Endogenous fluorescence lifetime of viable cells by flow cytometry

Jessica P. Houston; Mark Naivar; John C. Martin; Greg Goddard; Susan Carpenter; Judith R. Mourant; James P. Freyer

doi:10.1117/12.763816

29 February 2008 Endogenous fluorescence lifetime of viable cells by flow cytometry

Jessica P. Houston, Mark Naivar, John C. Martin, Greg Goddard, Susan Carpenter, Judith R. Mourant, James P. Freyer

Proceedings Volume 6859, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VI; 68590T (2008) https://doi.org/10.1117/12.763816
Event: SPIE BiOS, 2008, San Jose, California, United States

Abstract

Autofluorescence emission is commonly measured in flow cytometry and is used as a negative control in protocols that explore binding of exogenous fluorophores to cell receptors or other targets of interest. The presence of intrinsic fluorophores however may burden complex cytometry applications. For example it may be difficult to resolve fluorescence signals from multi-intensity and multi-color measurements when the de-convolved fluorescence in question falls close to the autofluorescence background. One possible solution to intensity and spectral overlap problems in flow cytometry is to acquire fluorescence decay kinetic measurements. To this end we focus on advancing time-dependent flow cytometry and conduct measurements of endogenous fluorescence lifetime. Instrument developments to a phase-sensitive flow cytometry (PSFC) system were coupled with lifetime measurements of intrinsic fluorophores from viable cell samples. The average lifetime of >300,000 individual rat fibroblast cells was measured at discrete wavelengths ranging from 457- to 785-nm using a 10-MHz intensity-modulated excitation beam. AC amplitude, DC, and phase-shift were resolved and the average lifetime from excitable endogenous species was measured. The lifetime results ranged from 1- to 6-ns over the broad spectral range. Cataloging lifetime values prefaces the use of phase-sensitive techniques in more complex systems and provides a priori measurements necessary for PSFC filtering known lifetime signals from Raman, or other emission and scattering events.

Citation Download Citation

Jessica P. Houston, Mark Naivar, John C. Martin, Greg Goddard, Susan Carpenter, Judith R. Mourant, and James P. Freyer "Endogenous fluorescence lifetime of viable cells by flow cytometry", Proc. SPIE 6859, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VI, 68590T (29 February 2008); https://doi.org/10.1117/12.763816

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available