Abstract
We present a new microscope integrating super-resolved imaging using structured illumination microscopy (SIM) with wide-field optically sectioned fluorescence lifetime imaging (FLIM) to provide optical mapping of molecular function and its correlation with biological nanostructure below the conventional diffraction limit. We illustrate this SIM + FLIM capability to map FRET readouts applied to the aggregation of discoidin domain receptor 1 (DDR1) in Cos 7 cells following ligand stimulation and to the compaction of DNA during the cell cycle.
Highlights
Super-resolved microscopy (SRM) is becoming an established tool for biomedical research that extends the ability to visualise structures and localise or colocalise proteins in cells beyond the Abbe limit of λ/(2NA)
An exemplar application of this approach is illustrated with the fluorescence lifetime imaging (FLIM) Förster resonant energy transfer (FRET) readout of aggregation of the receptor tyrosine kinase known as discoidin domain receptor 1 (DDR1)
Between Alexa488 as the donor and Alexa546 as the acceptor, resulting in a decreased lifetime of the Figure 2 shows structured illumination microscopy (SIM) and FLIM data of a Cos 7 cell expressing DDR1 labelled with AlexaFluor488 and AlexaFluor546 and stimulated with collagen for 60 min
Summary
Super-resolved microscopy (SRM) is becoming an established tool for biomedical research that extends the ability to visualise structures and localise or colocalise proteins in cells beyond the Abbe (diffraction) limit of λ/(2NA). Many techniques have been implemented to read out and quantify FRET [15,16] with the most widely used being spectral ratiometric imaging, where FRET is read out through the change in acceptor to donor intensity ratio, and fluorescence lifetime imaging (FLIM), where the increased de-excitation rate resulting from FRET results in a decrease in the donor fluorescence lifetime While the former approach typically requires fewer detected photons per pixel, quantitative spectrally resolved FRET measurements require calibration of the spectral response of the system (including sample and instrument) and an independent measurement of the actual FRET efficiency is required if the fractions of the FRETing donor and acceptor populations are needed [17]. Wide-field FLIM can be implemented in the time-domain or frequency domains and, for both approaches, the fluorescence lifetime images are calculated from series of gated fluorescence intensity images acquired at different relative delays following excitation
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