Fluorescence lifetime imaging of free and protein-bound NADH
- PMID: 1741380
- PMCID: PMC48431
- DOI: 10.1073/pnas.89.4.1271
Fluorescence lifetime imaging of free and protein-bound NADH
Abstract
We introduce a methodology, fluorescence lifetime imaging (FLIM), in which the contrast depends on the fluorescence lifetime at each point in a two-dimensional image and not on the local concentration and/or intensity of the fluorophore. We used FLIM to create lifetime images of NADH when free in solution and when bound to malate dehydrogenase. This represents a challenging case for lifetime imaging because the NADH decay times are just 0.4 and 1.0 ns in the free and bound states, respectively. In the present apparatus, lifetime images are created from a series of phase-sensitive images obtained with a gain-modulated image intensifier and recorded with a charge-coupled device (CCD) camera. The intensifier gain is modulated at the light-modulation frequency or a harmonic thereof. A series of stationary phase-sensitive images each obtained with various phase shifts of the gain-modulation signal, is used to determine the phase angle or modulation of the emission at each pixel, which is in essence the lifetime image. We also describe am imaging procedure that allows specific decay times to be suppressed, allowing in this case suppression of the emission from either free or bound NADH. Since the fluorescence lifetimes of probes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules, this method allows imaging of the chemical or property of interest in macroscopic and microscopic samples. The concept of FLIM appears to have numerous potential applications in the biosciences.
Similar articles
-
Fluorescence lifetime imaging.Anal Biochem. 1992 May 1;202(2):316-30. doi: 10.1016/0003-2697(92)90112-k. Anal Biochem. 1992. PMID: 1519759 Free PMC article.
-
Fluorescence lifetime imaging of calcium using Quin-2.Cell Calcium. 1992 Mar;13(3):131-47. doi: 10.1016/0143-4160(92)90041-p. Cell Calcium. 1992. PMID: 1576634 Free PMC article.
-
Fluorescence lifetime imaging of intracellular calcium.J Fluoresc. 1993 Sep;3(3):161-7. doi: 10.1007/BF00862736. J Fluoresc. 1993. PMID: 24234827
-
[Fluorescence lifetime imaging microscopy (FLIM) in biological and medical research].Postepy Biochem. 2009;55(4):434-40. Postepy Biochem. 2009. PMID: 20201357 Review. Polish.
-
NADH Autofluorescence-A Marker on its Way to Boost Bioenergetic Research.Cytometry A. 2019 Jan;95(1):34-46. doi: 10.1002/cyto.a.23597. Epub 2018 Sep 13. Cytometry A. 2019. PMID: 30211978 Review.
Cited by
-
Opportunities for Persistent Luminescent Nanoparticles in Luminescence Imaging of Biological Systems and Photodynamic Therapy.Nanomaterials (Basel). 2020 Oct 13;10(10):2015. doi: 10.3390/nano10102015. Nanomaterials (Basel). 2020. PMID: 33066063 Free PMC article. Review.
-
FLIM of NAD(P)H in Lymphatic Nodes Resolves T-Cell Immune Response to the Tumor.Int J Mol Sci. 2022 Dec 13;23(24):15829. doi: 10.3390/ijms232415829. Int J Mol Sci. 2022. PMID: 36555468 Free PMC article.
-
The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions.Plant Cell. 2006 Mar;18(3):688-98. doi: 10.1105/tpc.105.039354. Epub 2006 Feb 3. Plant Cell. 2006. PMID: 16461578 Free PMC article.
-
The Plasma Levels of 3-Hydroxybutyrate, Dityrosine, and Other Markers of Oxidative Stress and Energy Metabolism in Major Depressive Disorder.Diagnostics (Basel). 2022 Mar 26;12(4):813. doi: 10.3390/diagnostics12040813. Diagnostics (Basel). 2022. PMID: 35453861 Free PMC article.
-
Background suppression in frequency-domain fluorometry.Anal Biochem. 2000 Jan 1;277(1):74-85. doi: 10.1006/abio.1999.4346. Anal Biochem. 2000. PMID: 10610691 Free PMC article.
References
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Miscellaneous