Analytische Chemie
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2PM FlySCAN: Innovative approach for wide field high resolution imaging of skin tissue volumes
(2016)
Two Photon Microscopy (2PM) is an innovative Technology for high resolution imaging of human skin in vivo and ex vivo. Up to now the method is limited by its interdependence between the field of view and the attainable resolution. The new FlySCAN Technology overcomes these boundaries and enable images sizes of many millimeters by resolutions in the submicrometer range revealing very specific information on clinically and biologically relevant tissue and cell structures. The advantages of the new approach are demonstrated by excellent in vivo and ex vivo tissue images.
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.