Document Detail

Development of microscopic systems for high-speed dual-excitation ratiometric Ca2+ imaging.
MedLine Citation:
PMID:  18941899     Owner:  NLM     Status:  MEDLINE    
For quantitative measurements of Ca(2+) concentration ([Ca(2+)]), ratiometric dyes are preferable, because the use of such dyes allows for correction of uneven loading or partitioning of dye within the cell as well as variations in cell thickness. Although dual-excitation ratiometric dyes for measuring [Ca(2+)], such as Fura-2, Fura-Red, and ratiometric-pericam, are widely used for a variety of applications, it has been difficult to use them for monitoring very fast Ca(2+) dynamics or Ca(2+) changes in highly motile cells. To overcome this problem, we have developed three new dual-excitation ratiometry systems. (1) A system in which two laser beams are alternated on every scanning line, allowing us to obtain confocal images using dual-excitation ratiometric dyes. This system increases the rate at which ratio measurements can be made to 200 Hz and provides confocal images at 1-10 Hz depending on the image size. (2) A truly simultaneous dual-excitation ratiometry system that used linearly polarized excitation light and polarization detection, allowing us to obtain ratiometric images without any time lag. This system, however, is based on statistical features of the fluorescence polarization and is limited to samples that contain a large number of fluorophores. In addition, this method requires complicated calculations. (3) An efficient, nearly simultaneous dual-excitation ratiometry system that allows us to rapidly switch between two synchronized excitation-detection components by employing two high-power light-emitting diodes (LEDs) and two high-speed liquid crystal shutters. The open/close operation of the two shutters is synchronized with the on/off switching of the two LEDs. This system increases the rate at which ratio measurements are made to 1 kHz, and provides ratio images at 10-100 Hz depending on the signal intensity.
Takashi Fukano; Satoshi Shimozono; Atsushi Miyawaki
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2008-10-22
Journal Detail:
Title:  Brain cell biology     Volume:  36     ISSN:  1559-7113     ISO Abbreviation:  Brain Cell Biol     Publication Date:  2008 Aug 
Date Detail:
Created Date:  2008-11-26     Completed Date:  2009-02-04     Revised Date:  2009-12-11    
Medline Journal Info:
Nlm Unique ID:  101272887     Medline TA:  Brain Cell Biol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  43-52     Citation Subset:  IM    
Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan.
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MeSH Terms
Calcium / analysis,  metabolism*
Calcium Signaling / physiology
Fluorescence Polarization / instrumentation,  methods*
Fluorescent Dyes / metabolism
Green Fluorescent Proteins / metabolism
Microscopy, Confocal / instrumentation,  methods*
Microscopy, Fluorescence / instrumentation,  methods*
Reg. No./Substance:
0/Fluorescent Dyes; 147336-22-9/Green Fluorescent Proteins; 7440-70-2/Calcium

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