TY - JOUR
T1 - Microfluidic system for in-flow reversible photoswitching of near-infrared fluorescent proteins
AU - Lychagov, Vladislav V.
AU - Shemetov, Anton A.
AU - Jimenez, Ralph
AU - Verkhusha, Vladislav V.
N1 - Funding Information:
We thank Janne Ihalainen (University of Jyvaskylä,̈ Finland) for the DrBphP gene, Clark Lagarias (University of California at Davis) and Richard Vierstra (University of Wisconsin at Madison) for the plasmids for production of biliverdin in bacteria, and Brett Fiedler (JILA, University of Colorado at Boulder) for helpful discussions. This work was supported by the GM108579 and GM105997 grants from the U.S. National Institutes of Health (NIH) and by Grant No. ERC-2013-ADG-340233 from the EU FP7 program (to V.V.V.). R.J. is a staff member in the Quantum Physics Division of the National Institute of Standards and Technology (NIST). Certain commercial equipment, instruments, or materials are identified in this paper in order to specify the experimental procedure adequately. Such identification is not intended to imply recommendation or endorsement by the NIST, nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose.
PY - 2016/12/6
Y1 - 2016/12/6
N2 - 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.
AB - 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.
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U2 - 10.1021/acs.analchem.6b03499
DO - 10.1021/acs.analchem.6b03499
M3 - Article
C2 - 27807973
AN - SCOPUS:85045633076
SN - 0003-2700
VL - 88
SP - 11821
EP - 11829
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 23
ER -