| Deep-tissue multiphoton microscopy(MPM) enables noninvasive optical imaging into the deep regions of the tissue in animal models in vivo. Compared with the wavelength window of conventional Ti:Sa laser, multiphoton microscopy techniques, such as 3-photon and 4-photon fluorescence microscopy at the 1700-nm window, are emerging as promising imaging techniques for deeper penetration. For now, however, the potential of deep-tissue imaging at this window has not yet been fully excavated. Two of the pending issues facing MPM at 1700 nm includes signal optimization to further boost imaging depth, and heating at the focus due to absorption of laser power. Specifically speaking: 1.So far the imaging depth of three photon fluorescence imaging at 1700 nm is limited by the depletion of the signal, rather than the limit that the signal-to-background ratio(SBR) has reached unity commonly met in 2-photon fluorescence microscopy. As a result, how to further boost signal level is the key to achieving a larger imaging depth. Contrary to the previous thought that overfilling the back aperture of the objective yields the highest multiphoton fluorescence signal, we demonstrate that in case of biological tissue imaging, due to the fact that large-angle light suffers from more losses(due to absorption and scattering) than the paraxial light, they contribute less to signal generation. Therefore, it can be expected that the signal generation is maximized for certain underfilling of the back aperture of the objective lens; 2. Compared with excitation at other wavelengths, such as the commonly used Ti:Sa wavelength, water absorption at the 1700-nm window is much higher. As a result that most biological tissues, especially brain which is mainly composed of water, it can be expected that the brain tends to absorb more 1700-nm light than the 800-nm excitation light. One of the most significant effect on light absorption is the increase of tissue temperature, which will lead to tissue damage if temperature rise is too large. So far, there have been no theoretical estimation of the temperature rise at the focus at the 1700 nm window. In this thesis, we also performed detailed theoretical investigation of this issue. This thesis is organized as follows:1. A brief introduction of the basic theory of multiphoton microscopy is presented. We introduce the basic principles and the development of MPM. We also compare MPM to confocal fluorescence microscopy. Next we present both the advantages and the challenges facing multiphoton microscopy imaging techniques. At the end of this chapter, we introduced 1700-nm MPM, it enhancement in imaging depth, and signal optimization and temperature rise issues which are the main topics covered in this thesis.2. Based on the theoretical model of multi-photon microscopy and signal generation at 1700 nm window,we calculate the optic field distribution at the focus of three-photon and fourphoton microscopy by introducing underfilling of the back aperture of the objective lens. We calculate the filling factor dependence of signal generation in 3PM and 4PM, in the presence of attenuation of excitation light. Our results indicate for different ratios of imaging depth divided by attenuation length, there exists optimal filling factor for both plane wave and Gaussian beam illumination, which optimizes deep-tissues MPM signal.3. Base on the time-dependent heat conduction equation, we carried out quantitative theoretical calculation on the temperature change at the focus in MPM excited by the 1700-nm window. Through detailed investigation, we obtain the quantitative result of temperature rise for a variety parameters including numerical apertures of the objective lens, wavelength and time. These results will allow us to gain theoretical expectations for the temperature variation at the 1700 nm window in deep imaging process. It also provides theoretical guidance for excitation power selection, which avoids tissue damage caused by temperature changes induced by excessive excitation light absorption. |
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