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In vivo eye imaging at cellular levels is important to study eye diseases and laser surgery processes. We applied two-photon microscopy (TPM) to in-vivo and ex-vivo imaging of the cornea and lens of mouse eyes. TPM was used to image photodisruption spots of a femtosecond laser associated with cataract surgery and the size and shape of photodisruption spots were measured. In vivo imaging was performed by using a custom made mouse holder and cellular structures of individual corneal layers were visualized with less motion artifact.
Among the various immune responses of the body, the earliest is the inflammation response. Quantitative measurement of the inflammation response is important for the diagnosis of several diseases and evaluation of new drugs responses. In this study, we induced an acute inflammation response by topical application of LPS on the mouse ear and the resulting characteristics of acute inflammation response such as tissue swelling, blood vessel dilation, immune cell migration were imaged. To do so, we used noninvasive 3D imaging technologies: optical coherence tomography (OCT) and two-photon microscopy (TPM), label free. OCT displayed the increase in thickness of tissue and changes in blood vessel distribution due to the inflammation response. Meanwhile, TPM displayed molecular and cellular information such as immune cell infiltration. Taken together, OCT and TPM provided information about the inflammation response from this model.
Two-photon microscopy (TPM) has been used in plant research as a high-resolution high-depth 3D imaging modality. However, TPM is known to induce photo-damage to the plant in case of long time exposure, and optimal excitation wavelength for plant imaging has not been investigated. Longer excitation wavelength may be appropriate for in vivo two-photon imaging of Arabidopsis thaliana leaves, and effects of longer excitation wavelength were investigated in terms of imaging depth, emission spectrum. Changes of emission spectrum as a function of exposure time at longer excitation wavelength were measured for in vivo longitudinal imaging. Imaging depth was not changed much probably because photon scattering at the cell wall was a limiting factor. Chloroplast emission spectrum showed its intensity peak shift by 20 nm with transition of excitation wavelength from 849 nm or below to 850 nm or higher. Emission spectrum showed different change patterns with excitation wavelengths in longitudinal imaging. Longer excitation wavelengths appeared to interact with chloroplasts differently in comparison with 780 nm excitation wavelength, and may be good for in vivo imaging.
Information on the spatial distribution and organization of chloroplasts is very important to understand photosynthesis and other related mechanisms such as transpiration. During high irradiance, chloroplasts are mostly distributed on the upper side of the leave maximizing their light harvesting function but also protecting lower living tissues from photodamage. In order to monitor such movement effectively, we developed a two-photon microscopy (TPM) which allows to visualize in-vivo 3D imaging of chloroplasts. We monitored the change in organization and distribution of chloroplasts of maize plants grown at high radiation. During the successive imaging of the same area, the plants were kept in the darkroom. In addition, the change in chloroplasts size and distribution was compared between a young leaf and a senescent leaf. Such basic studies are expected to provide new insights in understanding photosynthesis efficiency by analyzing the change in distribution and organization of chloroplasts.
Two-photon microscopy (TPM) is minimally-invasive 3D fluorescence microscopy based on nonlinear excitation, and TPM can visualize cellular structures based on auto-fluorescence. Line-scanning TPM is one of high-speed TPM methods without sacrificing the image resolution by using spatial and temporal focusing. In this paper, we developed line-scanning TPM based on spatial and temporal focusing for auto-fluorescence imaging by exciting the tryptophan. Laser source for this system was an optical parametric oscillator (OPO) and it made near 570 ㎚ femtosecond pulse laser. It had 200fs pulse width and 1.72 ㎚ bandwidth, so that the achievable depth resolution was 2.4l ㎛ and field of view (FOV) is 1O.8 ㎛. From the characterization, our system has 3.0 ㎛ depth resolution and 12.3 ㎛ FOV. We visualized fixed leukocyte cell sample and compared with point scanning system.
We have developed two-photon microscope which is optimized for in-vivo deep tissue imaging. We have developed two-photon microscope which is optimized for in-vivo deep tissue imaging. This system was characterized by signal decay and PSF measurement. Axial resolution of this system increases with increasing depth. And light scattering by tissue limits the imaging depth of two-photon microscope. Using commercial microscope body cannot detect all of emission light through the objective lens. So, we are upgrading two-photon microscope for detecting all of fluorescence signals which are ballistic and scattered. This new system provides deeper and better for in-vivo deep tissue imaging.