Microfluidic System for In-Flow Reversible Photoswitching of Near-Infrared Fluorescent Proteins

We have developed a microfluidic flow cytometry system to screen reversibly photoswitchable fluorescent proteins for contrast and stability of reversible photoconversion between high- and low-fluorescent states. A two-color array of 20 excitation and deactivation beams generated with diffractive optics was combined with a serpentine microfluidic channel geometry designed to provide five cycles of photoswitching with real-time calculation of photoconversion fluorescence contrast. The characteristics of photoswitching in-flow as a function of excitation and deactivation beam fluence, flow speed, and protein concentration were studied with droplets of the bacterial phytochrome from Deinococcus radiodurans (DrBphP), which is weakly fluorescent in the near-infrared (NIR) spectral range. In agreement with measurements on stationary droplets and HeLa S3 mammalian cells expressing DrBphP, optimized operation of the flow system provided up to 50% photoconversion contrast in-flow at a droplet rate of few hertz and a coefficient of variation (CV) of up to 2% over 10 000 events. The methods for calibrating the brightness and photoswitching measurements in microfluidic flow established here provide a basis for screening of cell-based libraries of reversibly switchable NIR fluorescent proteins

A FRET-Facilitated Photoswitching Using an Orange Fluorescent Protein with the Fast Photoconversion Kinetics

Fluorescent proteins photoswitchable with noncytotoxic light irradiation and spectrally distinct from multiple available photoconvertible green-to-red probes are in high demand. We have developed a monomeric fluorescent protein, called PSmOrange2, which is photoswitchable with blue light from an orange (ex./em. at 546 nm/561 nm) to a far-red (ex./em. at 619 nm/651 nm) form. Compared to another orange-to-far-red photoconvertable variant, PSmOrange2 has blue-shifted photoswitching action spectrum, 9-fold higher photoconversion contrast, and up to 10-fold faster photoswitching kinetics. This results in the 4-fold more PSmOrange2 molecules being photoconverted in mammalian cells. Compared to common orange fluorescent proteins, such as mOrange, the orange form of PSmOrange has substantially higher photostability allowing its use in multicolor imaging applications to track dynamics of multiple populations of intracellular objects. The PSmOrange2 photochemical properties allow its efficient photoswitching with common two-photon lasers and, moreover, via Förster resonance energy transfer (FRET) from green fluorescent donors. We have termed the latter effect a FRET-facilitated photoswitching and demonstrated it using several sets of interacting proteins. The enhanced photoswitching properties of PSmOrange2 make it a superior photoconvertable protein tag for flow cytometry, conventional microscopy, and two-photon imaging of live cells

The effect of pH and ionic strength on sfGFP spectroscopic characteristics.

<p>Absorption spectra (<b><i>A</i></b>) and fluorescence spectra (<b><i>B</i></b>) of sfGFP in the solutions with different pH. Fluorescence was excited at 390 nm and normalized to total fluorescence at current pH value. The numbers on panels A and B are the values of pH in solution. Absorption spectra (<b><i>C</i></b>) and fluorescence spectra (<b><i>D</i></b>) of sfGFP in the buffered solution and in solutions with the equal ionic strength but containing 0.7 M Na₂SO₄ or 2.1 M NaCl. Fluorescence was excited at 390 nm and normalized in the same manner as data on panel B.</p

The effect of ionic denaturants and salts on the CD in the visible range of sfGFP.

<p>(<b><i>A</i></b>) CD spectra of sfGFP in the visible-UV region were recorded at final GTC concentrations of 0.0 (black line), 0.04 (red line), 0.2 (green line), and 0.5 M (blue line). (<b><i>B</i></b>) CD spectra of sfGFP in the visible-UV region were recorded at final NaSCN concentrations of 0.0 (black line), 0.05 (red line), 0.2 (green line), and 0.5 M (blue line). (<b><i>C</i></b>) CD spectra of sfGFP in the visible-UV region were recorded at final GdnHCl concentrations of 0.0 (black line), 0.25 (red line), 0.5 (green line), 2.0 M (blue line) and 2.5 M (pink line). (<b><i>D</i></b>) CD spectra of sfGFP in the visible-UV region were recorded at final NaCl concentrations of 0.0 (black line), 1.0 (red line), 1.25 (green line), 2.0 M (blue line) and 2.5 M (pink line).</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

Molar absorption spectra of neutral (pink), anionic (red) and complexed with Cl⁻ (green) states of sfGFP.

<p>Spectra decomposition was made on the basis of absorption spectra of sfGFP in the presence of studied agents. For details see the discussion section.</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

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

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