FLIM data analysis of NADH and Tryptophan autofluorescence in prostate cancer cells

Meghan J. O'Melia; Horst Wallrabe; Zdenek Svindrych; Shagufta Rehman; Ammasi Periasamy

doi:10.1117/12.2223985

14 March 2016 FLIM data analysis of NADH and Tryptophan autofluorescence in prostate cancer cells

Meghan J. O'Melia, Horst Wallrabe, Zdenek Svindrych, Shagufta Rehman, Ammasi Periasamy

Proceedings Volume 9712, Multiphoton Microscopy in the Biomedical Sciences XVI; 97122E (2016) https://doi.org/10.1117/12.2223985
Event: SPIE BiOS, 2016, San Francisco, California, United States

Abstract

Fluorescence lifetime imaging microscopy (FLIM) is one of the most sensitive techniques to measure metabolic activity in living cells, tissues and whole animals. We used two- and three-photon fluorescence excitation together with time-correlated single photon counting (TCSPC) to acquire FLIM signals from normal and prostate cancer cell lines. FLIM requires complex data fitting and analysis; we explored different ways to analyze the data to match diverse cellular morphologies. After non-linear least square fitting of the multi-photon TCSPC images by the SPCImage software (Becker & Hickl), all image data are exported and further processed in ImageJ. Photon images provide morphological, NAD(P)H signal-based autofluorescent features, for which regions of interest (ROIs) are created. Applying these ROIs to all image data parameters with a custom ImageJ macro, generates a discrete, ROI specific database. A custom Excel (Microsoft) macro further analyzes the data with charts and statistics. Applying this highly automated assay we compared normal and cancer prostate cell lines with respect to their glycolytic activity by analyzing the NAD(P)H-bound fraction (a2%), NADPH/NADH ratio and efficiency of energy transfer (E%) for Tryptophan (Trp). Our results show that this assay is able to differentiate the effects of glucose stimulation and Doxorubicin in these prostate cell lines by tracking the changes in a2% of NAD(P)H, NADPH/NADH ratio and the changes in Trp E%. The ability to isolate a large, ROI-based data set, reflecting the heterogeneous cellular environment and highlighting even subtle changes — rather than whole cell averages - makes this assay particularly valuable.

Citation Download Citation

Meghan J. O'Melia, Horst Wallrabe, Zdenek Svindrych, Shagufta Rehman, and Ammasi Periasamy "FLIM data analysis of NADH and Tryptophan autofluorescence in prostate cancer cells", Proc. SPIE 9712, Multiphoton Microscopy in the Biomedical Sciences XVI, 97122E (14 March 2016); https://doi.org/10.1117/12.2223985

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