In situ monitoring cytochrome c in dying RGC-5 cells by Raman microscopy

Abstract

 

Purpose

 

Label-free imaging is desirable for elucidating morphological and biochemical changes of neurons in vitro and in vivo. Raman microscopy provides high chemical contrast without requiring preprocessing or fluorescence staining of samples. In the present study, we demonstrated dynamic imaging of molecular distribution in unstained RGC-5 cells during the glutamine-induced death process using Raman microscopy.

 

Methods

 

Our originally developed slit-scanning Raman microscopy was used in this study. All the hyperspectral Raman scattering images were obtained with 532 nm excitation from a frequency-doubled Ti:Sapphire laser. Immortalized retinal ganglion cells (RGC-5) were used as a sample in all experiments. Prior to Raman imaging, the cells were seeded on a quartz substrate and cultured. For Raman imaging, the culture was maintained in a Hepes-buffered Tyrode’s solution and then was exposed to 400 mM glutamate. Raman scattering images were taken before and after administration of glutamate at 0, 30, 60, 120 minutes. The distribution of cytochrome c was reconstructed from the intensity distribution of the Raman peak at 753 cm-1 at each time points. Other cultures were also stained with Mitotracker or anti-cytochrome c antibody to compare the Raman images of cytochrome c.

 

Results

 

we imaged the molecular distributions of cytochrome c in unstained RGC-5 cells. After the administration of glutamate, the intensity of cytochrome c Raman peak decreased, and the Raman signal distribution was seen to diffuse into the cell body and the nucleus. Raman images resembled the image of immunostaining of Mitotracker and cytochrome c.

 

Conclusions

 

We revealed time-resolved distributions of cytochrome c in RGC-5 cells in the process of glutamate-induced cell death without the need for fluorescent labels or markers. Raman microscopy is useful to image the change of molecular distributions in dying RGC -5 cells with high temporal and spatial resolution.

 

 

Keywords: 551 imaging/image analysis: non-clinical • 615 neuroprotection • 690 retina: neurochemistry