Method of transmission filters to measure emission spectra in strongly scattering media

: We describe a method based on a pair of transmission filters placed in the emission path of a microscope to resolve the emission wavelength of every point in an image. The method can be applied to any type of imaging device that provides the light in the wavelength transmission range of the filters. Unique characteristics of the filter approach are that the light does not need to be collimated and the wavelength response does not depend on the scattering of the sample or tissue. The pair of filters are used to produce the spectral phasor of the transmitted light, which is sufficient to perform spectral deconvolution over a broad wavelength range. The method is sensitive enough to distinguish free and protein-bound NADH and can be used in metabolic studies.

[1]  A. Bader,et al.  Full-Harmonics Phasor Analysis: Unravelling Multiexponential Trends in Magnetic Resonance Imaging Data , 2020, The journal of physical chemistry letters.

[2]  Jianhua Xu,et al.  Dehydrogenase Binding Sites Abolish the "Dark" Fraction of NADH: Implication for Metabolic Sensing Via FLIM. , 2020, The journal of physical chemistry. B.

[3]  E. Gratton,et al.  Determination of the metabolic index using the fluorescence lifetime of free and bound nicotinamide adenine dinucleotide using the phasor approach , 2019, Journal of biophotonics.

[4]  E. Gratton,et al.  The DIVER Microscope for Imaging in Scattering Media , 2019, Methods and protocols.

[5]  E. Gratton,et al.  Hyperspectral imaging in highly scattering media by the spectral phasor approach using two filters. , 2018, Biomedical optics express.

[6]  Frank J Vergeldt,et al.  Multi-component quantitative magnetic resonance imaging by phasor representation , 2017, Scientific Reports.

[7]  M. Duchen,et al.  Investigating mitochondrial redox state using NADH and NADPH autofluorescence , 2016, Free radical biology & medicine.

[8]  E. Gratton,et al.  Measurements of absolute concentrations of NADH in cells using the phasor FLIM method. , 2016, Biomedical optics express.

[9]  E. Gratton,et al.  Model-free methods to study membrane environmental probes: a comparison of the spectral phasor and generalized polarization approaches , 2015, Methods and applications in fluorescence.

[10]  Enrico Gratton,et al.  Fluorescence lifetime imaging of endogenous biomarker of oxidative stress , 2015, Scientific Reports.

[11]  P. Urayama,et al.  Autofluorescence from NADH Conformations Associated with Different Metabolic Pathways Monitored Using Nanosecond-Gated Spectroscopy and Spectral Phasor Analysis. , 2015, Analytical chemistry.

[12]  E. Gratton,et al.  A deep tissue fluorescence imaging system with enhanced SHG detection capabilities , 2014, Microscopy research and technique.

[13]  E. Gratton,et al.  Laurdan fluorescence lifetime discriminates cholesterol content from changes in fluidity in living cell membranes. , 2013, Biophysical journal.

[14]  Enrico Gratton,et al.  Spectral phasor analysis of Pyronin Y labeled RNA microenvironments in living cells , 2012, Biomedical optics express.

[15]  Enrico Gratton,et al.  Phasor Fluorescence Lifetime Microscopy of Free and Protein-Bound NADH Reveals Neural Stem Cell Differentiation Potential , 2012, PloS one.

[16]  Enrico Gratton,et al.  Deep tissue fluorescence imaging and in vivo biological applications , 2012, Journal of biomedical optics.

[17]  Enrico Gratton,et al.  Metabolic trajectory of cellular differentiation in small intestine by Phasor Fluorescence Lifetime Microscopy of NADH , 2012, Scientific Reports.

[18]  Enrico Gratton,et al.  NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy. , 2012, Biophysical journal.

[19]  Hans C Gerritsen,et al.  Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images. , 2012, Optics express.

[20]  Enrico Gratton,et al.  Enhancement of imaging depth in turbid media using a wide area detector , 2011, Journal of biophotonics.

[21]  E. Gratton,et al.  Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue , 2011, Proceedings of the National Academy of Sciences.

[22]  H. S. de Bruijn,et al.  In vivo monitoring of protein-bound and free NADH during ischemia by nonlinear spectral imaging microscopy , 2011, Biomedical optics express.

[23]  K. Goodson,et al.  Non-invasive measurement of void fraction and liquid temperature in microchannel flow boiling , 2009 .

[24]  Dong Li,et al.  Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence. , 2008, Optics letters.

[25]  N. Ramanujam,et al.  In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia , 2007, Proceedings of the National Academy of Sciences.

[26]  Wei Zheng,et al.  Sensing cell metabolism by time-resolved autofluorescence. , 2006, Optics letters.

[27]  N. Ramanujam,et al.  Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH. , 2005, Cancer research.

[28]  S. de Vries,et al.  Optical spectroscopy of nicotinoprotein alcohol dehydrogenase from Amycolatopsis methanolica: a comparison with horse liver alcohol dehydrogenase and UDP-galactose epimerase. , 1998, Biochemistry.

[29]  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.

[30]  D. Jameson,et al.  Time-resolved fluorescence studies on NADH bound to mitochondrial malate dehydrogenase. , 1989, Biochimica et biophysica acta.

[31]  T. G. Scott,et al.  Synthetic spectroscopic models related to coenzymes and base pairs. V. Emission properties of NADH. Studies of fluorescence lifetimes and quantum efficiencies of NADH, AcPyADH, [reduced acetylpyridineadenine dinucleotide] and simplified synthetic models , 1970 .

[32]  A. Chorvatova,et al.  Time-resolved spectrometry of mitochondrial NAD(P)H fluorescence and its applications for evaluating the oxidative state in living cells. , 2015, Methods in molecular biology.