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

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

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

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 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

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

Changes of hydrodynamic dimensions of sfGFP induced by GTC.

<p>(<b><i>A</i></b>) Changes of elution profile of sfGFP at increasing denaturant concentration. Numerals at the curves specify applied denaturant concentration. (<b><i>B</i></b>) Changes of the position of elution peaks of compact and denatured molecules (red and blue circles, respectively) and the change of averaged elution volume of sfGFP (black triangles). The value of averaged elution volume (<<i>V</i>>) was calculated as , were <i>f_c</i>_(<i>d</i>) is portion of compact (denatured) molecules and <i>V_c</i>_(<i>d</i>) is elution volume of molecules in these states. The value of <i>f_c</i>_(<i>d</i>) is estimated as , were <i>S_c</i>_(<i>d</i>) represents the area under peak corresponding to compact (denatured) molecules.</p

Kinetics of GdnHCl-induced unfolding of EGFP (<i>A</i>) and sfGFP (<i>B</i>).

<p>The changes of chromophore fluorescence intensity at 494 nm during first 10 min of protein unfolding are shown. . Numbers at the curves indicate the denaturant concentration.</p

Conformational changes of sfGFP induced by GTC.

<p>(<b><i>A</i></b>) Changes of visible absorption spectra at increasing of GTC concentration as shown by arrows. Numbers at the curves indicate the denaturant concentration. (<b><i>B</i></b>) Changes of absorbance at 390 nm (gray circles and line) and 490 nm (black circles and line). (<b><i>C</i></b> and <b><i>D</i></b>) Changes of corrected fluorescence intensity of green chromophore at two wavelengths of excitation of 365 nm and 470 nm corrected to the change of chromophore absorption spectra. Red circles and line indicate unfolding, whereas blue circles and line represent refolding. Measurements were performed after 24 h incubation of native or denatured protein in the GTC presence. The values of chromophore intensity of sfGFP measured after incubation of native protein in the GTC presence during 1 h (gray circles and line), 45 h (black triangles), 69 h (reversed gray triangles) and 94 h (gray crosses) are also indicated. Inset to panel <b><i>C</i></b>: experimentally recorded (curve 1, gray), corrected to total density of solution as I/W, where (curve 2, pink), and corrected to the change of chromophore absorption spectra (see, panel <b>C</b>) on GTC concentration (curve 3, red) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048809#pone.0048809-Sulatskaya1" target="_blank">[47]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048809#pone.0048809-Kuznetsova4" target="_blank">[48]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048809#pone.0048809-Sulatskaya2" target="_blank">[49]</a>.</p