Hi everyone,
I am newbie on using the confocal and I am wondering if the setting on all the images I take should be the same? in order to compare between them?
because when I took the images I changed the setting between the channels in my different slides and now I think that I was not supposed to do that.
sorry for my "basic" question to all the experts out there
thank you!
Hey Delilah,
This will depend on how you plan to analyze the images, and what settings you are referring to. If you are simply counting some obvious structures or measuring some distance (e.g. nuclear width), then playing with the settings may not matter. However, if you are measuring relative fluorescence intensity between samples, or counting objects that only appear if you tweak the settings (whether brightness or focal depth), then you cannot compare between images without consistent settings.
When it comes to fluorescence intensity/brightness (voltage), I try to set the parameters such that I can see the structure of interest in the samples I expect to be most 'weakly fluorescent'. It helps to do some preliminary work to get an idea about how consistent your staining is within and among treatments. Keep in mind that you don't want to over-saturate the intensity either - you will lose detail and you can't adjust after the fact. Some programs allow you to see oversaturation as white areas, which can help you get some ideal settings.
Lauren
As Lauren E Des Marteaux mentioned it really depends on what you're planning on doing with them and how much time you're willing to spend on the microscope to get that data. A couple of tips that hold true in every scenario though is change the bit depth to 12 or 16 bit. The second is make sure your images are appropriately sampled (briefly described here https://www.microscopyu.com/tutorials/spatial-resolution-in-digital-imaging). Sometimes its impractical to image at the nyquist rate (for example if you're imaging a piece of tissue that's 2x2x2cm unless you''re using an objective with an NA < 0.2, you probably won't be nyquist sampling) but that is ideal and you should always be aiming to image as close to ideal conditions as you can
If you're comparing fluorescence intensity between slides then all your settings need to be consistent. But it helps to get your brightest sample and find the brightest region that's still representative of the whole sample and adjust the settings so that these images are just below saturation (if your images are 12 bit aim for the brightest pixels to be ~3800 to account for the fact that there might be brighter patches in other regions and you don't want saturated pixels). Line averaging will greatly improve the quality of your images and any measurements you take, try aiming for 3 or more if you can get away with it.
You're dullest samples aren't going to look great and in some cases you may have an almost black image. So to present this data it can sometimes help to have 2 of each image you've chosen to show for each condition. One image has no alterations and the other to show more detail using some sort of filtering. The caveat to this is that you must be very careful with what you are doing and make it clear what you have done.As an example I'll use adjusting the gamma which leaves the brightest regions relatively untouched while amplifying the lower pixel values. If you use a non-linear adjustment such as this it is very important to keep in mind that is for visual purposes only, you can't make any measurements on these images and you must explicitly state that you have done so in the methods and figure legends. If you were primarily looking at relative fluorescence intensity but wanted to show that the protein accumulates to the same region or something the gamma adjusted images would typically go in the supplementary material, again explicitly stating that you have done so. I've attached an example of gamma and non gamma adjusted images so illustrate where it might be useful.
If the relative abundance of the proteins aren't what you're aiming to measure and you aren't comparing relative intensity you can play around with the settings a bit more. Good examples of this are if you're looking at the structural organisation of something like the cytoskeleton or ECM proteins. In this scenario you can adjust the laser power and detector gain (within reason) to make full use of the dynamic range for every image.
Thank you so much Howard Vindin and Lauren E Des Marteaux for your detailed answers that I am sure will help me and any other new users of the confocal! I am actually just looking at phenotypic differences of multiple cell lines upon over expression/Knocking down my protein of interest. That is why I am not sure if I should keep the settings fixed all the way through.
Thank you again!
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