Video-rate Third-harmonic-generation Microscopy for in vivo Blood Cell Cytometry in Human Capillaries
Date Issued
2012
Date
2012
Author(s)
Chen, Chien-Kuo
Abstract
Complete Blood Count (CBC) is a routine examination running in the hospital. In clinical practices, CBC plays an important role in the diagnosis of diseases. The results of inspection will be a significant indicator of evaluating individual health status. The items of checking include: erythrocytes (red blood cells, RBCs) count, leukocytes (white blood cells, WBCs) count, thrombocytes (platelets) count, Hemoglobin, Hematocrit and so on. Although CBC is a common examination in laboratory medicines, it requires a draw of blood, which is an invasive method to patients. And it cost patients’ time to wait for the results. Hence, it is highly desired to have an on-site measurement method for blood cell counts without invasive draws of blood. That can save the medical resources and the time of patients.
Nowadays, laser scanning nonlinear optical microscopy, such as two-photon fluorescence microscopy (2PFM) and harmonic generation microscopy (HGM), has been widely used for in vivo biological studies with sub-micron three-dimensional (3D) spatial resolution. Owing to the nature of energy conservation of harmonic generation (HG), there is least energy deposition to tissues under observation. Compared with 2PFM, HGM is the least-invasive optical microscopic technique suitable for clinical applications. Moreover, the extra advantage of HGM is that the morphological information of tissues can be revealed without a need of extra labeling.
In this thesis, we developed the video-rate third-harmonic-generation (THG) microscopy for blood cell cytometry in human skin. The excitation laser source in our research was a home-built femtosecond Cr:forsterite laser with Kerr Lens Mode-locking. With its output wavelength covering the penetration windows of most biological tissues, the Cr:forsterite laser is an indispensable excitation laser source providing deeper penetration depth and reduced on-focus photodamage. To catch up the fast motion of blood cells, the high-speed scan rate is a critical issue. Combining a 16 kHz resonant mirror and a galvanometer mirror, the 2D scan rate could achieve 30 frames per second (FPS). Taking advantage of the high-speed frame grabber, the THG signal was synchronously sampled and reconstructed to an image with 512-by-512 pixels size.
With the aid of high-speed scan rate and HGM, the blood cells in human blood capillary could be clearly observed, especially for round and unstacked leukocytes. We could count the amount of white blood cells and evaluated the corresponding flux of blood volume. Thus evaluated number density agreed with the physiological range of white blood cell count. For clinical use, in the future, the technique is potential to be used to diagnose leucopenia for the patients under chemotherapy.
Subjects
third harmonic generation microscopy
nonlinear optics
cr:forsterite laser
blood cells
capillaries
Type
thesis
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