![]() Since you are looking at fluorescence, I assume you are using UV excitation. Also you may think about sonicating your lipid-water mixture to produce more repeatable droplet sizes by using different frequencies. You can get demountable curettes with different cross-sectional thicknesses and therefore allowing you to trap a constant thickness of liquid mixture for your experiment. You should control the thickness of your liquid mixture with demountable cuvettes ( ). Also your approach of plating a microcope slide with or without a cover slip gave you very unpredictable liquid thicknesses it’s uncontrolled and not easily repeatable. You didn’t mention the size range of the lipid droplets you are investigating. If anyone has any suggestions for a potential solution or an alternative method to create a triglyceride calibration curve using Nile Red and fluorescence microscopy it would be greatly appreciated! Thank you in advance! Essentially, I am looking for any ideas regarding a possible solvent (suspension media) that would allow the stained lipid droplets to remain suspended as spheres in the solution and not rise (or rise very slowly) to the surface of the liquid, causing them to lose their shape. When the images were attempted without a coverslip the droplets moved too much (or stuck to the sides of the slide well) to obtain good quality images for quantification. When we add a small volume of triglyceride to water, with the intent to simulate the “lipid droplets” found in the animals, plate the solution on a microscope slide and place a cover slip on top, the droplets naturally rise to the surface of the water and flatten out against the cover slip making it difficult to estimate their volume. In order to make the calibration curve we need to produce Nile Red stained spherical lipid droplets of various sizes. I am working on creating a calibration curve that relates the volume of Nile Red stained triglyceride droplets, in various tissues of small invertebrates, to a fluorescence intensity using a Leica microscope with a fluorescence detector.
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