PhD 4

Development of a directional freezing cryostage for cell imaging using multiphoton microscopy

Location: Institute for Multiphase Processes, Leibniz University Hannover, Germany

Main Supervisor: Prof. Birgit Glasmacher. For any questions, please contact Dr. Marc Müller, e-mail: mueller@imp.uni-hannover.de

Participating researchers: Dr. Marc Müller (LUH), Prof. Oleg Avrunin (NURE), Dr. Birgitta Stolze (LLS Rowiak), Dr. Tim Rittinghaus (LUH)

Objectives:

  1. Develop and validate a device for a multiphoton-cryo-microscope visualize phase changes in animal germplasm during directional freezing. In spontaneous freezing the growth of ice crystals is uncontrolled leading to a huge variation in the cryopreservation outcome. In this regard, development of a directional freezing cryostage will include its design, manufacturing and validation to ensure proper temperature control and applicability for multiphoton microscopy. The validation of the system will be conducted with the active support of LLS Rowiak. It will include application of reproductive cells and the respective fluorescent dyes (such as Hoechst33342, CellLight Plasma Membrane-CFP) to detect cell content integrity and membrane changes during cryopreservation.

  2. Develop sophisticated software for image analysis and processing. Images taken using a multiphoton cryo-microscope will be utilized to develop software for image processing and analysis. This particular objective will be conducted in cooperation with NURE.

  3. Investigate samples with multiphoton cryo-microscope during freezing and thawing process including image analysis. The effect of freezing parameters (temperature gradient, CPA/VA type, cooling rate) on the integrity of reproductive cells will be analyzed.

Expected Results:

  1. Improved understanding of the effects of ice formation, devitrification and ice recrystallization in sperm and oocytes during cooling and warming

  2. Functional and validated multiphoton-cryo-microscope with directional freezing cryostage

  3. Image analysis software for characterization of cooling and warming samples

Planned secondments:  (Host, Supervisor, timing, length, and purpose)

  1. LLS ROWIAK, Stolze, M12, 2 M, Training in multiphoton microscopy and recommendations for a cryostage

  2. NURE, Avrunin, M24, 2 M, Training in image analysis techniques

Enrollment in Doctoral Degree: Yes (LUH)