I want to use fluorescence microscopy to study fibroblasts grown on electrospun mats. Although such experiments are widely described in the literature, I faced a problem. We usually immerse the sample into a mounting medium (80% glycerol), place it between a glass slide and a coverslip, and seal using nail polish. This procedure works perfectly for cells on ordinary substrates, but the electrospun nonwovens are not wetted perfectly, and so they give rise to bubbles, cavities, and the overall distribution of the mounting medium is far from homogeneous. This problem is typical for various types of mats, made of PLA, PCL, and other polymers.
Are there any tricks to overcome this? Maybe I should change the mounting medium? Or add some sort of treatment to the mats?
Hello. Well, I wonder if you can consider:
-- switch to live cell imaging using confocal microscopy.
-- trim your mats/substrate materials to bite size (a few mm wide), pre-clean them.
-- then, please the substrate surface in a confocal dish. Like this,
https://us.vwr.com/store/product/21336515/vwr-35-mm-confocal-dish-sterile#:~:text=More%20Product%20Information-,Confocal%20Dishes%20are%20ideally%20suited%20for%20use%20in%20fluorescent%20microscope,and%20phase%2Dcontrast%20microscopic%20experiments.&text=living%20cell%20examinations-,Dish%20surfaces%20are%20smooth%20and%20free%20from,maximize%20usable%20area%20for%20growth.
-- Then, plate your fibroblasts in the fish as would for normal culture. (suggested density is 2x10^4 to 1x10^5 cells/mL, leave to recover/attach for 12-16 h before imaging).
In other words, you can image the cells live, in living form, and do without the hurdles of mounting, etc.. The more steps there are, the trickier it becomes to get good results.
-- The only issue for you becomes how to fashion your substrate materials into appropriate size and make sure they are clean and sterile.
Just wondering if a quick dip in xylene before mounting to fill the cavities will not affect the staining. The mounting medium should mix well with the xylene, which will eventually spread evenly into the cavities, and give no space for bubbles or cavities. I hope it works, can be tricky though.
The microscope is not magical ,without a good sample you won't a good sample you won't have images that are suitable for image analysis but a good design of your experiment could save you alot of time and money .Here are just highlight that have to be taken into consideration when you are preparing your sample for floresent imaging
(1)choice of coverslip
In general ,you should always use #1.5 coverslip (0.17mm thickness )as most microscope objectives are designated to work optimally with these .this is true for fixed samples .for live cell imaging ,lbidi as a very good choice of dishes and chambers .
(2)Choice of your florophores .
(3)Fixation and permeabilization e.g formaldehyde /paraformaldehyde methanol ,All these are what preserves cell morphology depending on specially .
Mounting and recording medium
Mounting medium helps preserves your sample and raises the refractive index to give good performance with oil performance.mountants often have scavengers which soak up free radicals and reduce photo bleaching these sometimes reduce initial briggteness of your samples.A glcerol PBS mixture is suitable for most fixed immunofluoresence and GEP -labelled samples .
Although they are also commercially products available e.g flouro amount -G ,Aqua/poly -mount ,pro long ,vest a shield immunity.
(4)Clearing methods
(6)Recording medium ,l will go in details in this.
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