NDLI: Recent developments in monitoring calcium and protein interactions in cells using fluorescence lifetime microscopy

Content Provider	Springer Nature Link
Author	Herman, Brian Wodnicki, Pawel Kwon, Seongwook Periasamy, Ammasi Gordon, Gerald W. Mahajan, Nupam Wang, Xue Feng
Copyright Year	1997
Abstract	Time-resolved fluorescence lifetime microscopy (TRFLM) allows the combination of the sensitivity of fluorescence lifetime to environmental parameters to be monitored in a spatial manner in single living cells, as well as providing more accurate, sensitive, and specific diagnosis of certain clinical diseases and chemical analyses. Here we discuss two applications of TRFLM: (1) the use of nonratiometric probes such as Calcium Crimson, for measuring Ca$^{2+}$; and (2) quantification of protein interaction in living cells using green and blue fluorescent protein (GFP and BFP, respectively) expressing constructs in combination with fluorescence resonance energy transfer microscopy (FRET). With respect to measuring Ca$^{2+}$ in biological samples, we demonstrate thatintensity-based measurements of Ca$^{2+}$ with single-wavelength Ca$^{2+}$ probes such as Calcium Crimson may falsely report the actual Ca$^{2+}$ concentration. This is due to effects of hydrophobicity of the local environment on the emission of Calcium Crimson as well as interaction of Calcium Crimson with proteins, both of which are overcome by the use of TRFLM. The recent availability of BFP (P4-3) and GFP (S65T) (which can serve as donor and acceptor, respectively) DNA sequences which can be attached to the carboxy-or amino-terminal DNA sequence of specific proteins allows the dual expression and interaction of proteins conjugated to BFP and GFP to be monitored in individual cells using FRET. Both of these applications of TRFLM are expected to enhance substantially the information available regarding both the normal and the abnormal physiology of cells and tissues.
Starting Page	85
Ending Page	91
Page Count	7
File Format	PDF
ISSN	10530509
Journal	Journal of Fluorescence
Volume Number	7
Issue Number	1
e-ISSN	15734994
Language	English
Publisher	Springer US
Publisher Date	1997-01-01
Publisher Place	Boston
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Fluorescence lifetime imaging microscopy calcium green fluorescent proteins blue fluorescent proteins Calcium Crimson fluorescence resonance energy transfer Biomedicine general Biophysics/Biomedical Physics Biotechnology Biochemistry Analytical Chemistry
Content Type	Text
Resource Type	Article
Subject	Sociology and Political Science Spectroscopy Law Biochemistry Clinical Biochemistry Clinical Psychology

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