Quantitative two-photon flow cytometry--in vitro and in vivo.
Flow cytometry is a powerful technique for quantitative characterization of fluorescence in cells. Quantitation is achieved by ensuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow. Two-photon excitation has the advantages that it enables simultaneous excitation of multiple dyes and achieves a very high SNR through simplified filtering and fluorescence background reduction. We demonstrate that two-photon excitation in conjunction with a targeted multidye labeling strategy enables quantitative flow cytometry even under conditions of nonuniform flow, such as may be encountered in simple capillary flow or in vivo. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling cells with targeted nanoparticles containing multiple fluorophores enables normalization of the fluorescence signal and thus quantitative measurements under nonuniform excitation. Flow cytometry using two-photon excitation is demonstrated for detection and differentiation of particles and cells both in vitro in a glass capillary and in vivo in the blood stream of live mice. The technique also enables us to monitor the fluorescent dye labeling dynamics in vivo. In addition, we present a unique two-beam scanning method to conduct cell size measurement in nonuniform flow.
Zhong
Cheng Frank
CF
University of Michigan, Electrical Engineering and Computer Science Department, Center for Ultrafast Optical Science, 2200 Bonisteel Boulevard, Ann Arbor, Michigan 48109-2099, USA.
Tkaczyk
Eric R
ER
Thomas
Thommey
T
Ye
Jing Yong
JY
Myc
Andrzej
A
Bielinska
Anna U
AU
Cao
Zhengyi
Z
Majoros
Istvan
I
Keszler
Balazs
B
Baker
James R
JR
Norris
Theodore B
TB
D000818:Animals; D002469:Cell Separation / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005260:Female; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D008819:Mice, Nude; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods