Fluorescence lifetime imaging microscopy principles and applications
Date Issued
2007
Date
2007
Author(s)
Kung, Chun-Ta
DOI
en-US
Abstract
In this thesis, we successfully implement the fluorescence lifetime imaging microscopy (FLIM), which maps the fluorescence lifetime distribution and thus provides a new dimension for observation in addition to frequency or wavelength. Disparate fluorescent molecules can be inherently discriminated with different fluorescence lifetime; with the help of additional time-resolved information, biological issues can be analyzed accurately. The inherent contrast in fluorescence lifetime is especially significant for the endogenous fluorescence, where different molecules often have highly overlapping spectra under the same excitation source.
According to many published studies, the Ti:sapphire laser is known to excite fluorescence from numerous kinds of molecules, which is advantageous in some applications but may form an obstacle for specific biological studies since unwanted fluorescence shows up as well. So an infrared Cr:forsterite laser is used instead and the endogenous fluorescence appears simpler and more definite in contrast with that from a Ti:sapphire laser. Besides, the Cr:forsterite provides good penetration depth and the least invasive observation due to low absorption at the wavelength by biological tissues. Its infrared working wavelength also enables us to integrate FLIM with our developed second and third harmonic generation microscopy; multi-modality information, including fluorescence, SHG and THG, are thus obtained simultaneously. With abundant optical information, the FLIM-integrated multi-modality laser scanning system has become a powerful tool for biological studies to solve certain relatively complicated issues
Fluorescence lifetime is a specific property of a fluorescent molecule, and therefore, molecular imaging can be realized with fluorescence lifetime imaging. As an initial study, several typical and essential molecules are selected to measure their fluorescence spectra and lifetime, which could be taken as a data base for molecular imaging. We then compare them with the autofluorescence in real human tissues, and possible fluorescent origins are found with the help of the fluorescence lifetime and spectra.
Cancer has been the leading cause of death for 25 years, and lung cancer and liver cancer are the first and the second place, respectively, in all kinds of cancer. Using the FLIM-integrated multi-modality system, we perform quantitative analysis on normal and abnormal samples and diagnose the occurrence of liver cancer with a high accuracy. In the case of lung cancer, the specific kind of adenocarcinoma is identified using fluorescence lifetime. In contrast to conventional tedious biopsy, the technique is very potential in clinical diagnosis of cancer and in serving as guidance for tumor removal surgery.
FLIM, combined with the developed harmonic generation microscopy in our group, provides multi-dimensional information, and creates a solid basis for cross analysis for biological studies. More complicated medical issues can be further studied using the FLIM-integrated multi-modality optical system, which could also be modified depending on different demands.
Subjects
螢光
螢光生命週期
肝細胞癌
肺腺癌
時域單光子計數
fluorescence
fluorescence lifetime
hepatocellular carcinoma
lung adenocarcinoma
TCSPC
SDGs
Type
thesis
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