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Takeshi Morimoto, Liang-da Chiu, Katsumasa Fujita, Hiroyuki Kanda, Kenta Hozumi, Kohji Nishida, Takashi Fujikado; In situ monitoring the redox dynamics of cytochrome c in glutamate-induced dying retinal cells by hybrid fluorescence-Raman imaging. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4688.
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Raman imaging is a powerful technique for label-free observation of biological molecules. Cytochrome c (Cyt c) is one of the major targets to be observed by a Raman microscopy. However, Raman imaging can detect only reduced form of Cyt c. Here we developed a hybrid fluorescence-Raman imaging technique by generating immortal retinal cells with monomeric teal fluorescent protein 1 (mTFP1) tagged Cyt c. And, we observed the redox dynamics of Cyt c in these cells during the glutamine-induced death process using this Raman microscopy
The mouse immortalized RGC-5 cell line was used in this study. A chimeric gene form through the combination of mTFP1 and human Cyt c coding sequences was created and cloned. Then the mTFP1-Cyt c gene was transfected into RGC-5 cells.Raman images were obtained using a home-built slit-scanning Raman microscope with 532 nm excitation from a frequency-doubled Nd: YVO4 laser.Prior to Raman imaging, the cells were seeded on a quartz substrate and were treated with 600 mM of L-glutamate for 0.5, 1.0, 1.5 or 2.0 hours before Raman imaging. For Raman imaging, the culture was maintained in a Hepes-buffered Tyrode’s solution. then Raman images were taken. After measurement, the Raman hyperspectral dataset was processed by singular value decomposition (SVD) for noise reduction. To evaluate the intensity of the Raman signals of cytochromes, the gray scale value (GSV) of the rectangular area at arbitrary selected three points in the cytoplasm was measured. The raman intensity of the Cyt c signal was determined as the amplitude between the peak and the base line at 751, 1127, 1313, 1585 cm-1 Raman shifts, respectively.
We successfully generated RGC-5 cells with mTFP-1 tagged Cyt c (Fig.1A).High concentrated glutamate rapidly induced celll death (Fig.1B-E).The decrease of Raman signals assigned to Cyt c was observed after the glutamate treatment. All four Raman signal intensities of Cyt c significantly decreased at 0.5 hours after administration of glutamate and continued to decrease after that (*P < 0.0001, one-way ANOVA followed by Dunnett test) (Fig.1F)
This hybrid fluorescence-Raman imaging could detect oxidized form of Cyt and the decrease of reduced form of Cyt c Raman signal. This method is a strong useful tool for the observation of various cells based on the Cyt c and provides the redox state of the cells.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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