1. Bio-Rad Labs. Chromatic Aberration Sources and Measurement

2. Bio-Rad Labs. Sources of Aberrations ä 1. The microscope objective. ä 2. Immersion oil ä 3. The filter blocks ä 4. The scan head mirrors. ä 5. The scan head eyepiece

3. Bio-Rad Labs. The objective and oil ä These ä These days, objectives are quite good BUT: ä If ä If you use the wrong oil, both chromatic aberration and image resolution will suffer.

4. Bio-Rad Labs. The scan head ä A small amount of aberration can be caused by the scan head if it is not properly aligned. ä When the scan head leaves the factory it is usually very well aligned. ä Dropping the scan head or “fiddling” with the mirrors will result in poor alignment.

5. Bio-Rad Labs. 10 Steps to Measuring Aberration ä To measure aberration you need to make a SINGLE Z series while completing a sequence at each layer in the series. This is EASY with the MRC 1024. ä A good value for chromatic aberration is around 300 nm or less. ä With a good microscope, correct oil and proper alignment, chromatic aberration can be zero.

6. Bio-Rad Labs. First Step ä Place a mirrored slide on the microscope stage and focus on it with a X60 or similar objective. Be sure to use the correct oil for the microscope. ä Ensure you have the correct lens magnification set in Lasersharp.

7. Bio-Rad Labs. Second Step ä Scan at normal speed using BLUE light. ä Set the aperture to 0.7 mm and adjust both M4 and the PMT to obtain an optimum image which DOES NOT saturate. ä Adjust the focus for the brightest image. ä Collect it with Epi 2. Do not use an emission filter. ä Save the settings as “blue”.

8. Bio-Rad Labs. Third step ä Repeat the second step using YELLOW light. ä Use Epi 1 to collect the image. ä Save the settings as “yellow”.

9. Bio-Rad Labs. Fourth step ä Repeat the second step using RED light. ä Use Epi 3 to collect the image. ä Save the settings as “red”.

10. Bio-Rad Labs. Fifth Step ä Turn ON the focus motor. ä Set its START position at +2.0 microns. ä Set its FINISH position at -2.0 microns. ä Set the distance per step to 0.1 microns. ä Set n = 1 so that one scan per section will be collected.

11. Bio-Rad Labs. Sixth Step ä Select Z series. ä Select sequence. ä Select “yellow” in mixer A. ä Select “blue” in mixer B. ä Select “red” in mixer C. ä Select a file name for the results file. The file name must have six characters or less.

12. Bio-Rad Labs. Seventh Step ä Start the sequence. ä The system will complete a sequence of yellow, blue, red for each section. ä There will be about 42 sections collected. ä This will take some time. Ensure the microscope and table are not bumped during the collection.

13. Bio-Rad Labs. Eighth Step ä Switch to the analysis screen and load the results file. ä Examine the image layer by layer, then edit it to remove the unwanted layers which do not contain any useful information. Use the “select rectangle and copy new” features.

14. Bio-Rad Labs. Ninth Step ä With the edited images, select “single section”. ä Choose a rotation or tilt of 90 degrees. ä Build a cross-section of the images.

15. Bio-Rad Labs. Final Step ä Select “line” and draw a line across the cross-section. ä Analyse the line intensity. ä Measure the offset of the peak value in each colour. ä The result is the chromatic aberration of the system.