15 Jun 2017
Technique offers alternative to currently used techniques and avoids complex sample preparation.
To date, imaging methods applied to the task have included the use of fluorescence microscopy along with both transmission- and scanning electron microscopy, although these methods involve more complex sample preparation than is desirable.
The project's photothermal microscopy platform uses a pump laser at 488-nm wavelength to photo-excite the mitochondria being analyzed, and then a 638-nm probe pulse to detect the resulting change in refractive index, which can be used to construct an image.
In its paper, the team note that the technique exploits the strong optical absorption of cytochrome c, a protein found within the mitochondria, and so does not require any added labeling operation that might distort the structures of interest, or other complicated sample preparation methods.
Seeing mitochondrial networks
Despite the importance of the morphological characteristics of skeletal muscle mitochondria to muscle function, the PT technique had not been applied to the question before now. The team believes that its work is the first example of PT label-free imaging and identification of the structure of skeletal muscle mitochondria, characteristically around one micron in size.
"The principle of the PT imaging technique is well known, but it has to date not been extensively used in actual applied imaging situations," Takayoshi Kobayashi of the University of Electro-Communications told Optics.org.
"Most of the small number of papers that have been published simply show some examples of the technique, and have almost no discussion about the physiology and pathology that can be detected. We, however, have been able to study both the physiology and pathology of mitochondrial networks in muscles, the brain, neurons, cancer cells and others."
The team's published findings showed that mitochondrial structures were clearly resolved in its PT images with higher contrast than in conventional bright field images. They also suggested that this instrumental method should be applicable to tissues in which there is a lower concentrations of cytochrome c than those involved in this study.
To prove the simplicity of sample preparation, the PT method was also applied to bare muscle pieces peeled from the thigh of a live rat, where structural detail of the mitochondria was still clearly visible in a sample of around 500-microns thickness.
"In our platform the high sensitivity and small laser spot size ensures that thermal diffusion is sufficient to remove any heat generated and avoid tissue damage," commented Takayoshi Kobayashi. "This does involve a certain trade-off with the resulting signal-to-noise ratio, but in comparison with fluorescence microscopy, this PT technique is much more robust."