Correction of chromatic shifts in three-dimensional (3D) multicolor fluorescence microscopy images is crucial for quantitative data analyses. This protocol is developed to measure and correct chromatic shifts in biological samples through acquisition of suitable reference images and processing with the open-source software, Chromagnon.
Marsh coral microscopy images contain Chromatic Shift in three dimensions. That impair colocalization analysis of high resolution images. It is generally difficult to remove Chromatic Shift originated from a biological sample itself, our method can correct chromatic shift in a biological sample using separately acquired reference images.
Demonstrating the procedure will be Takako Koujin a technician from my group. Begin by seeding 2.5 times 10 to the fifth healer cells on a 35 millimeter glass bottom dish and growing them in a carbon dioxide incubator. After 24 hours of incubation replace the medium with two milliliters of 3.7%formaldehyde in PBS and mix gently.
Fix the sales for 15 minutes at room temperature on a rotating platform. Wash the cells three times with two milliliters of PBS for five to 10 minutes. Permeabilize them with two milliliters of point 1%try the next 100 in PBS while rotating and repeat the washes with PBS.
Then stain the cells using a standard antibody staining protocol as described in the text manuscript. To obtain a reference image of crosstalk reference for widefield microscopy, add two milliliters of point five micrograms per milliliter Dappy to stay in the DNA. Leave the cells on the rotating platform for 30 minutes.
Then replace the solution with 100 microliters of mounting medium To obtain a reference image of biological calibration reference for any kind of microscopy, including confocal and super resolution microscopy. Fix the cells as previously described, and prepare the stock solution a fluorescent dye conjugated phalloidin in 1.5 milliliters of methanol. Add the phalloidin into PBS at a dilution of one to 100 for Alexa floor 405 one to 1000 for Alexa floor 488 and one to 200 for Alexa floor 594.
Add one milliliter of the solution to the cells and incubate them for 30 minutes while rotating. Wash the cells three to five times with two milliliters of PBS. Then replace the PBS with 100 microliters of mounting medium.
To acquire fluorescence images using widefield microscopy place the target sample on the microscope in the imaging software, open the design and run experiment window, set the appropriate Z step and select the blue, green and red channels. In the Run tab, add the image file name and begin imaging. To acquire crosstalk reference images for measuring Chromatic Shifts in the sample select the excitation light only for Dappy and make sure to use the exact same stage position and Z height as for the target sample.
Alternatively, to use a bright field image as a reference place to the target sample on the wide field microscope and acquire a fluorescence image of the target in blue, green and red channels. Then acquire bright field images of the target in blue, green and red channels at the exact same stage position and using the same Z height. To acquire fluorescence images using super resolution microscopy, such as 3D Sim place the target sample on the microscope and acquire a fluorescence sim image of the target in blue, green and red channels.
Then reconstruct the super resolved image. Next place the sample stain with multicolor phalloidin on a 3D sim microscope. And acquire multiple fluorescence sim images as the biological calibration reference images at point stage positions, then reconstruct the super resolved images.
Use a web browser to download the newest binary release of Chromagnon. Extract the program and put the executable file in a convenient location. Drag and drop the reference files of either crosstalk right field or biological calibration references to the reference box or click reference files to open a file selector dialog.
If the program asks to install a Java Development Kit, press Yes, and navigate to the downloaded page. Then download and install the JDK for the Operating System as instructed. Drag and drop the target files of antibody staining in the target box.
Choose an output image format and specify the suffix for the output file name. For crosstalk reference images with high signal to noise ratio. Choose projection from the choice list of local align and use a minimum window size of 60.
For a multiple biological calibration reference images check the average references option And choose none for local align, then click run all to start measurements. To make sure that the measurement was performed correctly, drag and drop the reference images into the reference box and target box. Then run the program and verify that the images are perfectly overlapped.
Chromatic Shift Correction in widefield microscopy can be performed using a crosstalk reference image. This image was obtained with a widefield microscope equipped with a single camera. Fluorescence emission from Dappy was used as a reference to correct the blue, green and red channels.
After alignment with Chromagnon the Chromatic Shift and the reference image was corrected. When the alignment parameter was applied to the target image by Chromagnon the DNA and the anaphase bridge, which is seen incorrectly outside of the nuclear envelope before alignment appears as expected inside the envelope. Chromatic Shift Correction of a 3D sim image can be performed using a biological calibration reference image.
Three 3D sim images of HeLa cells stained with phalloidins conjugated to blue, green and red dyes were averged and the Chromatic Shift was measured by Chromagnon. After alignment the Chromatic Shift and the reference image was corrected. The alignment parameter was applied to a target image.
The XZ view shows incorrect channel position along the Z and slightly along the X directions, which was corrected after alignment. For best performance it is important to obtain reference images under the same conditions and the timing as a target images.
