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

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 Ca2+; 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 Ca2+ in biological samples, we demonstrate thatintensity-based measurements of Ca2+ with single-wavelength Ca2+ probes such as Calcium Crimson may falsely report the actual Ca2+ 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.

[1]  S. Bolsover,et al.  The nucleus is insulated from large cytosolic calcium ion changes , 1994, Nature.

[2]  John Calvin Reed,et al.  Structure-function comparisons of the proapoptotic protein Bax in yeast and mammalian cells , 1996, Molecular and cellular biology.

[3]  M. Chalfie,et al.  Green fluorescent protein as a marker for gene expression. , 1994, Science.

[4]  P. Diliberto,et al.  Confocal imaging of Ca2+ in cells. , 1994, Methods in cell biology.

[5]  Robert M. Clegg,et al.  Fluorescence lifetime imaging microscopy (FLIM): Spatial resolution of microstructures on the nanosecond time scale , 1993 .

[6]  Á. Maquieira,et al.  Flow Injection Analysis of Seawater. Part II. Cationic Species , 1992 .

[7]  R. Tsien.  Intracellular signal transduction in four dimensions: from molecular design to physiology. , 1992, The American journal of physiology.

[8]  R. Tsien.  Fluorescent probes of cell signaling. , 1989, Annual review of neuroscience.

[9]  A. Periasamy,et al.  Fluorescence Lifetime Imaging Microscopy (FLIM): Instrumentation and Applications , 1992 .

[10]  R. Tsien,et al.  Fluorescence ratio imaging: a new window into intracellular ionic signaling , 1986 .

[11]  Hans C. Gerritsen,et al.  Fluorescence lifetime imaging using a confocal laser scanning microscope , 1992 .

[12]  R. Schreiber,et al.  Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death , 1990, Nature.

[13]  P. Selvin.  Fluorescence resonance energy transfer. , 1995, Methods in enzymology.

[14]  B. Herman,et al.  Plasma membrane architecture during hypoxic injury in rat hepatocytes measured by fluorescence quenching and resonance energy transfer imaging , 1993 .

[15]  R. Tsien.  Fluorescence Imaging Creates A Window On The Cell , 1994 .

[16]  H Szmacinski,et al.  Fluorescence lifetime imaging. , 1992, Analytical biochemistry.

[17]  R. Tsien,et al.  Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer , 1996, Current Biology.

[18]  Joseph R. Lakowicz,et al.  Calcium imaging using fluorescence lifetimes and long-wavelength probes , 1992, Journal of Fluorescence.

[19]  A Kusumi,et al.  Fluorescence lifetime imaging microscopy (flimscopy). Methodology development and application to studies of endosome fusion in single cells. , 1993, Biophysical journal.

[20]  Robert M. Clegg,et al.  Fluorescence lifetime imaging microscopy: pixel-by-pixel analysis of phase-modulation data , 1994 .

[21]  Richard P. Haugland,et al.  Handbook of fluorescent probes and research chemicals , 1996 .

[22]  B. Herman,et al.  Mitochondrial free calcium transients during excitation‐contraction coupling in rabbit cardiac myocytes , 1996, FEBS letters.

[23]  H Szmacinski,et al.  Fluorescence lifetime imaging of intracellular calcium in COS cells using Quin-2. , 1994, Cell calcium.

[24]  J. Lakowicz,et al.  Fluorescence lifetime imaging of free and protein-bound NADH. , 1992, Proceedings of the National Academy of Sciences of the United States of America.