Typical imaging depths with multiphoton microscopy (MPM) are limited to less than 300 µm in many tissues due to light scattering. Optical clearing significantly reduces light scattering by replacing water in the organ tissue with a fluid having a similar index of refraction to that of proteins. We demonstrate MPM of intact, fixed, cleared mouse organs with penetration depths and fields of view in excess of 2 mm. MPM enables the creation of large 3-D data sets with flexibility in pixel format and ready access to intrinsic fluorescence and second-harmonic generation. We present high-resolution images and 3-D image stacks of the brain, small intestine, large intestine, kidney, lung, and testicle with image sizes as large as 4096×4096 pixels.
A powerful advantage of multiphoton microscopy is its ability to image endogenous
fluorophores such as the ubiquitous coenzyme NADH in discrete cellular populations. NADH is
integral in both oxidative and non-oxidative cellular metabolism. NADH loses fluorescence
upon oxidation to NAD+; thus changes in NADH fluorescence can be used to monitor
metabolism. Recent studies have suggested that hypo metabolic astrocytes play an important
role in cases of temporal lobe epilepsy (TLE). Current theories suggest this may be due to
defective and/or a reduced number of mitochondria or dysfunction of the neuronal-astrocytic
metabolic coupling. Measuring NADH fluorescence changes following chemical stimulation
enables the quantification of the cellular distribution of metabolic anomalies in epileptic brain
tissue compared to healthy tissue. We present what we believe to be the first multiphoton
microscopy images of NADH from the human brain. We also present images of NADH
fluorescence from the hippocampus of the kainate-treated rat TLE model. In some experiments,
human and rat astrocytes were selectively labeled with the fluorescent dye sulforhodamine 101
(SR101). Our results demonstrate that multiphoton microscopy is a powerful tool for assaying
the metabolic pathologies associated with temporal lobe epilepsy in humans and in rodent
models.
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