Researcher/Grad Student UCI Ladera Ranch, California, United States
Introduction:: Imaging is done through microscopy which deals with utilizing microscopes to view objects not visible to naked eye. This method can be used to identify bacteria, viruses, microbes which in turn can help with diagnosis. A major problem with microscopy is light penetration as non-homogeneous parts affect the thickness which creates layers and causes light to scatter. A solution to these problems is tissue clearing as it renders the tissue transparent and improves imaging quality because of its spatial resolution and high signal to noise ratio. However, current methods of optical clearing take a long time, cause irreversible tissue deformation, and fluorescence quenching. A specific method commonly used and is utilized in Dr. Bernard Choi’s lab is iDISCO. This method is reliable and can clarify 1 mm thick brain slices within 4 hours. An alternative method is a faster optical clearing method (FOCM), but this method has only shown promising results within much thinner samples, such as 300μm, but can clear these tissues within two minutes. Furthermore, little research has been conducted on this approach for thicker sample sizes. Previously, I compared iDISCO to FOCM to determine whether the contrast-to-background ratios were similar. Now the focus is more open the pixel intensities produced with the two optical clearing methods during various period of time to determine whether FOCM does indeed come in comparison to iDISCO if the samples sat in the solution longer and whether it causes the images produced to reduce in pixel resolution over time.
Materials and Methods:: To conduct this experiment, lectin-DyLight649 is injected into the mice to label the vessels and then the brains are extracted from euthanized mice and cut into slices. The right side of the brain is cleared with FOCM which consists of dimethyl sulfoxide, urea, D-sorbitol, and glycerol, while the left side with iDISCO consisting of methanol, deionized water, dichloromethane, and dibenzyl ether. The samples are cleared in each respective method for 5 minutes, 60 minutes, and 48 hours. Once cleared, the samples are imaged with a laser and images are taken near the top of the samples. The images for each sample are then put into a MATLAB code where the median vessel and background intensities are computed.
Results, Conclusions, and Discussions:: Within each sample, there are three regions of interests taken, each being a 250x250 pixel sized square. At the topmost part of each region a threshold is calculated within MATLAB and is then applied to the entirety of the sample within that region. From there, the median vessel and background intensities are computed also through MATLAB. Once three intensities for the vessels and backgrounds are taken from all the samples, they are graphed and compared to determine whether time affects the image quality produced and whether it is better to leave the tissue samples within the clearing methods for longer periods of time to produce better results or if there is a point between the 60 minute and 48 hour that reduces the quality. Based off the results of the experiment this far, the two methods do have similar median vessel and background intensities, but the FOCM solution produced the best quality images at the 48-hour mark compared to the 5 minute and 60 minute which were previously claimed to clear the sample within by. However, with the sample size only being 9 brain samples right now, more tests and data need to be collected to further suggest this conclusion can be drawn. With FOCM taking less time, it can be concluded to be the more efficient data. Further data needs to be collected to prove this conclusion and determine the specific time FOCM takes to produce the best quality images. From there, comparing the methods at various thicknesses will also need to be conducted.
Acknowledgements (Optional): : This work was supported in part by the Arnold and Mabel Beckman Foundation, the National Institutes of Health (P41EB015890, R21AG066000), the Henry Samueli School of Engineering, and Summer Undergraduate Research Program at University of California, Irvine.
References (Optional): : Renier, Nicolas, et al. “IDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples
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Zhu, Xinpei, et al. “Ultrafast Optical Clearing Method for Three-Dimensional Imaging with Cellular Resolution.”
Proceedings of the National Academy of Sciences, vol. 116, no. 23, 2019, pp. 11480–11489.,
