研究目的
To evaluate the usefulness of high-pressure freezing (HPF) for examination of the rapid reaction of Drosophila photoreceptor cells to light.
研究成果
HPF with direct insertion of living Drosophila under light exposure enabled the examination of rapid ultrastructural changes in photoreceptor cells, specifically the movement of pigment granules towards the rhabdomere base within seconds of light stimulation, demonstrating the method's utility for capturing dynamic biological processes.
研究不足
The technique may have limitations in evaluating very deep areas beyond the frozen surface, and infiltration of epoxy resin into pigment granules was poor, potentially affecting observation. Future modifications to carrier shape are needed for deeper area examination.
1：Experimental Design and Method Selection:
The study used high-pressure freezing (HPF) to cryofix living anesthetized Drosophila directly inserted into the HPF unit, followed by freeze substitution and transmission electron microscopy to observe ultrastructural changes in photoreceptor cells under light exposure.
2：Sample Selection and Data Sources:
Drosophila were grown in a specialized room at Shinshu University, anesthetized with diethyl ether, and used as specimens.
3：List of Experimental Equipment and Materials:
Equipment includes Leica EM HPM100 HPF machine, specimen carriers, flashlight (about 6,000 lx), acetone with 2% paraformaldehyde, epoxy resin (Plain Resin; Nissin EM Co.), ultramicrotome with diamond knife, electron microscopes (JEM-1400 and JEM-2100F with EDX microanalyzer JED-2300T), and staining materials (uranyl acetate, lead citrate, toluidine blue).
4：Experimental Procedures and Operational Workflow:
Anesthetized Drosophila were placed on the HPF carrier, exposed to light for 10 seconds, frozen via HPF, freeze-substituted in acetone at various temperatures, embedded in epoxy resin, sectioned for light and electron microscopy, stained, and observed. EDX microanalysis was performed for elemental mapping.
5：Data Analysis Methods:
The localization of cytoplasmic small pigment granules in photoreceptor cells was measured from the rhabdomere base, with statistical analysis using Mann-Whitney U-tests.
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