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 reso- lution information generated by the single-photon detectors. To fill this gap, we developed an open-source multi-channel time-tagging module (TTM) based on a field-programmable-gate-array (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) bidimensional 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.

scholarly journals Image Scanning Microscopy with Single-Photon Detector Array

2018 ◽  
Author(s):  
Marco Castello ◽  
Giorgio Tortarolo ◽  
Mauro Buttafava ◽  
Takahiro Deguchi ◽  
Federica Villa ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Single Photon ◽  
Laser Scanning Microscopy ◽  
Scanning Microscopy ◽  
Detector Array ◽  
Photon Detector ◽  
Single Photon Detector ◽  
Image Scanning ◽  
Reconstruction Methods ◽  
The Way

Image scanning microscopy (ISM) improves the spatial resolution of conventional confocal laser-scanning microscopy (CLSM), but current implementations reduce versatility and restrict its combination with fluorescence spectroscopy techniques, such as fluorescence lifetime. Here, we describe a natural design of ISM based on a fast single-photon detector array, which allows straightforward upgrade of an existing confocal microscope, without compromising any of its functionalities. In contrast to all-optical ISM implementations, our approach provides access to the raw scanned images, opening the way to adaptive reconstruction methods, capable of considering different imaging conditions and distortions. We demonstrate its utility in the context of fluorescence lifetime, deep, multicolor and live-cell imaging. This implementation will pave the way for a transparent and massive transition from conventional CLSM to ISM.confocal microscopy | time-resolved spectroscopy | image scanning microscopy | single-photon detector array

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