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9 February 2012 Two-photon cryomicroscope
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We report on a new two-photon cryomicroscope which consist of a compact laser-scanning microscope combined with a motorized heating and freezing stage. Samples can be cooled down to -196 °C (77 K) and heated up to 600 °C (873 K) with adjustable heating/freezing rates between 0.01 K / min and 150 K / min. Two-photon imaging is realized by near infrared femtosecond-laser pulse excitation. The abilities of the two-photon cryomicroscope are illustrated in several measurements: imaging of fluorescent microspheres inside a piece of ice illustrates the feasibility of deep-microscopic imaging inside frozen sample. The temperature-dependent structural integrity of collagen is monitored by detection of second harmonic generation signals from porcine cornea. The measurements reveal also the dependence of the collagendenaturation temperature on hydration state of the cornea collagen. Furthermore, the potential of the two-photon cryomicroscope for optimization of freezing and thawing procedures as well as to evaluate the viability of frozen cells and tissue is discussed.
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H. G. Breunig, C. Köhler, and K. König "Two-photon cryomicroscope", Proc. SPIE 8225, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues X, 822516 (9 February 2012);

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