Phasor S-FLIM: a new paradigm for fast and robust spectral fluorescence lifetime imaging

Scipioni, Lorenzo; Rogora, Alessandro; Tedeschi, Giulia; Gratton, Enrico

doi:10.1038/s41592-021-01108-4

Cited by 70 publications

(71 citation statements)

References 58 publications

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“…Multiplexed fluorescence microscopy based on fluorescence lifetime is an attractive approach to image multiple targets simultaneously. It requires only one spectral channel and therefore frees up other channels for further multiplexing 1 – 5 . To facilitate the use of fluorescence lifetime multiplexing in living cells, we aimed to establish a method in which different variants of a self-labeling protein (SLP) tag are labeled with a single fluorophore but become distinguishable based on their fluorescence lifetime.…”

Section: Mainmentioning

confidence: 99%

Engineered HaloTag variants for fluorescence lifetime multiplexing

et al. 2021

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Self-labeling protein tags such as HaloTag are powerful tools that can label fusion proteins with synthetic fluorophores for use in fluorescence microscopy. Here we introduce HaloTag variants with either increased or decreased brightness and fluorescence lifetime compared with HaloTag7 when labeled with rhodamines. Combining these HaloTag variants enabled live-cell fluorescence lifetime multiplexing of three cellular targets in one spectral channel using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. Additionally, the brightest HaloTag variant showed up to 40% higher brightness in live-cell imaging applications.

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Section: Mainmentioning

confidence: 99%

Engineered HaloTag variants for fluorescence lifetime multiplexing

et al. 2021

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“…Combining the lifetime measurement with a spectral detector, one effectively has a 5-dimensional space in which to characterize each pixel. On top of the spatio-temporal coordinates (x,y,z,t), each pixel now carries information in five additional coordinates: its intensity value (however many photons arrived at that pixel), the two phasor coordinates for the lifetime phasor transform, and the two phasor coordinates for the spectral phasor transform 34 . A typical image, on the order of 10 6 pixels, obtained with this method provides 10 6 points in this 5D space 34 .…”

Section: Resultsmentioning

confidence: 99%

Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis

Vallmitjana

et al. 2022

Nat Commun

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Multiplexed mRNA profiling in the spatial context provides new information enabling basic research and clinical applications. Unfortunately, existing spatial transcriptomics methods are limited due to either low multiplexing or complexity. Here, we introduce a spatialomics technology, termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA), that integrates in situ labeling of mRNA and protein markers in cells or tissues with combinatorial fluorescence spectral and lifetime encoded probes, spectral and time-resolved fluorescence imaging, and machine learning-based decoding. We demonstrate MOSAICA’s multiplexing scalability in detecting 10-plex targets in fixed colorectal cancer cells using combinatorial labeling of five fluorophores with facile error-detection and removal of autofluorescence. MOSAICA’s analysis is strongly correlated with sequencing data (Pearson’s r = 0.96) and was further benchmarked using RNAscopeTM and LGC StellarisTM. We further apply MOSAICA for multiplexed analysis of clinical melanoma Formalin-Fixed Paraffin-Embedded (FFPE) tissues. We finally demonstrate simultaneous co-detection of protein and mRNA in cancer cells.

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“…HP1 is responsible for the methylation of lysine 9 of the H3, which is a posttranslational modification known to induce heterochromatin formation, while ECFP and YPet are the FRET pair. Cells were transfected with the chromatin compaction biosensor (H3Kme3) and in hypo-osmolar or hyper-osmolar conditions to induce chromatin decompaction and compaction, respectively [ 11 ]. Phasor S-FLIM allows to compute the FRET efficiency with a four to five times increased number of photons compared to conventional FLIM, which results in much sharper images and distributions, revealing small differences in FRET efficiency at the single cell level.…”

Section: Figurementioning

confidence: 99%

Genetically Encoded Fluorescent Biosensors for Biomedical Applications

et al. 2021

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One of the challenges of modern biology and medicine is to visualize biomolecules in their natural environment, in real-time and in a non-invasive fashion, so as to gain insight into their physiological behavior and highlight alterations in pathological settings, which will enable to devise appropriate therapeutic strategies. Genetically encoded fluorescent biosensors constitute a class of imaging agents that enable visualization of biological processes and events directly in situ, preserving the native biological context and providing detailed insight into their localization and dynamics in cells. Real-time monitoring of drug action in a specific cellular compartment, organ, or tissue type; the ability to screen at the single-cell resolution; and the elimination of false-positive results caused by low drug bioavailability that is not detected by in vitro testing methods are a few of the obvious benefits of using genetically encoded fluorescent biosensors in drug screening. This review summarizes results of the studies that have been conducted in the last years toward the fabrication of genetically encoded fluorescent biosensors for biomedical applications with a comprehensive discussion on the challenges, future trends, and potential inputs needed for improving them.

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Phasor S-FLIM: a new paradigm for fast and robust spectral fluorescence lifetime imaging

Cited by 70 publications

References 58 publications

Engineered HaloTag variants for fluorescence lifetime multiplexing

Engineered HaloTag variants for fluorescence lifetime multiplexing

Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis

Genetically Encoded Fluorescent Biosensors for Biomedical Applications

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