Wide-field time-domain fluorescence lifetime imaging microscopy Dhruv Sud

ISBN: 9780549994992

Published:

NOOKstudy eTextbook

123 pages


Description

Wide-field time-domain fluorescence lifetime imaging microscopy  by  Dhruv Sud

Wide-field time-domain fluorescence lifetime imaging microscopy by Dhruv Sud
| NOOKstudy eTextbook | PDF, EPUB, FB2, DjVu, talking book, mp3, RTF | 123 pages | ISBN: 9780549994992 | 4.30 Mb

Steady-state fluorescence imaging is routinely employed to obtain physiological information but is susceptible to artifacts such as absorption and photobleaching. FLIM provides an additional source of contrast oblivious to these but is affected byMoreSteady-state fluorescence imaging is routinely employed to obtain physiological information but is susceptible to artifacts such as absorption and photobleaching.

FLIM provides an additional source of contrast oblivious to these but is affected by factors such as pH, gases, and temperature. Here we focused on developing a resolution-enhanced FLIM system for quantitative oxygen sensing. Oxygen is one of the most critical components of metabolic machinery and affects growth, differentiation, and death.

FLIM-based oxygen sensing provides a valuable tool for biologists without the need of alternate technologies. We also developed novel computational approaches to improve spatial resolution of FLIM images, extending its potential for thick tissue studies.-We designed a wide-field time-domain UV-vis-NIR FLIM system with high temporal resolution (50 ps), large temporal dynamic range (750 ps -- 1 mus), short data acquisition/processing times (15 s) and noise-removal capability.

Lifetime calibration of an oxygen-sensitive, ruthenium dye (RTDP) enabled in vivo oxygen level measurements (resolution = 8 muM, range = 1 -- 300 muM). Combining oxygen sensing with endogenous imaging allowed for the study of two key molecules (NADH and oxygen) consumed at the termini of the oxidative phosphorylation pathway in Barretts adenocarcinoma columnar (SEG-1) cells and Esophageal normal squamous cells (HET-1). Starkly higher intracellular oxygen and NADH levels in living SEG-1 vs. HET-1 cells were detected by FLIM and attributed to altered metabolic pathways in malignant cells.-We performed FLIM studies in microfluidic bioreactors seeded with mouse myoblasts.

For these systems, oxygen concentrations play an important role in cell behavior and gene expression. Oxygen levels decreased with increasing cell densities and were consistent with simulated model outcomes. In single bioreactor loops, FLIM detected spatial heterogeneity in oxygen levels as high as 20%.-We validated our calibration with EPR spectroscopy, the gold standard for intracellular oxygen measurements.

Differences between FLIM and EPR results were explained by cell lysate-FLIM studies. We proposed a new protocol for estimating oxygen levels by using a reference cell line and cellular lysate analysis. Lastly, we proposed and compared two different image restoration approaches, direct lifetime vs. intensity-overlay. Both approaches improve resolution while maintaining veracity of lifetime.



Enter the sum





Related Archive Books



Related Books


Comments

Comments for "Wide-field time-domain fluorescence lifetime imaging microscopy":


whitefishmedia.pl

©2014-2015 | DMCA | Contact us