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Stimulated emission reduced fluorescence microscopy: a concept for extending the fundamental depth limit of two-photon fluorescence imaging

Wei, Lu and Chen, Zhixing and Min, Wei (2012) Stimulated emission reduced fluorescence microscopy: a concept for extending the fundamental depth limit of two-photon fluorescence imaging. Biomedical Optics Express, 3 (6). pp. 1465-1475. ISSN 2156-7085. PMCID PMC3370985. https://resolver.caltech.edu/CaltechAUTHORS:20181119-102040395

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Abstract

Two-photon fluorescence microscopy has become an indispensable tool for imaging scattering biological samples by detecting scattered fluorescence photons generated from a spatially confined excitation volume. However, this optical sectioning capability breaks down eventually when imaging much deeper, as the out-of-focus fluorescence gradually overwhelms the in-focal signal in the scattering samples. The resulting loss of image contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation efficiency. Herein we propose to extend this depth limit by performing stimulated emission reduced fluorescence (SERF) microscopy in which the two-photon excited fluorescence at the focus is preferentially switched on and off by a modulated and focused laser beam that is capable of inducing stimulated emission of the fluorophores from the excited states. The resulting image, constructed from the reduced fluorescence signal, is found to exhibit a significantly improved signal-to-background contrast owing to its overall higher-order nonlinear dependence on the incident laser intensity. We demonstrate this new concept by both analytical theory and numerical simulations. For brain tissues, SERF is expected to extend the imaging depth limit of two-photon fluorescence microscopy by a factor of more than 1.8.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1364/boe.3.001465DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3370985/PubMed CentralArticle
ORCID:
AuthorORCID
Wei, Lu0000-0001-9170-2283
Min, Wei0000-0003-2570-3557
Additional Information:© 2012 Optical Society of America. Received 27 Apr 2012; revised 19 May 2012; accepted 19 May 2012; published 22 May 2012. We thank Ya-Ting Kao, Xinxin Zhu, Louis Brus, Rafael Yuste, Darcy Peterka, Virginia Cornish, Christophe Dupre and Miguel Jimenez for helpful discussions. W. M. acknowledges the startup funds from Columbia University, and grant support from Kavli Institute for Brain Science.
Funders:
Funding AgencyGrant Number
Columbia UniversityUNSPECIFIED
Kavli Institute for Brain ScienceUNSPECIFIED
Issue or Number:6
Classification Code:OCIS codes: (180.4315) Nonlinear microscopy; (180.2520) Fluorescence microscopy; (190.4180) Multiphoton processes; (170.4090) Modulation techniques
PubMed Central ID:PMC3370985
Record Number:CaltechAUTHORS:20181119-102040395
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20181119-102040395
Official Citation:Lu Wei, Zhixing Chen, and Wei Min, "Stimulated emission reduced fluorescence microscopy: a concept for extending the fundamental depth limit of two-photon fluorescence imaging," Biomed. Opt. Express 3, 1465-1475 (2012)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:91019
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:19 Nov 2018 19:04
Last Modified:23 Oct 2019 20:47

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