June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
In situ monitoring cytochrome c in dying RGC-5 cells by Raman microscopy
Author Affiliations & Notes
  • Takeshi Morimoto
    Applied Visual Science, Osaka Univ Graduate Sch of Med, Suita, Japan
  • Hiroyuki Kanda
    Applied Visual Science, Osaka Univ Graduate Sch of Med, Suita, Japan
  • Liang-da Chiu
    Applied Physics, Osaka Univ Graduate Sch of Eng, Suita, Japan
  • Katsumasa Fujita
    Applied Physics, Osaka Univ Graduate Sch of Eng, Suita, Japan
  • Satoshi Kawata
    Applied Physics, Osaka Univ Graduate Sch of Eng, Suita, Japan
  • Makoto Nakamura
    Ophthalmology, Kobe Univ Graduate Sch of Med, kobe, Japan
  • Kohji Nishida
    Ophthalmology, Osaka Univ Graduate Sch of Med, Suita, Japan
  • Takashi Fujikado
    Applied Visual Science, Osaka Univ Graduate Sch of Med, Suita, Japan
  • Footnotes
    Commercial Relationships Takeshi Morimoto, None; Hiroyuki Kanda, Nidek Co., Ltd. (P); Liang-da Chiu, None; Katsumasa Fujita, None; Satoshi Kawata, None; Makoto Nakamura, None; Kohji Nishida, Alcon (C), Alcon (F), HOYA (F), Senju (F), Pfizer (F), Santen (F), Osaka University (P); Takashi Fujikado, Nidek (P)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 2320. doi:
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    • Get Citation

      Takeshi Morimoto, Hiroyuki Kanda, Liang-da Chiu, Katsumasa Fujita, Satoshi Kawata, Makoto Nakamura, Kohji Nishida, Takashi Fujikado; In situ monitoring cytochrome c in dying RGC-5 cells by Raman microscopy. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2320.

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      © ARVO (1962-2015); The Authors (2016-present)

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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  
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