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Five-color FLIM-STED with One Depletion Laser

Webinar on five-color STED with a single depletion laser and fluorescence lifetime phasor separation.

Five-color FLIM-STED  5-color_FLIM-STED.jpg

Stimulated emission depletion (STED) microscopy achieves super-resolution by exciting a diffraction-limited volume and then suppressing fluorescence in its outer parts by depletion with a doughnut-shaped STED laser.

The choice of a STED laser and fluorophores depends on the spectral characteristics of the latter. A single STED wavelength is preferable for multicolor analyses, but this limits the number of usable spectral channels. To further increase the number of distinguishable fluorophores, in addition to spectra, their lifetimes can be used. A challenge is to find suitable fluorophore combinations since lifetime information is often not available.

What to expect in the webinar

Key Learnings

  • The benefits of using a single STED laser in STED microscopy
  • Five-color FLIM-STED imaging using a single STED laser
  • The possibilities of using fluorescence lifetime to expand the number of fluorophores in applications requiring confocal and STED multicolor imaging

Doubling the number of distinguishable colours

Using cultured cells, common staining protocols, and commercially available fluorochromes and microscopes, we exploit that the number of fluorochromes in STED or confocal microscopy can be increased by phasor-based fluorescence lifetime imaging microscopy (FLIM) separation of two dyes with similar emission spectra but different fluorescent lifetimes.

In our multicolor FLIM-STED approach, two fluorochromes in the near red (exc. 594 nm, em. 600–630 nm) and two in the far-red channel (exc. 633 nm, em. 641–680 nm), supplemented by a single further redshifted fluorochrome (exc. 670 nm, em. 701–750 nm) were all depleted with a single laser at 775 nm. The analysis, based on the phasor plot, granted an easy and quick way to visualize the quality of the stainings, the lifetime of the fluorophores, and the tools for an easy unmixing. We also provide evidence that eight-color FLIM-STED with a single depletion laser would be possible if suitable fluorochromes were identified and we confirm that a fluorochrome may have different lifetimes depending on the molecules to which it is coupled.

Generally, this approach doubles the number of distinguishable colors in laser scanning microscopy. The phasor-based analysis simplifies the whole procedure, making it possible to be used by a large number of scientists after a short training.

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