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Pushing the boundaries of microscopy applying FLIM-STED: Five color superresolution microscopy

Stimulated emission depletion (STED) microscopy emerged over the last decades as a technique that allows microscopic observations with several fluorescent colors at unprecedented resolution. This is achieved by first exciting fluorochromes in the smallest possible volume (diffraction limited spot) with one color of laser light. Second, fluorescence emission in the outer parts of this volume is suppressed by depleting the energy of the excited fluorochromes with another color of laser light. By technical tricks, this depletion laser beam is shaped around the excited spot like a doughnut. For depletion to work, the color of the depletion beam has to be part of the natural emission spectrum of the fluorochrome used. This limits the number of spectrally distinguishable colors that can be used with one depletion laser color.

Several depletion lasers can be used to increase the number of available colors, but multiple depletion lasers need to be exactly aligned, a condition that may be difficult to achieve at the required precision. Even more disturbing is that the depletion laser with the smaller wavelength will bleach the fluorochromes with the longer wavelength. Hence, a single depletion laser color is much preferable for multi-color analyses.

In a recently published article, the team from the Core Facility Bioimaging at the BMC raised the new record for the number of fluorochromes in STED microscopy with a single depletion laser to five. Samples were labeled with standard immunofluorescence procedures with commercially available fluorochromes so that other groups should be able to easily duplicate the approach. The trick to achieve five color STED images was to separate fluorochromes not only by their color, but also by their lifetime, i.e. the number of nanoseconds it takes the fluorochrome to emit fluorescence after it was excited. This Fluorescence Lifetime Imaging (FLIM) is implemented on the Leica STED microscope available at the Core Facility. In contrast to older FLIM systems, the available FLIM control software is fully integrated in the regular microscope control software so that usage is a lot easier to learn than in previous FLIM implementations.

Generally, the FLIM separation approach doubles the number of fully distinguishable colors in laser scanning microscopy, not only in STED, but also in regular confocal microscopy

Publication: Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation. Mariano Gonzalez Pisfil, Iliya Nadelson, Brigitte Bergner, Sonja Rottmeier, Andreas W. Thomae, Steffen Dietzel. Scientific Reports 12, 14027 (2022).