I posted previously about automated acquisition and stitching of tiled fluorescence images in Micro-Manager. Today I want to talk about how to properly flat-field correct them. In the previous post I mentioned that I have been developing tools for flat-fielding images with independent correction images in each channel. However, if you looked at the linked stitched image from the previous post you will notice that there is still some uncorrected shading in the images, which manifests itself as the checkerboard pattern in the final stitched image.
I suspected that this was because the correction image was not a good match to the true shading image. Normally, we measure flat-field correction images using 1 mm thick fluorescent plastic slides. Chroma gives these out at conferences, and they’re easy to use, but you might expect that a 1 mm thick fluorescent slide is not a good way to measure the correction image for a 20 μm thick tissue section. To test this, I measured correction images from one of these fluorescent plastic slide and from concentrated and dilute solutions of dye (fluorescein or rhodamine). To image the dye samples a drop of dye was placed between a coverslip and slide to produce a thin layer of dye. The dilute dye solutions produced poor correction images due to high variability in intensity from position to position. The concentrated dye solutions (a spatula-full of dye dissolved in 5 mL of PBS) produced good correction images. These were tested by tiling image acquisition to look for uniformity in the stitched image. The results are shown below.
A 6×4 stitched image of a mouse kidney section with no shading correction applied.
The same mouse kidney section, flat-field corrected using shading images recorded from a 1mm thick fluorescent plastic slide.
The same mouse kidney section, flat-field corrected using shading images acquired from concentrated solutions of fluorescein and rhodamine.
The entire kidney imaged with the best shading correction and stitched together in Fiji can be seen in this Gigapan.
As you can see, the fluorescent plastic slide performs substantially better than no correction, but the correction using the dye solution is much better. It’s a little bit more work to do the correction with the dye solution, but I’m hopeful that the corrections will be stable over time so that we don’t have to re-measure them very often. Fluorescein and rhodamine work well for acquiring correction image for the green and red channels; I’m hopeful that 7-diethylamino-4-methylcoumarin will work well for the DAPI channel. All of these are available cheaply. For the Cy5 channel I don’t know of any cheap dye, so we’ll probably just use Cy5 or Alexa 647. I didn’t measure the concentration of the dye solutions, but here’s a picture of them so you can get a sense of the concentration.
Concentrated rhodamine (left) and fluorescein (right) solutions.
The shading correction is implemented as (Image – Background) / Shading, where Background is an average image with no light reaching the camera, and Shading is the average image recorded from the dye solution (I average 30-50 images recorded at different positions to average out dust particles and other spatial fluctuations). The Shading image is itself background subtraction. This is all implemented in a plugin for Micro-Manager, MultiChannelShading, which will be available in nightly builds soon.