TY - JOUR
T1 - Microfluidic flow cytometer for quantifying photobleaching of fluorescent proteins in cells
AU - Lubbeck, Jennifer L.
AU - Dean, Kevin M.
AU - Ma, Hairong
AU - Palmer, Amy E.
AU - Jimenez, Ralph
PY - 2012/5/1
Y1 - 2012/5/1
N2 - 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, nontemporally coincident excitation beams. Here, we characterize the design and operation of a cytometer with a three-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 to 179 kW/cm 2). The measured percent photobleaching (ratio of fluorescence intensities excited by the first and third beams: S beam3/S beam1) 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 multichannel fluorescence cytometry and provides a means for flow-cytometry-based screening of directed libraries of fluorescent protein photobleaching.
AB - 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, nontemporally coincident excitation beams. Here, we characterize the design and operation of a cytometer with a three-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 to 179 kW/cm 2). The measured percent photobleaching (ratio of fluorescence intensities excited by the first and third beams: S beam3/S beam1) 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 multichannel fluorescence cytometry and provides a means for flow-cytometry-based screening of directed libraries of fluorescent protein photobleaching.
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U2 - 10.1021/ac202825z
DO - 10.1021/ac202825z
M3 - Article
C2 - 22424298
AN - SCOPUS:84860442533
SN - 0003-2700
VL - 84
SP - 3929
EP - 3937
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 9
ER -