Shading correction (online) for fluorescent images in epifluorescent microscopy

 The issue: Many users have complained over the years about the presence of tilling artifacts when they decide to make mosaic images. Namely, it is possible to see lines or boxes through the length and width of the tiled image they acquired. When using brightfield microscopy, this is easily fixed by acquiring an empty image with the same settings of light and focus distance, and then use that image as the reference for shading correction in the program they are using. With fluorescence, this is more complicated because the user needs a fluorescent homogeneous background to focus on. I tried buying fluorescent slides for that purpose, but the results are widely variable, depending on the quality and ease of focus of the slides used. 

Solution: I recently acquired calibration beads from Invitrogen (InSpeck Green Microscope Image Intensity Calibration Kit, wavelength 505/515 nm, 2.5 micrometer size, reference I7219) that I was intending to use for another purpose. I generally follow the standard procedure given by the supplier, but I accidently found out that if you use methyl salicylate as a solvent instead of the recommended mounting medium that comes with the kit, the beads will dissolve uniformly in the medium and create a relatively homogeneous fluorescent background under the coverglass. You can also adjust which level of fluorescent intensity you want to use by using different bead intensities and bead concentration. 

Once you prepared your slide, you can use it to take the shading correction reference image. 

If you are using a Zeiss microscope (we use a Zeiss Axio.Imager.Z1) and ZEN software (we have blue ZEN version 2.3), than this step is possible by choosing the acquisition menu and selecting the channels of interest by using the Smart Setup button. Once you define the channels, you can put the reference slide under the objective that you are interested in using for your observation, and adjust the illumination levels and exposure time. Select a field of view that is free of artifacts and as homogeneous as possible. Focus on its fluorescent-coated surface. Tick the Show All alternative on the top of the submenu Channels (see figure under), so that all options are visible to the user..Take a picture of this field of view by pressing the button Define (indicated by a green arrow in the figure 1 under). Choose the Global alternative in the switch that is on the right side (so that the program applies the same reference image to all channels) and check the Shading Correction tick on the left side of the button.  

Fig 1. - ZEN 2.3 submenus under the Acquisition Mode menu.

Once you have done this, the program will always use the image you acquired as its reference for online shading correction. 

In the following figures, you can compare the difference between the same images taken without and with shading correction applied (figures 2 and 3 under). 

Fig 2. - Single tile image without shading correction. 

Fig 3. - Same image as the one above, but with shading correction selected.

This process is more dramatic for mosaic (tiled) pictures, as you can see in the images under (figures 4 and 5). You can see that the image with the online shading correction option on is more uniform and the seams between the tiles are less visible. From my experience, a reference picture taken in one channel (I used the AF488 channel settings for the reference picture) can be applied to all channels (therefore the choice of the 'Global' option in the menu shown in figure 1) with satisfactory results. 

 

Fig. 4 - Tiled image (2x2) taken without shading correction. 

Fig. 5 - Same tiled image as above, but with online shading correction option selected.