Confocal Imaging Tips for LSM 7/8 systems: Z-stack Refractive Index Correction

Here we cover Refractive Index Correction as one of the tools in ZEN when acquiring z stacks. Basic knowledge on LSM is recommended, in particular

Setting up z-stacks

Refractive indices of your embedding medium, objective lens and immersion

The effective refractive index of the sample is rarely known exactly. Moreover, it exhibits some dependence on temperature and presence of dissolved salts or impurities. One should normally expect a mismatch between the refractive indices of the used immersion, glass and embedding medium. As a result, the optical path for light rays inside the sample with the refractive index    (n _spec) may differ from the path inside the immersion oil (n _imm). If there is a match between these indices, the total optical path will be equal to the total physical path, as shown on the left part of the drawing below. In case there is a mismatch, total optical path will be smaller or larger, depending on the n _spec  / n _imm ratio, see the right side of the drawing below

The refractive index (RI) correction is well documented in the system operation manual with a few numerical examples

Example 1: Water embedded specimen (n), oil immersion objective (n’). With n = 1.33 – 1.43 and n’ = 1.518, the correction factor is r = n/n’ = 0.88 - 0.94

Example 2: Glycerol embedded specimen (n), water immersion objective (n’). With n = 1.48 and n’ = 1.34, the correction factor is r = n/n’ = 1.10

Example 3: Glycerol embedded specimen (n), air objective (n’). With n = 1.48 and n’ = 1.0, the correction factor is r = 1.48

As one can see, using air (dry) objectives with fixed samples having embedding medium RI close the one of glycerol/oil will result in appearing specimens flatter than they really are unless the correction isn’t done accordingly to the example 3

At the illustration above we can see how a glycerol-like embedded sample which has a characteristic feature size of 10 µm has been imaged using a dry objective 20x/0.8. It appears much flatter if no correction is made (software will always use r = 1.0 by default, left image), as oppose to the more realistic sample representation with the correction factor set as r = 1.5

In general, we can determine the refractive index of a specimen if its size in z is known. This can be done by using fluorescent beads. The image below shows a 5 µm sized sphere fluorescent beads imaged with a 20x/0.8 objective (mounted in a glycerol-like medium). It is markedly rounder and displays the size and shape much closer to the expected when the refractive index correction is set to be 1.5

Additional remarks

Refractive index correction visible in ZEN when “View à Show all” is active. It can be found in under “Acquisition” tab in the Z-Stack menu à Correction

The used refractive index correction factor cannot be re-used, e.g. one should right down its value for a certain sample types and keep separately

Please remember, when imaging embedded samples using air objectives you may want to setup correction factor close to 1.5 to see true shape of objects