Multiphoton microscopy for the in-situ investigation of cellular processes and integrity in cryopreservation.
In this study we demonstrate a new noninvasive imaging method to monitor freezing processes in biological samples and to investigate life in the frozen state. It combines a laser scanning microscope with a computer-controlled cryostage. Nearinfrared (NIR) femtosecond laser pulses evoke the fluorescence of endogenous fluorophores and fluorescent labels due to multiphoton absorption.The inherent optical nonlinearity of multiphoton absorption allows 3D fluorescence imaging for optical tomography of frozen biological material in-situ. As an example for functional imaging we use fluorescence lifetime imaging (FLIM) to create images with chemical and physical contrast.
Doerr
Daniel
D
Fraunhofer Institut Biomedizinische Technik IBMT, St. Ingbert, Germany.
Stark
Martin
M
Ehrhart
Friederike
F
Zimmermann
Heiko
H
Stracke
Frank
F
D000818:Animals; D001709:Biotechnology / Q000295:instrumentation / Q000379:methods*; D015925:Cryopreservation / Q000295:instrumentation* / Q000379:methods*; D004867:Equipment Design; D005456:Fluorescent Dyes / Q000494:pharmacology; D005615:Freezing; D031321:Hydrocharitaceae; D021621:Imaging, Three-Dimensional / Q000379:methods; D007834:Lasers; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods*; D018515:Plant Leaves / Q000378:metabolism; D019265:Spectroscopy, Near-Infrared / Q000379:methods; D013696:Temperature
D005456:Fluorescent Dyes