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 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 Assessment of Fluorochromes for Two-Photon Laser Scanning Microscopy of Biofilms

2002 ◽  
Vol 68 (2) ◽  
pp. 901-909 ◽  
Author(s):  
Thomas R. Neu ◽  
Ute Kuhlicke ◽  
John R. Lawrence
Keyword(s):  
Nucleic Acid ◽  
Laser Scanning ◽  
Single Photon ◽  
Selective Excitation ◽  
Laser Scanning Microscopy ◽  
Scanning Microscopy ◽  
Excitation Wavelength ◽  
Laser Microscopy ◽  
Two Photon ◽  
Photon Excitation

ABSTRACT A major limitation for the use of two-proton laser scanning microscopy (2P-LSM) in biofilm and other studies is the lack of a thorough understanding of the excitation-emission responses of potential fluorochromes. In order to use 2P-LSM, the utility of various fluorochromes and probes specific for a range of biofilm constituents must be evaluated. The fluorochromes tested in this study included classical nucleic acid-specific stains, such as acridine orange (AO) and 4",6"-diamidino-2-phenylindole (DAPI), as well as recently developed stains. In addition, stains specific for biofilm extracellular polymeric substances (EPS matrix components) were tested. Two-photon excitation with a Ti/Sapphire laser was carried out at wavelengths from 760 to 900 nm in 10-nm steps. It was found that autofluorescence of phototrophic organisms (cyanobacteria and green algae) resulted in strong signals for the entire excitation range. In addition, the coenzyme F420-related autofluorescence of methanogenic bacteria could be used to obtain images of dense aggregates (excitation wavelength, 780 nm). The intensities of the emission signals for the nucleic acid-specific fluorochromes varied. For example, the intensities were similar for excitation wavelengths ranging from 780 to 900 nm for AO but were higher for a narrower range, 780 to 810 nm, for DAPI. In selective excitation, fading, multiple staining, and combined single-photon-two-photon studies, the recently developed nucleic acid-specific fluorochromes proved to be more suitable regardless of whether they are intended for living or fixed samples. Probes specific for proteins and glycoconjugates allowed two-photon imaging of polymeric biofilm constituents. Selective excitation-emission was observed for Calcofluor White M2R (780 to 800 nm) and SyproOrange (880 to 900 nm). In addition, fluor-conjugated concanavalin A lectins were examined and provided acceptable two-photon emission signals at wavelengths ranging from 780 to 800 nm. Finally, CellTracker, a fluorochrome suitable for long-term labeling of microbial eucaryote cells, was found to give strong emission at wavelengths ranging from 770 to 810 nm. If fluorochromes have the same two-photon excitation cross section, they are suitable for multiple staining and multichannel recording. Generally, if an appropriate excitation wavelength and fluorochrome were used, it was possible to obtain more highly resolved images for thick biofilm samples with two-photon laser microscopy than with conventional single-photon laser microscopy. Due to its potential for higher resolution in light-scattering tissue-like material, such as biofilms, and extremely localized excitation, 2P-LSM is a valuable addition to conventional confocal laser scanning microscopy with single-photon excitation. However, further development of the method and basic research are necessary to take full advantage of nonlinear excitation in studies of interfacial microbial ecology.

scholarly journals Single-photon-counting detector for increased sensitivity in two-photon laser scanning microscopy

2008 ◽  
Vol 33 (24) ◽  
pp. 2895 ◽  
Author(s):  
Richard K. P. Benninger ◽  
William J. Ashby ◽  
Elisabeth A. Ring ◽  
David W. Piston
Keyword(s):  
Laser Scanning ◽  
Single Photon ◽  
Photon Counting ◽  
Laser Scanning Microscopy ◽  
Scanning Microscopy ◽  
Single Photon Counting ◽  
Photon Counting Detector ◽  
Two Photon ◽  
Increased Sensitivity

scholarly journals Confocal-based fluorescence fluctuation spectroscopy with a SPAD array detector

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Eli Slenders ◽  
Marco Castello ◽  
Mauro Buttafava ◽  
Federica Villa ◽  
Alberto Tosi ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Single Photon ◽  
Function Analysis ◽  
Pair Correlation ◽  
Correlation Spectroscopy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Array Detector ◽  
Fluorescence Lifetime Imaging ◽  
Fluorescence Fluctuation Spectroscopy

AbstractThe 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.

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

2021 ◽  
pp. 100025
Author(s):  
Eli Slenders ◽  
Eleonora Perego ◽  
Mauro Buttafava ◽  
Giorgio Tortarolo ◽  
Enrico Conca ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Light Dose ◽  
Laser Scanning Microscopy ◽  
Scanning Microscopy ◽  
Array Detector ◽  
Low Light

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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