Optimizing the fluorescent yield in two-photon laser scanning microscopy with dispersion compensation.
A challenge for nonlinear imaging in living tissue is to maximize the total fluorescent yield from each fluorophore. We investigated the emission rates of three fluorophores-rhodamine B, a red fluorescent protein, and CdSe quantum dots-while manipulating the phase of the laser excitation pulse at the focus. In all cases a transform-limited pulse maximized the total yield to insure the highest signal-to-noise ratio. Further, we find evidence of fluorescence antibleaching in quantum dot samples.
Field
Jeffrey J
JJ
Center for Microintegrated Optics for Advanced Bioimaging and Control, and Department of Physics, Colorado School of Mines, Golden, CO 80401, USA. jjfield@mines.edu
Carriles
Ramón
R
Sheetz
Kraig E
KE
Chandler
Eric V
EV
Hoover
Erich E
EE
Tillo
Shane E
SE
Hughes
Thom E
TE
Sylvester
Anne W
AW
Kleinfeld
David
D
Squier
Jeff A
JA
D001695:Biology / Q000295:instrumentation; D019187:Cadmium Compounds / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry*; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D008962:Models, Theoretical; D038761:Photobleaching; D059828:Plant Cells; D045663:Quantum Dots; D012235:Rhodamines / Q000737:chemistry*; D018036:Selenium Compounds / Q000737:chemistry; D000097573:Red Fluorescent Protein
D019187:Cadmium Compounds; D008164:Luminescent Proteins; D012235:Rhodamines; D018036:Selenium Compounds; D000097573:Red Fluorescent Protein; C058667:cadmium selenide; C029773:rhodamine B