Microfluidic system for in-flow reversible photoswitching of near-infrared fluorescent proteins

Vladislav V. Lychagov, Anton Shemetov, Ralph Jimenez, Vladislav Verkhusha

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)11821-11829
Number of pages9
JournalAnalytical Chemistry
Volume88
Issue number23
DOIs
StatePublished - Jan 1 2016

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Microfluidics
Infrared radiation
Diffractive optics
Phytochrome
Proteins
Flow cytometry
Luminance
Screening
Fluorescence
Cells
Color
Geometry

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Microfluidic system for in-flow reversible photoswitching of near-infrared fluorescent proteins. / Lychagov, Vladislav V.; Shemetov, Anton; Jimenez, Ralph; Verkhusha, Vladislav.

In: Analytical Chemistry, Vol. 88, No. 23, 01.01.2016, p. 11821-11829.

Research output: Contribution to journalArticle

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