Comparison of the effect of culture medium on unfixed rat pancreatic tissue in two-photon excitation microscopy

Purpose: Autofluorescence (AF) is the fluorescence of naturally occurring substances, such as nicotinamide adenine dinucleotide (NADH) and collagen. Two-photon microscopy (2PM) allows for the evaluation of living organs or tissues by excitation of tissue AF. Methods: In the present study, we compared AF intensities of pancreatic tissues in different culture mediums [Roswell Park Memorial Institute (RPMI) 1640, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and phosphate buffered saline (PBS) ] to determine the optimal conditions for observing unfixed and unstained tissues with 2PM. Results: Tissues incubated in RPMI 1640 showed the highest overall fluorescence intensity, followed by those in HEPES and PBS. Comparing the fluorescence intensities of the respective wavelengths, two broad peaks (approximately 760 nm and 860 nm) were recognized. At approximately 760 nm, acinar cells and ductal cells were observed below the surface. This wavelength primarily detects NADH. At approximately 860 nm, dense fibrous tissue was observed on the pancreatic surface, suggesting the presence of a connective tissue surrounding the pancreas. Conclusion: 2PM imaging using AF of the murine pancreas is a promising technique to provide new insights on structure and morphology.