Microfluidic flow cytometer for quantifying photobleaching of fluorescent proteins in cells.
Microfluidic flow cytometer for quantifying photobleaching of fluorescent proteins in cells.
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DOI：
10.1021/ac202825z
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发表时间：
2012-05-01
影响因子：
7.4
通讯作者：
Jimenez, Ralph
中科院分区：
文献类型：
--
作者：
Lubbeck, Jennifer L.;Dean, Kevin M.;Ma, Hairong;Palmer, Amy E.;Jimenez, Ralph
Traditional flow cytometers are capable of rapid cellular assays on the basis of fluorescence intensity and light scatter. Microfluidic flow cytometers have largely followed the same path of technological development as their traditional counterparts, however the significantly smaller transport distance and resulting lower cell speeds in microchannels provides for the opportunity to detect novel spectroscopic signatures based on multiple, non-temporally-coincident excitation beams. Here, we characterize the design and operation of a cytometer with a 3-beam, probe/bleach/probe geometry, employing HeLa suspension cells expressing fluorescent proteins. The data collection rate exceeds 20 cells/s under a range of beam intensities (5 kW – 179 kW/cm2). The measured percent photobleaching (ratio of fluorescence intensities excited by the first and third beams: Sbeam3/Sbeam1) partially resolves a mixture of four red fluorescent proteins in mixed samples. Photokinetic simulations are presented and demonstrate that the percent photobleaching reflects a combination of the reversible and irreversible photobleaching kinetics. By introducing a photobleaching optical signature, which complements traditional fluorescence intensity-based detection, this method adds another dimension to multi-channel fluorescence cytometry, and provides a means for flow-cytometry-based screening of directed libraries of fluorescent protein photobleaching.
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影响因子：
2.8
作者：
Chen, Chun H.;Cho, Sung Hwan;Tsai, Frank;Erten, Ahmet;Lo, Yu-Hwa
通讯作者：
Lo, Yu-Hwa
影响因子：
1.6
作者：
BONNER, WA;SWEET, RG;HERZENBERG, LA
通讯作者：
HERZENBERG, LA
影响因子：
48
作者：
Donnert, Gerald;Eggeling, Christian;Hell, Stefan W.
通讯作者：
Hell, Stefan W.
影响因子：
64.8
作者：
Dickson, RM;Cubitt, AB;Moerner, WE
通讯作者：
Moerner, WE
DOI：
10.1073/pnas.97.22.11996
发表时间：
2000-10-24
影响因子：
11.1
作者：
Heikal, AA;Hess, ST;Webb, WW
通讯作者：
Webb, WW