Figure 5 | Scientific Reports

Figure 5

From: Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation

Figure 5

Three fluorochromes in the same spectral range with different lifetimes cannot be separated with high quality by the phasor approach. (a) Confocal raw image with Abberior STAR 635P labeled vimentin, Alexa Fluor Plus 647 labeled mitochondria and ATTO 647N labeled actin. Scale bar 2 µm. Magenta circle highlights some delicate vimentin fibers. (b) Phasor plot of this image. The lifetime components are indicated by circles: ATTO 647N-actin red, Abberior STAR 635P-vimentin cyan, Alexa Fluor 647-mitochondria orange. A background component (yellow circle) was also included for phasor separation to suppress noise. In pixels with a low photon count, noise from reflection and other sources will determine the average lifetime of the pixel, making it likely to be sorted with the background component. (c) Phasor separated image, same color code. Note the poor preservation of delicate vimentin fibers in the circle in (a). The respective pixels were not correctly assigned to the vimentin channel.

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