Morphodynamic properties of flowingleukocytes under bright-field microscope

• Main Authors: CHIA-HUNG LIN, 林家宏
• Other Authors: Tzu-Ming Liu
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/32133220507427409647

碩士 === 國立臺灣大學 === 醫學工程學研究所 === 104 === Blood test is one of the most important indicators of physical examination. The amount and proportion of every role in blood can be direction of medical assessment. For instant, the amount of red blood cell could be the sign of Anemia. Also, the variety of amount of white blood cell could be a guide of HIV, cancer, and other disease. In more detail, traditionally, biopsy is a relatively common inspection, but it wastes many time and artificially efforts. Thus, in order to achieve automatically cell counting goal, in the past decades, scientists have beginning to develop CBC (Complete Blood Count), and Blood analysis technology. In the moment, the most accurate and precise machine is Flow cytometer (FCM). Compare to traditional biopsy inspection, FCM can not only count blood cell with higher speed but also can sort specific cell and particles. FCM was divided into four parts: microfluidic system, optical system, electric system, and sorting system. The intensity of forward scattering, side scattering, and fluorescence serve as the index to distinguish different kinds of cells. Nevertheless, in order to achieve higher accuracy, it frequently is required to add particular biomarker, which probably damage intact cell, moreover, increase the cost for laser maintenance. Therefore, currently our Lab devote to develop cell sorting with higher quality depended on the characteristics of cell image in many ways. Previously, our Lab differentiates Neutrophils, Monocytes, Lymphocytes by the intensity of Third-Harmonic Generation. From the cell image, we can find the intensity of Third-Harmonic Generation of Neutrophils is higher than Monocytes and Lymphocytes. Besides, Neutrophils included many small nucleuses. With the intensity of Third-Harmonic Generation, auto-fluorescence and size and size, we can divide these three kinds of leukocytes into three groups. However, both Flow Cytometry and Harmonic Generation technique need to depend on laser source, so this thesis devote to develop an innovative way to distinguish different kinds of leukocytes without the maintenance of laser source and the waste of biomarker. To achieve our goal, the main approach in this thesis is that by size of leukocytes and morphodynamic properties of leukocytes under bright-field to sort different kinds of leukocytes without biomarker. First, we observe the morphology of stationary leukocytes on cover glass by Leica TCS SP5 II confocal microscope with laser source, resonant scanner and Leica DMI 3000B Fluorescence microscope with CCD under bright-field. Secondly, we pμmp Neutrophils, Monocytes and Lymphocytes into microfluidic channel and observe the deformation of cells suffer from normal stress and shear stress under different flow field. From the result, we can find the lymphocytes deform significantly under higher speed. Moreover, the difference of size of kinds of leukocytes is significantly obvious that means bright-field image is able to be applied to observe blood cells in high speed. In near future, our lab plan to use laser light sheet microscopy to observe cell image with higher speed, which can increase the sorting precision of Flow cytometry without biomarker. In the long run, the value of this thesis not only helps increase the quality and accuracy of blood sorting machine in vitro, but also can be served as haemodynamic reference for in vivo developing non-invasive blood inspection.