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Proceedings Paper

Improving precision in time-gated FLIM for low-light live-cell imaging
Author(s): Ching-Wei Chang; Mary-Ann Mycek
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Paper Abstract

Fluorescence Lifetime Imaging Microscopy (FLIM) is a molecular imaging technique that is useful for biological studies in living cells and tissues [1, 2]. When high-intensity light sources such as lasers are used for fluorescence excitation, it is important to ensure that live-cell systems remain viable and do not become significantly stressed. Error analysis helps to achieve precision in lifetime determination with low-light live-cell imaging [3-5]. We have combined error analysis and Monte Carlo simulations to develop a temporal approach to enhance the precision of time-gated FLIM. This approach can involve both optimal gating and curve fitting. We have compared the precision associated with various lifetime determination techniques, and then searched parameter space in order to find optimal gating conditions in terms of minimal achievable relative standard deviation. Precision and accuracy were investigated via Monte Carlo simulations that included Poisson noise. The results were validated with fluorescence lifetime standards and fluorescent beads. Because time-gated FLIM produces images for each gating, another way to improve precision in low-light FLIM is to utilize spatial information from the gated images to remove noise. Total variation (TV) models are commonly used denoising algorithms [6, 7]. We have considered several TV denoising models to improve the precision of lifetime determination with low-light FLIM. These methods remove electronic-related noise from FLIM images and hence can increase the precision with which the lifetimes are determined. Since the temporal and spatial methods apply to different dimensions, we assume that they work independently and their precision improvements are additive. We test this assumption when the pixel-to-pixel variation due to noise in one image is high enough to cause possible unexpected nonlinear effects. We demonstrate that the precision improvements from the temporal and spatial techniques are independent and additive in a regime pertinent to livecell FLIM studies.

Paper Details

Date Published: 1 July 2009
PDF: 6 pages
Proc. SPIE 7370, Molecular Imaging II, 737009 (1 July 2009); doi: 10.1117/12.831712
Show Author Affiliations
Ching-Wei Chang, Univ. of Michigan (United States)
Mary-Ann Mycek, Univ. of Michigan (United States)

Published in SPIE Proceedings Vol. 7370:
Molecular Imaging II
Kai Licha; Charles P. Lin, Editor(s)

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