Extended cavity laser enhanced two-photon flow cytometry.
We demonstrate enhanced sensitivity in two-photon flow cytometry with an extended cavity laser excitation source. At low power, the home-built 20-MHz oscillator was able to detect a significantly larger fraction, in either phosphate buffered saline (PBS) or whole blood, of green fluorescent protein (GFP)-expressing MCA-207 cells cross-labeled with the membrane-binding lipophilic dye DiD. A geometrical model is used to explain unique features of the signals resulting from the different spatial distribution of DiD and GFP. These unique features include sub-square law scaling of unsaturated two-photon signal, a sigmoidal sensitivity curve for detection under varying powers for cell detection thresholds as low as a single photon, and uncorrelated signal strengths in two detection channels.
Tkaczyk
Eric R
ER
University of Michigan, Center for Ultrafast Optical Science, Electrical Engineering & Computer Science Department, Ann Arbor, Michigan 48109-2099, USA. etkaczyk@umich.edu
Tkaczyk
Alan H
AH
Katnik
Steve
S
Ye
Jing Yong
JY
Luker
Kathryn E
KE
Luker
Gary D
GD
Myc
Andrzej
A
Baker
James R
JR
Norris
Theodore B
TB
D004867:Equipment Design; D019544:Equipment Failure Analysis; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods; D007834:Lasers; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods; D010783:Photometry / Q000295:instrumentation*; D017785:Photons; D011829:Radiation Dosage; D011874:Radiometry / Q000295:instrumentation*