Two-photon microscopy of living cells by simultaneously exciting multiple endogenous fluorophores and fluorescent proteins

Wei Zheng; Dong Li; Jianan Y. Qu

doi:10.1117/12.841428

24 February 2010 Two-photon microscopy of living cells by simultaneously exciting multiple endogenous fluorophores and fluorescent proteins

Wei Zheng, Dong Li, Jianan Y. Qu

Author Affiliations +

Proceedings Volume 7568, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VIII; 756806 (2010) https://doi.org/10.1117/12.841428
Event: SPIE BiOS, 2010, San Francisco, California, United States

Abstract

Endogenous fluorophores, such as reduced nicotinamide adenine dinucleotide (NADH), keratin, and tryptophan, have been used as contrast agents for imaging metabolism and morphology of living cells and tissues. Multilabeling which maps the distribution of different targets is an indispensable technique in many biomedical and biochemical studies. Therefore, two-photon excitation fluorescence (TPEF) microscopy of endogenous fluorophores combining with in vivo fluorescence labeling techniques such as genetically encoded fluorescent protein could be a powerful tool for imaging living cells and tissues. However, the challenge is that the excitation and emission wavelengths of these endogenous fluorophores and fluorescence labels are very different. A multi-color ultrafast source is required for the excitation of multiple fluorescence molecules. In this study, we developed a two-photon imaging system with excitations from the pump femtosecond laser and the selected Supercontinuum generated from a photonic crystal fiber (PCF). Multiple endogenous fluorophores and fluorescent proteins such as NADH, tryptophan, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) were excited in their optimal wavelengths alternately or simultaneously. A time- and spectral-resolved detection system was used to record the TPEF signals. This detection technique separated the TPEF signals from multiple sources in time and spectral domains. Cellular organelles such as nucleus, mitochondria, microtubule and Endoplasmic Reticulum (ER), were clearly revealed in the TPEF images.

Citation Download Citation

Wei Zheng, Dong Li, and Jianan Y. Qu "Two-photon microscopy of living cells by simultaneously exciting multiple endogenous fluorophores and fluorescent proteins", Proc. SPIE 7568, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VIII, 756806 (24 February 2010); https://doi.org/10.1117/12.841428

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

Members: $17.00

Non-members: $21.00 ADD TO CART

PROCEEDINGS
6 PAGES

DOWNLOAD PAPER SAVE TO MY LIBRARY

GET CITATION

RIGHTS & PERMISSIONS

Get copyright permission Get copyright permission on Copyright Marketplace

KEYWORDS

Luminescence

Green fluorescent protein

Fluorescent proteins

Femtosecond phenomena

Signal detection

Microscopy

Mirrors

Show All Keywords

Keywords/Phrases

Search In:

Publication Years