Abstract
Fluorescence microspectroscopy (FMS) with environmentally sensitive dyes provides information about local molecular surroundings at microscopic spatial resolution. Until recently, only probes exhibiting large spectral shifts due to local changes have been used. For filter-based experimental systems, where signal at different wavelengths is acquired sequentially, photostability has been required in addition. Herein, we systematically analyzed our spectral fitting models and bleaching correction algorithms which mitigate both limitations. We showed that careful analysis of data acquired by stochastic wavelength sampling enables nanometer spectral peak position resolution even for highly photosensitive fluorophores. To demonstrate how small spectral shifts and changes in bleaching rates can be exploited, we analyzed vesicles in different lipid phases. Our findings suggest that a wide range of dyes, commonly used in bulk spectrofluorimetry but largely avoided in microspectroscopy due to the above-mentioned restrictions, can be efficiently applied also in FMS.
Highlights
Fluorescence microscopy has boosted advances in life sciences within the last several decades due to its high sensitivity, applicability to live-cell experiments and ability to visualize the sample [1]
Within this work we systematically evaluated our recently developed experimental and analytical fluorescence microspectroscopy (FMS) methods
We showed that accuracy and precision of peak position determination were largely maintained even for photosensitive dyes if bleaching correction algorithms were applied
Summary
Fluorescence microscopy has boosted advances in life sciences within the last several decades due to its high sensitivity, applicability to live-cell experiments and ability to visualize the sample [1]. To localize this information within the investigated sample, many combined microspectroscopic techniques have emerged that enable molecular characterization at optical spatial resolution Among such hybrid methods, spectral imaging, or fluorescence microspectroscopy (FMS), has seen considerable development in recent years [3,4]. This approach improved spectrum peak position resolution well below filter-width and wavelength sampling step in a similar manner as lateral position determination in particle tracking [16]. Probe photobleaching should not be considered a disadvantage; instead, when properly recorded and analyzed, it represents additional valuable information about local molecular environment that can be exploited for bleach rate imaging [24,25,26]
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