Fig 1.
Typical array of laser wavelengths available on most flow cytometers.
The array of the cytometry lasers is overlaid with the major groups of the GFP-like fluorescent proteins (from blue to far-red) and of the bacterial phytochrome based family of iRFP fluorescent proteins (near-infrared).
Table 1.
Comparison of near-infrared iRFPs engineered from bacterial phytochromes with several far-red FPs of the GFP-like family as probes for deep-tissue imaging.
Fig 2.
Fluorescence spectra for five purified iRFP proteins.
Fluorescence excitation (a) and emission (b) spectra are shown for iRFP670, iRFP682, iRFP702, iRFP713 and iRFP720 fluorescent proteins.
Fig 3.
Fluorescence intensity of iRFP expressing cells with varying laser wavelengths and detection filters.
The intensity of iRFP670 (a), iRFP682 (b), iRFP702 (c), iRFP713 (d) and iRFP720 (e) expressing samples are shown. Laser wavelength is plotted on the x-axis, and detection filter wavelength and window width is plotted on the y-axis. Fluorescence intensity on the z-axis is expressed as a staining index (SI), a relative value proportional to sample fluorescence versus background as described in the Materials and Methods.
Fig 4.
Fluorescence emission of iRFP expressing cells using dual red and NIR laser excitation.
(a) Simultaneous excitation of all five iRFPs with spatially separated red 620 nm and either NIR 685 nm (top panel) or 705 nm (bottom panel) lasers, using detection using 660/20 nm and 740/13 nm filters respectively. Note that all FPs except iRFP713 and iRFP720 can be distinguished from each other. (b) Simultaneous excitation of all five iRFPs with red 620 nm and NIR 705 nm laser, with detection using 680/30 nm and 740/13 nm filters respectively. Note that all FPs can be distinguished from each other. No compensation or correction for fluorescence overlap was applied in this analysis.
Fig 5.
Simultaneous analysis of iRFP expressing samples using 620 nm and 685 nm lasers.
(a) Laser and filter combinations. (b) Analysis of iRFP670 and iRFP702 (left), iRFP713 (middle) or iRFP720 (right) using the above lasers and filters. (c) Analysis of iRFP682 and iRFP702 (left), iRFP713 (middle) or iRFP720 (right) using the above lasers and filters. Data are compensated. The compensation values showing the subtraction of fluorescence overlap from each iRFP into the other is shown on each scatterplot.
Fig 6.
Simultaneous analysis of iRFP expressing samples using 620 nm and 705 nm lasers.
(a) Laser and filter combinations. (b) Analysis of iRFP670 and iRFP702 (left), iRFP713 (middle) or iRFP720 (right) using the above lasers and filters. (c) Analysis of iRFP682 and iRFP702 (left), iRFP713 (middle) or iRFP720 (right) using the above lasers and filters. Data are compensated. The compensation values showing the subtraction of fluorescence overlap from each iRFP into the other is shown on each scatterplot.
Fig 7.
Spectral flow cytometry analysis of iRFP expressing cells.
(a) Individual spectra for each iRFP using Sonly SP6800 spectral cytometer. (b) Analysis of all five iRFPs with data derived from spectral deconvolution of individual FP data.