Subcellular Biochemical Investigation of PurkinjeNeurons Using Synchrotron Radiation Fourier Transform Infrared SpectroscopicImaging with a Focal Plane Array Detector

摘要

Coupling Fourier transform infrared spectroscopy with focal plane array detectors at synchrotron radiation sources (SR-FTIR-FPA) has provided a rapid method to simultaneously image numerous biochemical markers in situ at diffraction limited resolution. Since cells and nuclei are well resolved at this spatial resolution, a direct comparison can be made between FTIR functional group images and the histology of the same section. To allow histological analysis of the same section analyzed with infrared imaging, unfixed air-dried tissue sections are typically fixed (after infrared spectroscopic analysis is completed) via immersion fixation. This post fixation process is essential to allow histological staining of the tissue section. Although immersion fixation is a common practice in this filed, the initial rehydration of the dehydrated unfixed tissue can result in distortion of subcellular morphology and confound correlation between infrared images and histology. In this study, vapor fixation, a common choice in other research fields where postfixation of unfixed tissue sections is required, was employedin place of immersion fixation post spectroscopic analysis. This methodprovided more accurate histology with reduced distortions as the dehydratedtissue section is fixed in vapor rather than during rehydration inan aqueous fixation medium. With this approach, accurate correlationbetween infrared images and histology of the same section revealedthat Purkinje neurons in the cerebellum are rich in cytosolic proteinsand not depleted as once thought. In addition, we provide the firstdirect evidence of intracellular lactate within Purkinje neurons.This highlights the significant potential for future applicationsof SR-FTIR-FPA imaging to investigate cellular lactate under conditionsof altered metabolic demand such as increased brain activity and hypoxiaor ischemia.