Fluorescence lifetime imaging (FLIM) using ps-pulsed diode lasers in laser scanning microscopes

Angelika C. Ruck; Frank Dolp; Claudia Happ; Rudolf Steiner; Michael Beil

doi:10.1117/12.477828

19 June 2003 Fluorescence liftime imaging (FLIM) using ps-pulsed diode lasers in laser scanning microscopes

Angelika C. Ruck, Frank Dolp, Claudia Happ, Rudolf Steiner, Michael Beil

Proceedings Volume 4962, Manipulation and Analysis of Biomolecules, Cells, and Tissues; (2003) https://doi.org/10.1117/12.477828
Event: Biomedical Optics, 2003, San Jose, CA, United States

Abstract

A setup consisting on a laser scanning microscope equipped with appropriate detection units was developed for time-resolved intracellular fluorescence spectroscopy and fluorescence lifetime imaging (FLIM) for on-line detection of structural changes of various biomolecules. Short-pulsed excitation was performed with a diode laser which emits pulses at 398 nm with 70 ps duration. The laser was coupled to the laser scanning microscope. For time resolved spectroscopy a setup consisting of an Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in "spot scan" mode. In addition, a time-correlated single photon counting module was used to determine the fluorescence lifetime from single spots and to record lifetime images (τ-mapping). The time-resolved fluorescence characteristics of 5-ALA (5-aminolevulinic-acid), as well as 5-ALAhe (5-aminolevulinic-acid-hexylester)- induced protoporphyrine IX (PPIX) were investigated before and during PDT with subcellular resolution. For cells which were incubated with 5-ALA, a component with a fluorescence lifetime of about 7 ns was correlated with a structured fluorescence, which probably coincides with mitochondria, whereas a shorter lifetime was found in the cytoplasm. In the case of 5-ALAhe the lifetime of PPIX was longer, which could be due to different localization. During PDT the component with the longer lifetime completely vanished, whereas the shorter liftime was retained. It seems that FLIM is a valuable method to selectively identify and localize the photodynamically active photosensitizer.

Citation Download Citation

Angelika C. Ruck, Frank Dolp, Claudia Happ, Rudolf Steiner, and Michael Beil "Fluorescence liftime imaging (FLIM) using ps-pulsed diode lasers in laser scanning microscopes", Proc. SPIE 4962, Manipulation and Analysis of Biomolecules, Cells, and Tissues, (19 June 2003); https://doi.org/10.1117/12.477828

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