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A wide-field microscope with epi-fluorescence and selective plane illumination was combined with a single-photon avalanche diode (SPAD) array camera to enable live-cell fluorescence lifetime imaging (FLIM) using time-correlated single-photon counting (TCSPC). The camera sensor comprised of 192 × 128 pixels, each integrating a single SPAD and a time-to-digital converter. Jointly, they produced a stream of single-photon images of photon arrival times with ≈ 38 ps accuracy. The photon arrival times were subject to systematic delays and nonlinearities, which were corrected by a Monte-Carlo algorithm. The SPAD camera was then applied to FLIM where histogramming the resulting photon arrival times in each pixel resulted in decays compatible with common data processing pipelines for fluorescence lifetime analysis. The capabilities of the TCSPC camera-based FLIM microscope were demonstrated by imaging living unicellular photosynthetic algae and artificial lipid vesicles. Epi-fluorescence illumination enabled rapid fluorescence lifetime imaging of living cells and selective-plane illumination enabled 3-dimensional FLIM of stationary samples.

The removal of varnish from the surface is a key step in painting conservation. Varnish removal is traditionally monitored by examining the painting surface under ultraviolet illumination. We show here that by imaging the fluorescence lifetime instead, much better contrast, sensitivity, and specificity can be achieved. For this purpose, we developed a lightweight (4.8 kg) portable instrument for macroscopic fluorescence lifetime imaging (FLIM). It is based on a time-correlated single-photon avalanche diode (SPAD) camera to acquire the FLIM images and a pulsed 440 nm diode laser to excite the varnish fluorescence. A historical model painting was examined to demonstrate the capabilities of the system. We found that the FLIM images provided information on the distribution of the varnish on the painting surface with greater sensitivity, specificity, and contrast compared to the traditional ultraviolet illumination photography. The distribution of the varnish and other painting materials was assessed using FLIM during and after varnish removal with different solvent application methods. Monitoring of the varnish removal process between successive solvent applications by a swab revealed an evolving image contrast as a function of the cleaning progress. FLIM of dammar and mastic resin varnishes identified characteristic changes to their fluorescence lifetimes depending on their ageing conditions. Thus, FLIM has a potential to become a powerful and versatile tool to visualise varnish removal from paintings.

We present a novel imaging system combining total internal reflection fluorescence (TIRF) microscopy with measurement of steady-state acceptor fluorescence anisotropy in order to perform live cell Förster Resonance Energy Transfer (FRET) imaging at the plasma membrane. We compare directly the imaging performance of fluorescence anisotropy resolved TIRF with epifluorescence illumination. The use of high numerical aperture objective for TIRF required correction for induced depolarization factors. This arrangement enabled visualisation of conformational changes of a Raichu-Cdc42 FRET biosensor by measurement of intramolecular FRET between eGFP and mRFP1. Higher activity of the probe was found at the cell plasma membrane compared to intracellularly. Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants. Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor.

We present a web engine boosted fluorescence in-situ hybridization (webFISH) algorithm using a genome-wide sequence similarity search to design target-specific single-copy and repetitive DNA FISH probes. The webFISH algorithm featuring a user-friendly interface (http://www.webfish2.org/) maximizes the coverage of the examined sequences with FISH probes by considering locally repetitive sequences absent from the remainder of the genome. The highly repetitive human immunoglobulin heavy chain sequence was analyzed using webFISH to design three sets of FISH probes. These allowed direct simultaneous detection of class switch recombination in both immunoglobulin-heavy chain alleles in single cells from a population of cultured primary B cells. It directly demonstrated asynchrony of the class switch recombination in the two alleles in structurally preserved nuclei while permitting parallel readout of protein expression by immunofluorescence staining. This novel technique offers the possibility of gaining unprecedented insight into the molecular mechanisms involved in class switch recombination.

Antibodies are assembled by a highly orchestrated series of recombination events during B cell development. One of these events, class switch recombination, is required to produce the IgG, IgE and IgA antibody isotypes characteristic of a secondary immune response. The action of the enzyme activation induced cytidine deaminase is now known to be essential for the initiation of this recombination event. Previous studies have demonstrated that the immunoglobulin switch regions acquire distinct histone modifications prior to recombination. We now present a high resolution analysis of these histone modifications across the IgE switch region prior to the initiation of class switch recombination in primary human B cells and the human CL-01 B cell line. These data show that upon stimulation with IL-4 and an anti-CD40 antibody that mimics T cell help, the nucleosomes of the switch regions are highly modified on histone H3, accumulating acetylation marks and tri-methylation of lysine 4. Distinct peaks of modified histones are found across the switch region, most notably at the 5' splice donor site of the germline (I) exon, which also accumulates AID. These data suggest that acetylation and K4 tri-methylation of histone H3 may represent marks of recombinationally active chromatin and further implicates splicing in the regulation of AID action.

This article reports on a conference workshop conducted at CYTO 2019. This workshop centered on an online directory for non-commercial cytometry innovations called CYTO Lab Hacks. The CYTO Lab Hacks website is being developed to become a curated platform to collate and to promote cytometry related materials developed by the wider scientific community. The website will present brief summaries and links to repositories with experimental protocols, descriptions of hardware changes, document templates, software code, and other innovations. The workshop outcomes, summarized in this manuscript, cover the topics of the website functionality and user experience, organization of the volunteer task force, and understanding the needs of the cytometry community in respect to sharing innovations.

This article reports on a conference workshop conducted at CYTO 2018. During the workshop a new Open Science forum \"CYTO Lab Hacks\" has been launched within the International Society for the Advancement of Cytometry (ISAC). Its goal is to serve as an open, transparent, sustainable and accessible forum for innovation-exchange in cytometry. Here we report the captured status quo, the perceived requirements of the members in relation to open innovation sharing and dissemination and publicize the format of \"CYTO Lab Hacks\".

This article reports on a conference workshop conducted at CYTO 2018. During the workshop a new Open Science forum \"CYTO Lab Hacks\" has been launched within the International Society for the Advancement of Cytometry (ISAC). Its goal is to serve as an open, transparent, sustainable and accessible forum for innovation-exchange in cytometry. Here we report the captured status quo, the perceived requirements of the members in relation to open innovation sharing and dissemination and publicize the format of \"CYTO Lab Hacks\".

This article reports on a conference workshop conducted at CYTO 2018. During the workshop a new Open Science forum \"CYTO Lab Hacks\" has been launched within the International Society for the Advancement of Cytometry (ISAC). Its goal is to serve as an open, transparent, sustainable and accessible forum for innovation-exchange in cytometry. Here we report the captured status quo, the perceived requirements of the members in relation to open innovation sharing and dissemination and publicize the format of \"CYTO Lab Hacks\".

A modular instrument was developed to measure the fluorescence yield in plants subject to a combination of two harmonically-oscillating blue lights with independently controlled frequencies and phases.It uses the pulse-amplitude-modulation (PAM) method to measure the fluorescence yield independently of the plant irradiance. Compared to existing commercial instruments, it uses a higher measuring frequency (≈ 60 kHz) and higher measuring flash irradiance. This enables averaging over a number of subsequent measurement data points to achieve a higher signal-to-noise ratio. The manuscript describes the design, testing, and characterization of the operational limits of the instrument. It identifies its current weaknesses and makes recommendations for improvements. It is accompanied by supplementary materials containing the electronic schematics and the source code. The instrument was used to study a plant response to a mixture of two oscillating lights. It resulted in an excellent signal-to-noise ratio of the measured fluorescent yield. The measurements clearly demonstrated that the fluorescence yield of a plant subject to a combination of two harmonically-oscillating lights is not the same as the sum of the responses to the two oscillating lights applied independently. The observed non-linearity leads to the important conclusion that the time- and frequency-domain cannot be connected by a Fourier transform. Therefore, the frequency-domain approach will yield novel information that is not redundant to the well-established time-domain measurements.