PMID- 16690123
OWN - NLM
STAT- MEDLINE
DCOM- 20060907
LR  - 20131121
IS  - 0143-4160 (Print)
IS  - 0143-4160 (Linking)
VI  - 40
IP  - 1
DP  - 2006 Jul
TI  - Quantitative two-photon Ca2+ imaging via fluorescence lifetime analysis.
PG  - 73-9
AB  - Two-photon microscopy (TPM) revolutionized Ca2+ imaging by allowing recordings in 
      the depth of intact tissue and live organisms. A serious limitation in TPM, 
      however, is the lack of an accurate and straightforward approach for the 
      quantification of Ca2+ signals, an ability that became an invaluable tool in 
      fluorescence microscopy. Here, we present time-correlated fluorescence lifetime 
      imaging (tcFLIM) as a ratiometric method for the quantification of Ca2+ signals 
      in TPM. The fluorescence lifetime of the Ca2+-indicator dye Oregon Green BAPTA-1 
      (OGB-1) can be recorded using the approximately 80 MHz excitation pulses utilized 
      in TPM. It shows a Ca2+ dependence that can be explained by the Ca2+-affinity, 
      spectral properties and purity of the dye. Pixel-wise lifetime recordings, 
      controlled by a laser-scanning microscope, allowed quantitative Ca2+ imaging in 
      full-frame and linescan mode. Although we focused on the high-affinity Ca2+ 
      indicator OGB-1, our tcFLIM-based quantification is applicable to other Ca2+ dyes 
      and to fluorescence indicators in general.
FAU - Wilms, Christian D
AU  - Wilms CD
AD  - Leipzig University, Carl-Ludwig-Institute for Physiology, Liebigstr. 27, 04103 
      Leipzig, Germany. christian.wilms@medizin.uni-leipzig.de
FAU - Schmidt, Hartmut
AU  - Schmidt H
FAU - Eilers, Jens
AU  - Eilers J
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20060509
PL  - Netherlands
TA  - Cell Calcium
JT  - Cell calcium
JID - 8006226
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - *Calcium
MH  - Calcium Signaling/physiology
MH  - Microscopy/*instrumentation/*methods
MH  - Microscopy, Confocal
MH  - Microscopy, Fluorescence
MH  - Photons
EDAT- 2006/05/13 09:00
MHDA- 2006/09/08 09:00
CRDT- 2006/05/13 09:00
PHST- 2006/01/04 00:00 [received]
PHST- 2006/03/10 00:00 [revised]
PHST- 2006/03/16 00:00 [accepted]
PHST- 2006/05/13 09:00 [pubmed]
PHST- 2006/09/08 09:00 [medline]
PHST- 2006/05/13 09:00 [entrez]
AID - S0143-4160(06)00047-9 [pii]
AID - 10.1016/j.ceca.2006.03.006 [doi]
PST - ppublish
SO  - Cell Calcium. 2006 Jul;40(1):73-9. doi: 10.1016/j.ceca.2006.03.006. Epub 2006 May 
      9.