Development of high-speed two-photon microscopy for biological and medical applications
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2005.
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| Format: | Thesis |
| Language: | eng |
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Massachusetts Institute of Technology
2008
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| Online Access: | http://hdl.handle.net/1721.1/32383 |
| _version_ | 1826191974718242816 |
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| author | Kim, Ki Hean |
| author2 | George Barbastathis, Bevin P. Englward, Ian W. Hunter and Peter T.C. So. |
| author_facet | George Barbastathis, Bevin P. Englward, Ian W. Hunter and Peter T.C. So. Kim, Ki Hean |
| author_sort | Kim, Ki Hean |
| collection | MIT |
| description | Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2005. |
| first_indexed | 2024-09-23T09:04:13Z |
| format | Thesis |
| id | mit-1721.1/32383 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T09:04:13Z |
| publishDate | 2008 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/323832020-04-02T21:52:54Z Development of high-speed two-photon microscopy for biological and medical applications Kim, Ki Hean George Barbastathis, Bevin P. Englward, Ian W. Hunter and Peter T.C. So. Massachusetts Institute of Technology. Dept. of Mechanical Engineering. Massachusetts Institute of Technology. Department of Mechanical Engineering Mechanical Engineering. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2005. Includes bibliographical references (p. 135-144). Two-photon microscopy (TPM) is one of the most powerful microscopic technologies for in-vivo 3D tissue imaging up to a few hundred micrometers. It has been finding important applications in neuronal imaging, tumor physiology study, and optical biopsy. A practical limitation of TPM is its slow imaging speed (0.3 1 frames/s). We designed high-speed two-photon microscopes (HSTPMs) whose imaging speed is more than 10 times faster than traditional TPM, while their imaging depths, image contrast are comparable to those of TPM. The first high speed system is HSTPM based on polygonal mirror scanner. The scanning speed reaches 13 frames/s for typical tissues using a polygonal mirror scanner. This system is based on single-focus scanning and single-pixel signal collection. The usage of higher input power is required to compensate for the signal reduction due to higher scanning speed. However, since fluorescence signal is ultimately limited by the saturation of fluorophores due to their finite lifetimes, is the signal to noise ratio (SNR) of single focus scanning systems are also ultimately limited at high speed. This problem is circumvented in a second system based on parallelization by scanning specimens with multiple foci of excitation light and collecting signals with spatially resolved detectors. The imaging speed is increased proportional to the number of foci and similar excitation laser power per focus circumventing the problem of fluorophore saturation. However, it has been recognized that this method is severely limited for deep tissue imaging due to photon scattering. (cont.) We quantitatively measured the photon scattering effect and demonstrated that its image resolution is the same as conventional TPM but its image contrast is degraded to the faster signal decay with the increase of imaging depth. We designed a new MMM based on multi-anode photomultiplier tube (MAPMT) which utilizes the advantage of MMM in terms of parallelization but overcomes the emission photon scattering problem by optimizing the design detector geometry. This method achieved equivalent SNR as conventional TPM with imaging speed more than 10 times higher than TPM. We applied these HSTPMs to a number of novel biomedical applications focusing on studying biological problems that needs to resolve the high speed kinetics processes or or the imaging of large tissue sections with subcellular resolution to achieve the requisite statistical accuracy. In the study of transdermal drug delivery mechanisms with chemical enhancers,, large section imaging enables microscopic transport properties to be measured even in skin which is highly topographical heterogeneous. This methodology allowed us to identify the novel transport pathways through the stratum corneum of skin. In the study of tumor physiology, microvasculature in tumor tissue deep below the surface was characterized to be densely distributed and tortuous compared to that of normal tissue. The interaction of leukocyte and endothelium in tumor tissue was measured by imaging the kinetics of leukocyte interaction with blood vessel wall in tumor tissues using HSTPM. The capability of large section imaging was further applied to develop a 3D tissue cytometer with the advantages that cell-cell and cell- extracellular matrix interaction can be quantified in tissues. (cont.) The statistical accuracy of this instrument was verified by quantitatively measuring cell population ratios in engineered tissue constructs composed of a mixture of two cell subpopulations. Further, this 3D tissue cytometer was applied to screen and to identify rare recombination events in transgenic mice that carry novel fluorescent genetic reporters. by Ki Hean Kim. Ph.D. 2008-03-26T20:32:37Z 2008-03-26T20:32:37Z 2005 2005 Thesis http://hdl.handle.net/1721.1/32383 61523600 eng M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission. http://dspace.mit.edu/handle/1721.1/7582 144 p. application/pdf Massachusetts Institute of Technology |
| spellingShingle | Mechanical Engineering. Kim, Ki Hean Development of high-speed two-photon microscopy for biological and medical applications |
| title | Development of high-speed two-photon microscopy for biological and medical applications |
| title_full | Development of high-speed two-photon microscopy for biological and medical applications |
| title_fullStr | Development of high-speed two-photon microscopy for biological and medical applications |
| title_full_unstemmed | Development of high-speed two-photon microscopy for biological and medical applications |
| title_short | Development of high-speed two-photon microscopy for biological and medical applications |
| title_sort | development of high speed two photon microscopy for biological and medical applications |
| topic | Mechanical Engineering. |
| url | http://hdl.handle.net/1721.1/32383 |
| work_keys_str_mv | AT kimkihean developmentofhighspeedtwophotonmicroscopyforbiologicalandmedicalapplications |