Biology subjects

Koho, S. V.

Publications and source records attributed to Koho, S. V..

4 recordsLinked to original sources

bioRxiv2021

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

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 revolutions 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.

bioengineering↗

bioRxiv2019

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

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.

bioengineering↗

bioRxiv2019

Easy Two-Photon Image Scanning Microscopy with SPAD Array and Blind Image Reconstruction

Two-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.

bioengineering↗

bioRxiv2019

Fourier ring correlation simplifies image restoration in fluorescence microscopy

Fourier ring correlation (FRC) has recently gained some popularity among (super-resolution) fluorescence microscopists as a straightforward and objective method to measure the effective resolution of a microscopy image. 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 novel 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, with no need for prior knowledge of the instrument or sample characteristics. The deconvolution is shown to work exquisitely with both two- and three-dimensional images. We also show how FRC can be used as a powerful metric to observe the progress of iterative deconvolution. While developing the image restoration methods, we also addressed two important limitations in FRC that are of more general interest: how to make FRC work with single images and with three-dimensional images with anisotropic resolution.

bioinformatics↗