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

Abstract

Stimulated emission depletion (STED) microscopy achieves super-resolution by exciting a diffraction-limited volume and then suppressing fluorescence in its outer parts by depletion. Multiple depletion lasers may introduce misalignment and bleaching. Hence, a single depletion wavelength is preferable for multi-color analyses. However, this limits the number of usable spectral channels. 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 separation of two dyes with similar emission spectra but different fluorescent lifetimes. In our multi-color FLIM-STED approach two fluorochromes in the near red (exc. 594 nm, em. 600-630) and two in the far red channel (633/641-680), supplemented by a single further redshifted fluorochrome (670/701-750) were depleted with 775 nm. To the best of our knowledge, these are the first published five color STED images. Generally, this approach doubles the number of fully distinguishable colors in laser scanning microscopy. We 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.

Identifying STEDable BF2-Azadipyrromethene Fluorophores

“…In most instruments, the laser wavelengths for the depletion beam are restricted to 595, 660, or 775 nm with the latter being the one used in this study [ 7 ]. While this DL wavelength was beyond the spectral absorbance of all four fluorophores, an overlap existed to varying extents for each of their emissions ( Figure 3 ).…”

“…Fluorophores 1 and 2 were imaged using CLSM and FLIM-STED (DL 30%). TauSTED mode (τ-strength 80, denoise 100, time gate 0.5–6 ns) was used for the HyD detector to filter pixels using the phasor signature produced for each STED image [ 7 , 17 ]. For 2 , to ensure this did not include any photons produced from anti-Stokes, expert mode was used to set the phasor signature.…”

“…The DL de-excites fluorophores in the coinciding regions through a stimulated emission at the wavelength of the DL, with a higher resolution image provided from the residual non-overlapped area. With the development of the 775 nm near-infrared DL, longer wavelengths for STED imaging are now possible, potentially enabling improved resolution imaging of live cells [ 7 ].…”

“…To develop organic dyes for far-red STED imaging, rigorous physical and biocompatibility requirements must be met, including high photostability, brightness, and no light or dark toxicity [ 2 ]. Recent investigations have shown that rhodamine derivatives, π-extended-BODIPYs, and Nile red fluorophores are STED compatible when used with a 775 nm DL ( Figure 1 ) [ 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. Achievements have been made in defining subcellular structures, such as the cristae of mitochondria [ 8 , 9 , 10 ] and vimentin filaments [ 13 ] using these dye classes.…”

“…Achievements have been made in defining subcellular structures, such as the cristae of mitochondria [ 8 , 9 , 10 ] and vimentin filaments [ 13 ] using these dye classes. With a continually increasing demand for imaging in this region some commercial dyes have also become available [ 7 ].…”