Expand this Topic clickable element to expand a topic
Skip to content
Optica Publishing Group

Role of three-dimensional bleach distribution in confocal and two-photon fluorescence recovery after photobleaching experiments

Not Accessible

Your library or personal account may give you access

Abstract

The quantitative analysis of fluorescence perturbation experiments such as fluorescence recovery after photobleaching (FRAP) requires suitable analytical models to be developed. When diffusion in 3D environments is considered, the description of the initial condition produced by the perturbation (i.e., the photobleaching of a selected region in FRAP) represents a crucial aspect. Though it is widely known that bleaching profiles approximations can lead to errors in quantitative FRAP measurements, a detailed analysis of the sources and the effects of these approximations has never been conducted until now. In this study, we measured the experimental 3D bleaching distributions obtained in conventional and two-photon excitation schemes and analyzed the deviations from the ideal cases usually adopted in FRAP experiments. In addition, we considered the non-first-order effects generated by the high energy pulses usually delivered in FRAP experiments. These data have been used for finite-element simulations mimicking FRAP experiments on free diffusing molecules and compared with FRAP model curves based on the ideal bleach distributions. The results show that two-photon excitation more closely fits ideal bleaching patterns even in the event of fluorescence saturation, achieving estimations of diffusion coefficients within 20% accuracy of the correct value.

© 2007 Optical Society of America

Full Article  |  PDF Article
More Like This
Experimental and theoretical investigation of fluorescence photobleaching and recovery in human breast tissue and tissue phantoms

Sharad Gupta, Bhawna, Pallab Goswami, Asha Agarwal, and Asima Pradhan
Appl. Opt. 43(5) 1044-1052 (2004)

Straightforward FRAP for quantitative diffusion measurements with a laser scanning microscope

Hendrik Deschout, Joel Hagman, Sophia Fransson, Jenny Jonasson, Mats Rudemo, Niklas Lorén, and Kevin Braeckmans
Opt. Express 18(22) 22886-22905 (2010)

Simultaneous FRAP, FLIM and FAIM for measurements of protein mobility and interaction in living cells

James A. Levitt, Penny E. Morton, Gilbert O. Fruhwirth, George Santis, Pei-Hua Chung, Maddy Parsons, and Klaus Suhling
Biomed. Opt. Express 6(10) 3842-3854 (2015)

Cited By

You do not have subscription access to this journal. Cited by links are available to subscribers only. You may subscribe either as an Optica member, or as an authorized user of your institution.

Contact your librarian or system administrator
or
Login to access Optica Member Subscription

Figures (8)

You do not have subscription access to this journal. Figure files are available to subscribers only. You may subscribe either as an Optica member, or as an authorized user of your institution.

Contact your librarian or system administrator
or
Login to access Optica Member Subscription

Tables (1)

You do not have subscription access to this journal. Article tables are available to subscribers only. You may subscribe either as an Optica member, or as an authorized user of your institution.

Contact your librarian or system administrator
or
Login to access Optica Member Subscription

Equations (12)

You do not have subscription access to this journal. Equations are available to subscribers only. You may subscribe either as an Optica member, or as an authorized user of your institution.

Contact your librarian or system administrator
or
Login to access Optica Member Subscription

Select as filters


Select Topics Cancel
© Copyright 2024 | Optica Publishing Group. All Rights Reserved