Using Third Harmonic Generation Microscopy to Observe and Identify The Type of Leukocytes
Date Issued
2014
Date
2014
Author(s)
Huang, Shi-Hong
Abstract
Blood is an important index of medical examination. The percent composition of red blood cells, white blood cells and platelets in blood is related to health. In the hospital, drawing blood has been a dominant way of health exam. The method is an invasive medical practice, and vitro specimen tends to be affected by the external factors, which is possible to affect the accuracy of the estimation. To overcome the problems mentioned, we use nonlinear optical microscopy to gather statistics and analysis of all types of blood cells with non-invasive detection in the future. My research focuses on the differences of characteristics of blood cells observed by nonlinear optical microscopy and broadening the conditions of identification. Equipped with sub-micron grade three-dimensional analysis, non-linear optical microscopy can be applied to get deep optical images and causes no biological damages. In addition, no dye is included in the research, so the blood cells remain almost intact. In the research, we use Cr:forsterite laser as the excitation source and further to collect THG signals emitted by target objects. Then, we capture the images generated by the transition from analogical signals to digital signals processed by the FPGA (ALTERA DE4). Finally, the optimized images, generated by an image software (programmed by visual studio) are adopted to observe the characteristics of blood cells. In the third harmonic generation images, the red blood cells and platelets show a strong third harmonic generation signal. It is easy to identify them because of the size difference between the two. As for Leukocyte, it can be further divided into three types: neutrophil, lymphocyte and monocyte. Unlike red blood cells and platelets, the THG signal of Leukocyte is weaker. Because of the characteristics observed from optimized images, we can easily identify the specific type of Leukocyte. It is observed that neutrophil THG signal is stronger than lymphocyte and monocyte. Neutrophil has multiple small nucleus, and the cell membrane structure is obvious. In terms of the size of lymphocyte, it is the smallest and its circle nuclear is bigger, and oftentimes, it can be identified because of a circular bright spot. Monocyte, being the largest in size, has an irregular-shaped nuclear. Because of the optimized images, identification of cells become easy. In order to achieve more rapid identification. The size of cells, and the intensity of THG and the intensity of fluorescence condition identification of specific types of cells. In terms of the intensity of THG, Neutrophil exhibits the strongest, while monocyte and lymphocyte shows no significant difference. Concerning the intensity of fluorescence, neutrophil exhibits the strongest and lymphocyte the weakest. With the distinctive features of cells mentioned above, the three types of white blood cells can be effectively identified and further divided into three groups. Although much remains to be done, but this study is still valuable in that the empirical results enable the flow cytometry to sort blood cells effectively. Sorting blood cells through flow cytometry is usually conducted with dye; the use of dye is consuming and the most importantly, it is possible to cause cell mutation. With the result of this study, the broadened conditions on blood cells sorting conducted by flow cytometry and the features observed by the optimized images surely improve the accuracy of blood cells sorting.
Subjects
Neutrophil
Monocyte
Lymphocyte
Harmonic Generation
Flow Cytometry
Type
thesis
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