Multiparametric Flow Cytometry Using Near-Infrared Fluorescent Proteins Engineered from Bacterial Phytochromes

Engineering of fluorescent proteins (FPs) has followed a trend of achieving longer fluorescence wavelengths, with the ultimate goal of producing proteins with both excitation and emission in the near-infrared (NIR) region of the spectrum. Flow cytometers are now almost universally equipped with red lasers, and can now be equipped with NIR lasers as well. Most red-shifted FPs of the GFP-like family are maximally excited by orange lasers (590 to 610 nm) not commonly found on cytometers. This has changed with the development of the iRFP series of NIR FPs from the protein family of bacterial phytochromes. The shortest wavelength variants of this series, iRFP670 and iRFP682 showed maximal excitation with visible red lasers. The longer wavelength variants iRFP702, iRFP713 and iRFP720 could be optimally excited by NIR lasers ranging from 685 to 730 nm. Pairs of iRFPs could be detected simultaneously by using red and NIR lasers. Moreover, a novel spectral cytometry technique, which relies on spectral deconvolution rather than optical filters, allowed spectra of all five iRFPs to be analyzed simultaneously with no spectral overlap. Together, the combination of iRFPs with the advanced flow cytometry will allow to first image tissues expressing iRFPs deep in live animals and then quantify individual cell intensities and sort out the distinct primary cell subpopulations ex vivo.Peer reviewe

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.

<p>^aDefined as a product of extinction coefficient and quantum yield. The molecular brightness values are from [<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122342#pone.0122342.ref019" target="_blank">19</a></b>] for five iRFPs, [<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122342#pone.0122342.ref010" target="_blank">10</a></b>] for mNeptune, [<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122342#pone.0122342.ref011" target="_blank">11</a></b>] for E2-Crimson and [<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122342#pone.0122342.ref013" target="_blank">13</a></b>] for eqFP650 and eqFP670.</p><p>^bData are from [<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122342#pone.0122342.ref019" target="_blank">19</a></b>]. The signal-to-background ratios were measured for equal amounts of purified FPs imaged in a planar epifluorescence mode on an IVIS Spectrum instrument (PerkinElmer, Waltham, MA) inside a XFM-2 fluorescent phantom mouse (PerkinElmer) at the indicated depths from the mouse surface using the optimal filter channels for each FP.</p><p>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.</p

Typical array of laser wavelengths available on most flow cytometers.

<p>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).</p

Fluorescence emission of iRFP expressing cells using dual red and NIR laser excitation.

<p>(<b>a</b>) 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>b</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.</p

Spectral flow cytometry analysis of iRFP expressing cells.

<p>(<b>a</b>) Individual spectra for each iRFP using Sonly SP6800 spectral cytometer. (<b>b</b>) Analysis of all five iRFPs with data derived from spectral deconvolution of individual FP data.</p

Simultaneous analysis of iRFP expressing samples using 620 nm and 705 nm lasers.

<p>(<b>a</b>) Laser and filter combinations. (<b>b</b>) Analysis of iRFP670 and iRFP702 (left), iRFP713 (middle) or iRFP720 (right) using the above lasers and filters. (<b>c</b>) 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.</p

Simultaneous analysis of iRFP expressing samples using 620 nm and 685 nm lasers.

<p>(<b>a)</b> Laser and filter combinations. (<b>b</b>) Analysis of iRFP670 and iRFP702 (left), iRFP713 (middle) or iRFP720 (right) using the above lasers and filters. (<b>c</b>) 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.</p

Fluorescence spectra for five purified iRFP proteins.

<p>Fluorescence excitation (<b>a</b>) and emission (<b>b</b>) spectra are shown for iRFP670, iRFP682, iRFP702, iRFP713 and iRFP720 fluorescent proteins.</p