I’ve finished my testing of concentrated dye solutions for flat-fielding images. As described previously (1, 2), we’re using concentrated dye solutions to collect shading correction images, following the work of Michael Model. Following his protocol, we use 100 mg/ml fluorescein, rose bengal, and acid blue 9 for correcting the FITC, Cy3, and Cy5 channels, respectively. Additionally, we’ve found that 50 mg/ml 7-diethylamino-4-methylcoumarin is a good dye for collecting shading images for the DAPI channel.
A detailed protocol for collecting the shading images is posted on the NIC wiki, but in brief we first collect a dark image with no light going to the camera, and then collect multiple images of each dye at different positions, and calculate the median of these images to eliminate any spatial nonuniformities (e.g. dust particles) in the dye itself. Example dark and flat-field images are shown below.
We have measured flat-field images for the DAPI, FITC, Cy3, and Cy5 objectives, and for 10x / 0.45, 20x / 0.75, 40x / 0.95, and 100x / 1.4 objectives. The resulting flat-field images are compared in the montage below:
The correction images don’t vary as much from channel-to-channel as they do from objective-to-objective, and interestingly, the 100x objective appears to be flatter than the lower magnification objectives. We can further quantitate this by comparing the deviation from a uniform image for each flat-field image as well as the deviation from a mean image for each channel or a mean image for each objective:
As can be seen, the objective has a bigger effect on the shading correction than the channel. The mean error when using a single correction per channel is 5.2%; using a single correction per objective, it is 3.8%. By contrast, using a single mean image to correct results in a mean error of 5.8%. These compare to a 15% error for no correction at all. Although incorporating both objective and channel is necessary to achieve the best correction, using a single correction for all images taken with one objective will perform pretty well.
Fortunately, recording the correction images doesn’t take too long, and the appear to be stable for many days. We haven’t yet followed the performance over time to see how often flat-field images need to be remeasured but I’m hopeful that they will be stable for months and so only need to be remeasured when the microscope is adjusted or realigned.
Finally, here are some examples of our favorite kidney section (Molecular Probes slide #3) stitched at 10x with and without correction (these have been downscaled by 50% to save space):