Multi-target spectrally resolved fluorescence lifetime imaging microscopy

Abstract

We introduce a pattern-matching technique for efficient identification of fluorophore ratios in complex multidimensional fluorescence signals using reference fluorescence decay and spectral signature patterns of individual fluorescent probes. Alternating pulsed laser excitation at three different wavelengths and time-resolved detection on 32 spectrally separated detection channels ensures efficient excitation of fluorophores and a maximum gain of fluorescence information. Using spectrally resolved fluorescence lifetime imaging microscopy (sFLIM), we were able to visualize up to nine different target molecules simultaneously in mouse C2C12 cells. By exploiting the sensitivity of fluorescence emission spectra and the lifetime of organic fluorophores on environmental factors, we carried out fluorescence imaging of three different target molecules in human U2OS cells with the same fluorophore. Our results demonstrate that sFLIM can be used for super-resolution multi-target imaging by stimulated emission depletion (STED).

Super-multiplex vibrational imaging

“…Even though existing methods have been used successfully to explore structural-functional relationships in nervous systems, profile RNA in situ , reveal tumor microenvironment heterogeneity or study dynamic macromolecular assembly 1–4 , it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a “color barrier” due to the intrinsically broad (~1500 cm −1 ) and featureless nature of fluorescence spectra 5 that limits the number of resolvable colors to 2 to 5 (or 7-9 if using complicated instrumentation and analysis) 6–8 . Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width ~10 cm −1 ) and thus doesn’t suffer this problem, but its feeble signals make many demanding bio-imaging applications impossible.…”

Spectral imaging to visualize higher-order genomic organization

“…Indeed, a similar strategy has been demonstrated recently for protein-based structures in different cellular compartments. 57 The data presented here indicates that obvious differences exist between inter-and intra-chromosomal gene associations that require further investigation. This is a flexible, high-resolution visualization tool for the dissection of higher-order genome dynamics which cannot typically be quantified using highthroughput genomic sequencing or automated deep imaging methodologies.…”

Fluorescence lifetime imaging of fluorescent proteins as an effective quantitative tool for noninvasive study of intracellular processes

“…35 Furthermore, the potential of FLIM techniques for multiplex imaging was demonstrated. 37 The algorithm for signal di®erentiation from up to¯ve°uor-ophores with similar°uorescence emission spectra was introduced recently. 37 In that study,¯ve°uo-rescent probes, including EGFP, ATTO-488, ATTO 488 azide, Alexa Fluor 488 and ATTO490LS were used in the cultured cells for labeling of nuclear envelope, cytoskeleton, nascent DNA, Golgi Apparatus and the nucleoli, respectively.…”

“…Moreover, the ability to visualize up to nine targets using spectrally resolved FLIM microscopy (sFLIM) was recently demonstrated. 37 In another study, FLIM was applied for investigating spatial distribution of nucleocapsid protein (NCp7) of the Human immunode¯ciency virus type 1 (HIV-1) in HeLa cells. 38 In this study, NCp7 was tethered with EGFP and the cellular RNA was labeled with Sytox Orange, to probe the FRET between both°uorophores.…”

“…This feature of FPs can be used for sensing changes in their environment such as intracellular pH, 39-41 temperature 42 or protein concentration. 43,44 As value of these parameters in speci¯c cellular compartments plays an important role in regulation of many intracellular processes, the reliable, accurate and noninvasive methods for Source: Reprinted by permission from Macmillan Publisher Ltd: Nature Methods, 37 Copyright 2016. determination of intracellular parameters are highly desirable in molecular biology and biomedicine. In this regard, genetically encoded FPs-based sensors have a big advantage, as they can be targeted to almost any cellular compartment without loading procedure that is required for conventional dyes.…”