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 non-ratiometric 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 that intensity-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.
Bibliographical noteFunding Information:
This work was supported by Grants AGO7218, AG10104, and AG13637 from the National Institute of Health, the Gustavus and Louise Pfeiffer Research Foundation, and Grants CB-85B and FRA 383 from the American Cancer Society, as well a instrumentation grants from the National Sciences Foundation, American Cancer Society, North Carolina Biotechnology Center, and American Heart Association. We also wish to acknowledge gratefully the support from Hamamatsu Photonic Systems, Olympus Co., and ISS Co., Dr. Roger Tsien for supplying us with the cDNA for GFP and BFP, and Dr. John Reed for the Bax cDNA.
- Blue fluorescent proteins
- Calcium Crimson
- Fluorescence lifetime imaging microscopy
- Fluorescence resonance energy transfer
- Green fluorescent proteins