Bio-Rad Labs. Chromatic Aberration Sources and Measurement.

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Presentation transcript:

Bio-Rad Labs. Chromatic Aberration Sources and Measurement

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

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.

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.

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 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.

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.

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”.

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”.

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”.

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.

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.

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.

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.

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.

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.