NAD(P)H fluorescence lifetime measurements in fixed biological tissues

Area of Science:

• Biophotonics
• Molecular Imaging
• Histopathology

Background:

• Autofluorescence imaging (AFI) and AF-FLIM are primarily used for live samples to study metabolic fluxes.
• Chemical fixation of tissues, crucial for morphology and preservation, raises concerns about the reliability of metabolic imaging.
• Limited studies exist on interpreting metabolic imaging in fixed tissues.

Purpose of the Study:

• To evaluate the feasibility of extracting microenvironment information from fixed tissues using endogenous fluorescence.
• To assess the preservation of metabolic signatures, such as those from reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), after chemical fixation.
• To determine if AF-FLIM can provide reliable metabolic interpretations in fixed tissues.

Main Methods:

• Utilized autofluorescence-based fluorescence lifetime imaging microscopy (AF-FLIM).
• Investigated endogenous fluorescence from reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) in chemically fixed tissue samples.
• Analyzed changes in fluorescence lifetime and metabolic contrast post-fixation.

Main Results:

• Chemical fixation, including formaldehyde, was found to preserve lifetime-based metabolic contrast in tissues.
• Despite an increase in fluorescence lifetime due to fixatives, intrinsic metabolic signatures remained detectable.
• Fixed tissues retained their metabolic signatures, demonstrating the viability of AF-FLIM for such samples.

Conclusions:

• AF-FLIM can successfully extract microenvironment information from chemically fixed tissues.
• Metabolic interpretations are possible in fixed tissues using NAD(P)H autofluorescence, even after prolonged storage.
• This study validates the use of AF-FLIM on archived unstained histopathology tissues, opening new avenues for research.