Ultrafast supercontinuum sculpting for two-photon spectroscopy and microscopy of ratiometric fluorescent indicators

被引:1
|
作者
Chebotarev, Artem S. [1 ]
Raevsky, Roman I. [2 ,3 ]
Linovsky, Georgy N. [1 ]
Kostyuk, Alexander I. [2 ,3 ]
Belousov, Vsevolod V. [2 ,3 ,4 ]
Fedotov, Andrei B. [1 ]
Bilan, Dmitry S. [2 ,3 ]
Lanin, Aleksandr A. [1 ]
机构
[1] Moscow MV Lomonosov State Univ, Phys Dept, Moscow 111992, Russia
[2] Russian Acad Sci, MM Shemyakin & Yu A Ovchinnikov Inst Bioorgan Chem, Moscow 117997, Russia
[3] Pirogov Russian Natl Res Med Univ, Moscow 117997, Russia
[4] Fed Ctr Brain Res & Neurotechnol, Fed Med Biol Agcy, Moscow 119330, Russia
基金
俄罗斯科学基金会;
关键词
HYDROGEN-PEROXIDE; ABSORPTION; H2O2; STANDARDS; PROBE;
D O I
10.1063/5.0197580
中图分类号
O59 [应用物理学];
学科分类号
摘要
We present a compact laser system for quantitative two-photon excitation spectra measurements and ratiometric two-photon imaging of fluorescent protein indicators. The fundamental of the system is a short segment of photonic crystal fiber (PCF), which supports a nonlinear transformation of low-power ultrashort pulses by preserving temporal coherence, and this generates an ultrafast almost octave-spanning supercontinuum (SC). Accurate sculpting of the SC by its amplitude and phase modulation provides implementation of the spectroscopic and microscopic modalities. The spectroscopic one was exhibited by two-photon action cross section spectra measuring for the genetically encoded fluorescent sensing proteins of the vital biochemical parameters: acidity (SypHer3s), concentration of hydrogen peroxide (HyPer3 and HyPer7), redox status of NADH and glutathione (RexYFP and Grx1-roGFP2), hypohalous acids and their derivatives (Hypocrates). For the microscopy, we investigated and optimized the intensity pump pulse profiles under the high numerical objective by dispersion scan technique. We conducted real-time monitoring of the dynamics of hydrogen peroxide in HeLa cells with subcellular spatial resolution by means of ratiometric two-photon imaging of Hyper7 sensors. The presented hybrid laser system provides an ideal optical toolbox in order to develop ratiometric fluorescent sensors, which can be visualized in vivo using two-photon microscopy.
引用
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页数:7
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