Developing and implementing a l-openspim platform using in-air optics and measurement of tissue temperature changes
Oluniran, Gideon
Oluniran, Gideon
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Publication Date
2023-10-07
Type
Thesis
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Abstract
Relative to other microscopy techniques, light sheet fluorescence microscopy is widely accepted due to its ability to provide optical sectioning, higher speed, larger field of view, lesser photobleaching, and physiological support for timelapse imaging. However, biologists interested in harnessing these benefits may not have access to commercial systems possibly due to limited funding. In cases where they do, the rapid development in research will ultimately render such platforms obsolete. Resulting in the need to consistently purchase new microscopes or upgrade modules off the shelf. At best, biologists will be forced to limit investigations to microscope capabilities. Do-it-yourself (DIY) versions exist and when combined with air objectives give greater flexibility. Biologists are therefore encouraged to learn the art of building their own microscope, starting off with platforms built off air optics, while gaining the skill required to consistently make changes when the need arises. openSPIM DIY versions arguably have the most detailed and published build instructions, but they are more tailored towards in-vivo imaging and samples that fit within the field of view. I further expand on this by documenting stepwise instructions and building a LopenSPIM platform using air objectives for in-vivo and in-vitro imaging. More practical insights into finer light sheet alignment, choice of image acquisition software, and efficient data acquisition and saving tips are documented. I show the ability of our SPIM platform to produce quality 3D images of a millimetre sized Varroa destructor, and to distinctly reveal cells of the optically transparent head of a Hydractinia symbiolongicarpus. Details needed to extend the microscope capacity to centimetre sized samples at a high isotropic raw image resolution are stated. This work primarily aims to aid biologists with limited technical experience, and I further describe a protocol for investigating thresholds on light sheet microscopes that limit laser induced heating effects and ensure tissue bio-fidelity. I show that 10 mW, 20 mW, 30 mW, 40 mW, 50 mW, and 60 mW of laser power corresponding to intensity values of 1182 mW/mm2 , 2363 mW/mm2 , 3545 mW/mm2 , 4726 mW/mm2 , 5908 mW/mm2 , and 7090 mW/mm2 should not be applied for more than 26 s, 10 s, 5 s, 4 s, 4 s, and 3 s to 1 mm2 of mammal tissue, respectively, to limit temperature rise to ~ <1 0C.
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Publisher
NUI Galway