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- Two photon microscopy (2)
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In a standard high magnification two-photon microscopy (2PM) setup the scan is achieved by moving the excitation beam inside the beam path limits of the microscope. Thus, the scanning area of the focus is limited by the field of view of the objective lens. As a consequence, the maximum scanning area is optically restricted to a diameter of 0.5 to 0.7 mm which impairs the application in biological and biomedical research.
Herein, wide-field 2PM was used to image every dimension of the human hair follicle in whole tissue samples in situ. Therefore, a 2P microscope with a new approach called 2PM FlySCAN was applied. The 2P microscope moves the scan lens in a flying optics setup: The focus of the excitation laser beam is always concentric and collinear with the scan lens. The laser focus is in a fixed axial position to the scan lens. This approach allows to scan a large field of view (7×10 mm2) up to a resolution of 500 nm2 and a scan depth ~ 500 µm without vignetting or distortion.
Further, a new en bloc longitudinal imaging mode was implemented which allows to image hair follicles in full length at a glance in a histomorphological correlation without further processing or sectioning. In situ labelling was successfully applied to investigate certain cell populations in the hair follicle which is of highly clinical relevance for various issues.
Our results demonstrate that the here used wide-field 2P microscope combined with the en bloc longitudinal imaging mode is a promising approach for the preclinical assessment of the human hair follicle addressing the demand for an appropriate sample preparation and analysis techniques.
Two Photon Microscopy (2PM) generates microscopic images out of depth of biological samples. Up to now the method is restricted by narrow limitations of the field of view, the imaging depth and the orientation of the image field. A new approach overcomes these boundaries and delivers high resolution images revealing very specific information on clinically and biologically relevant tissue and cell structures. The status of the 2PM technology is critically reviewed and the options are discussed. The advantages of the new approach demonstrated by excellent tissue images.