How to Correct Aberration in Stereo Microscopy by Using the Right Objective Lenses

Eliminating Refractive Index Mismatch for Liquid-Immersed or Embedded Samples/Specimens

August 21, 2015

For samples/specimens immersed in a liquid or embedded in a polymer, high quality microscopic observation can be hindered as a result of spherical aberration. The refractive index mismatch between air and the liquid or embedding media of the sample is the reason for this aberration which leads to image distortion [1]. The aberration may make it difficult for certain features of the sample to be accurately observed. For users observing immersed or embedded samples with optical microscopy (stereo or compound), an objective which can correct for refractive index mismatch allows images with greatly reduced spherical aberration and sharper focus to be obtained.

Background

One drawback of stereo microscopes with increased resolving power is that it becomes more difficult to observe and document samples/specimens which are not directly exposed to air, for example, ones embedded in a polymer (e.g. electronic components) or immersed in a liquid (e.g. aquatic organisms). When observing samples immersed in an aqueous solution, fine structures and features of the sample tend to look blurred, especially at high magnification values.

This blurring or image distortion is due to spherical aberration. The aberration is caused by the difference in the refractive indices of air (n = 1) and water (n = 1.3), called the refractive index mismatch [1]. Interesting or important structures could easily be missed or overlooked due to spherical aberrations that may occur with "normal objectives" for stereo microscopes which are optimized for use with samples exposed directly in air (no immersion or embedding media). For 3D composite images created using a "z-stacking" software function, the aberration is especially noticeable, because the structures appear elongated in the z-direction.

The same phenomenon also occurs for compound microscopes, however there are liquid immersion compound objectives which can be used with a liquid layer between the lens and sample. Typically, the immersion liquid is water or oil and these help to reduce significantly the image aberration mentioned above.

The Leica Plan Apo 2x objective for stereo microscopy was introduced in 2010. It achieved a resolution of 1,050 line pairs/mm (952 nm with visible light) due to an effective numerical aperture of 0.35. This gain in resolution compared to earlier microscope systems was possible in part due to FusionOptics [2]. The FusionOptics technology utilizes asymmetric zoom lenses providing stereo microscope users the best 3D perception of an observed sample (through the eyepieces) with the highest resolution and corresponding depth of field possible. Unfortunately, when using the Plan Apo 2x objective to image immersed or embedded media, the problem of spherical aberration can occur.

Studies of aquatic organisms which are used as model systems for vertebrate embryo development, like the zebrafish, still generate demand for higher resolving power, better light transmission, and better signal-to-noise (S/N) performance. This demand is especially true for fluorescence applications with stereo microscopy. When using such a highly resolving objective which is optimized for samples exposed to air, obviously embedded and immersed samples, as well as those with cover slips, are being observed and documented under sub-optimal conditions.

Eliminating image aberration

There are microscope objectives with "correction collars" that allow adjustments for mismatch in refractive index between the lens immersion media and sample immersion or embedding media. Adjustments to the correction collar cause a shift in the position of a group of lenses within the objective. An illustration of how the objective collar corrects for refractive index mismatch is shown in Figure 1.

The correction collar of the objective enables users to eliminate spherical aberration and obtain an image of an embedded (polymer or glass) or liquid-immersed (water) sample/specimen with even sharper, crisper focus. After correcting for refractive index mismatch with the objective collar, it seems as if the immersion or embedding medium is not present. Proper adjustment of the objective correction collar improves both spatial resolution and the S/N ratio of the image.

An example of a stereo microscope objective with a correction collar is the Leica Plan Apo 2x Corr objective. With the Leica Plan Apo 2x Corr, even thick embedded samples or samples immersed in a deep aqueous solution (5 mm) can be imaged with little or no aberration. 

Fig. 1: Microscope observation of an immersed or embedded sample/specimen; tracing the rays of light (yellow) through the optical system (blue dashed line is the optical axis).
– Non-corrected case: the refractive index mismatch between the sample immersion/embedding medium and air lead to spherical aberration and elongation of the image along the vertical direction [1].
– Corrected case: an objective which is capable of adjusting for the refractive index mismatch can largely eliminate the aberration.

Application examples

Many users inspect or document embedded and liquid-immersed samples/specimens with stereo microscopes. The examples below demonstrate the improved image quality when using a stereo microscope with the Leica 2x Plan Apo Corr objective (assuming proper set up and adjustment).

Sample/Specimen immersed in aqueous solution

Bovine pulmonary artery endothelial (BPAE) cells are used as a model system for the study of cardiovascular function and disease [3]. Images of Bovine Pulmonary Artery Endothelial (BPAE) cells using the fluorophores FluoCells®, MitoTracker® Red, CMXRos, BODIPY® FL phallacidin, and DAPI (Molecular Probes, Leiden, Netherlands) are seen in Figure  2. They were acquired with a Leica M205 FA stereo microscope, Leica 2x Plan Apo objective or Leica 2x Plan Apo Corr objective, Leica DFC3000G digital camera, and LAS X software.

In Figure 2A, a fluorescence image with only the phallacidin channel and a bright field image captured with the Leica 2x Plan Apo objective (no correction collar). The BODIPY® FL phallacidin stains selectively the F-actin filaments. Image Z-stacks were acquired and the sharpest image of each stack is shown. No image processing algorithms have been applied, thus these images show the raw data.

In Figure 2B, the images were captured with a Leica 2x Plan Apo Corr objective where the correction collar was set to 0.25 mm to compensate for the embedding media and coverslip. The best position of the correction collar was identified by visually observing and comparing a sequence of Z-stacks acquired with the correction collar at slightly different settings. The image shows clearly the difference a correct setting on the correction collar makes to the image quality. Actin filament structures in the cells become much more visible and the S/N ratio is improved. The BPAE cell sample is extremely thin in comparison to most types.

Fig. 2: A: Fluorescence image of BPAE cells embedded onto a slide with coverslip and captured with a Leica M205 FA stereo microscope where a 2x Plan Apo objective with no correction collar was used. The actin filaments in the cells (red arrow) are visible, however appear blurry. There is also a slight blurring (white arrow) at the boundaries of the cells. B: Same sample where the Leica 2x Plan Apo Corr objective with correction collar was used. The actin filaments (red arrow) become even more visible. The blurring (white arrow) at the boundaries of the cells is significantly reduced. Structures in the nuclei are better resolved.

A larger effect can be seen with thicker samples, such as the larva of zebrafish immersed in an aqueous solution up to several millimeters deep [4]. The videos below (Figure 3) were acquired with a Leica M165 FC stereo microscope, Leica 2x Plan Apo objective or Leica 2x Plan Apo Corr objective, and Hamamatsu Orca-R2 digital camera. The zebrafish larva is expressing green fluorescent protein (GFP) in its blood vessels.

Fig. 3: Fluorescence video imaging of a zebrafish larva expressing green fluorescent protein (GFP) immersed in an aqueous solution. The images were captured with a Leica M165 FC stereo microscope where a Leica 2x Plan Apo objective with (right image) and without (left image) correction collar was used. The heart of the zebrafish larva is seen beating in both videos. The larva appears blurrier in the non-corrected image on the right. Courtesy of M. J. Hamm and W. Herzog, Angiogenesis Laboratory, Max Planck Institute for Molecular Biomedicine and Westfälische Wilhelms University in Münster, Germany.

Sample embedded in polymer

Light-emitting diodes sold commercially tend to be embedded in polymers for practical usage. Images of a LED embedded in a polymer are shown in Figure 4. They were acquired with a Leica M205 FA stereo microscope, Leica 2x Plan Apo objective or Leica 2x Plan Apo Corr objective, DFC450 digital camera, and LAS X software.

In Figure 4A, a bright field image captured with the Leica 2x Plan Apo objective (no correction collar). The individual segments of the LED are seen. Image Z-stacks were acquired and the sharpest image of each stack is shown. No image processing algorithms have been applied, thus these images show the raw data.

In Figure 4B, the image is captured with the Leica 2x Plan Apo Corr objective where the correction collar was set to 2 mm to compensate for the plastic. As before, the best position of the correction collar was identified by visually observing and comparing a sequence of Z-stacks acquired with the correction collar at slightly different settings. A clear difference in the image quality is seen. The LED segments are in crisper focus and the S/N ratio is improved.

Fig. 4: A: Bright field image of a LED embedded in a polymer captured with the Leica M205 FA stereo microscope where a Leica 2x Plan Apo objective with no correction collar was used. The individual LED segments are visible, but appear blurry. B: Same sample where the Leica 2x Plan Apo Corr objective with correction collar was used. The LED segments are now sharper and crisper. The blurring is significantly reduced.

Summary

Spherical aberration due to refractive index mismatch between air and the liquid or embedding media (e.g. polymer) of a sample or specimen can reduce the quality of microscopic observation [1]. Specific features of the sample can appear distorted during observation. Optical microscope (stereo or compound) users observing immersed or embedded samples can avoid the aberration and image distortion by using an objective lens which corrects for refractive index mismatch.

Acknowledgements

We would like to thank Mailin J. Hamm and Wiebke Herzog of the Angiogenesis Laboratory at the Max Planck Institute (MPI) for Molecular Biomedicine and the Westfälische Wilhelms University in Münster, Germany for supplying the videos of zebrafish larva (Figure 3).

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