Monday, 18 July 2016

The microscope objective - the key issue for best image performance

Different samples require different microscopes. This rule refers to the fact that an opaque, bulky sample with a reflective surface needs another treatment than a transparent, unstained smear from the cavitas oris. The microscope stand offers the necessary space for a correct positioning of the sample and all options for the appropriate illumination method.

 The microscope objective is an even more specific item. Here we talk about the required resolution power (= numerical aperture), but also about cover glass correction, immersion method and working distance.

1. The standard upright microscope for transmitted light is constructed for glass slides with a 0.17mm cover slip. This restriction is indicated on the objective sleeve:

Cover slip thickness indicated on Motic's Plan UC Fluor objectives

The cover slip has to be placed on top of the sample. A slight pressing of a dissecting needle will help to avoid too much embedding medium (water, etc.) between sample and cover slip. The embedding medium in this case works as an
additional layer and thus simulates a thicker cover slip. A wrong covering of the sample reduces the N.A. power, means detail information and contrast. So please take care to mount your section properly.

Cover slip mounting with the help of a dissecting needle

For “dry” objectives with high numerical apertures (≥ 0.7) it is helpful to use cover slips with a minimized tolerance in thickness. The lab suppliers offer glasses with a tolerance of +/- 0.005mm. Together with a “flat” embedding of the sample, preconditions for a good image result are given.

2. In some medical work flows it is common to omit the covering of the sample. In this case Non-Cover-Glass objectives without cover slip correction are necessary, especially for objectives with an N.A. ≥ 0.30. The higher the N.A., the more the correct objective (0.17 vs. 0) has an influence on the image quality. Non-covered blood smears are typical samples.

Motic's non-cover glass objectives EC Plan NCG

Sample without cover glass

3. These NCG objectives may not be mixed up with objectives for incident light applications with non-covered specimen.

Motic's LM Plan objectives for incident light

Besides performing the first step of magnification in a compound microscope, means following an imaging purpose, in incident light applications for uncovered specimen this kind of objectives is also used for illumination purposes. Traced from the light source, the light passes these objectives on its way to the sample, thus requesting another kind of anti-reflex coating within the objective. A typical setup for that kind of samples looks like this:

Example of incident light application

4. For maximum resolution power, immersion objectives are the best option. Mostly it is immersion oil with a defined refractive index (1,51) to be in use, but please realize that water or glycerin in some cases are preferred. The necessary immersion medium is indicated on the objective sleeve. Immersion for that kind of objectives is not an option, it is an imperative!

Motic's EC-H Plan objectives indicating the immersion medium (oil)

5. Objectives for Polarization microscopes just have one purpose. Here we do not talk about color fidelity (in any case Plan Apos will be best) or transmission rates (Fluorite objectives with a reasonable quantity of glass built in are mostly recommended). Here we talk about strain-free glass elements mounted without tension within the objective. Once this target is achieved, the purpose of these objectives is fulfilled: maximum extinction when polarizer and analyzer are crossed.

Motic's EC Plan objectives for polarization (RED color code)

6. Following another international coding, Phase contrast objectives are marked with a green inscription. Phase contrast is a contrast method for transmitted light, but the respective objectives can also be used in bright field. A slight reduction of image quality is system immanent.

Motic's EC-H Plan objectives for Phase Contrast (GREEN color code)

7. For metallurgical applications, dark field (DF) in incident light is quite common. The necessary objectives have to have a larger diameter to incorporate the DF illumination ring: a mirror system built around the centrally positioned bright field objective. Analog to the situation in transmitted light, a central stop in the Epi illuminator (some people call this device Epi condenser!) stops the direct light entering the objective. So the periphery of such an objective works as an illuminator, the central part as an imager.

Motic's LM BD objectives for Dark field incident light

Dark field objectives for incident light applications

8. For inverted microscopes in bio/medical applications, especially higher magnifications need a Long-Working-Distance construction. The LWD feature works of course at the expense of resolution power.

Motic's LWD Long Working Distance objectives for inverted microscopes

The idea behind such a construction is quite clear. The samples for an inverted microscope are positioned in a petri dish, a flask or any other “high volume” vessel. Adherend cells at the bottom of the vessel have to be treated with an objective of 1.1mm cover slip correction (in this case the bottom of the vessel is equivalent to the cover slip). Floating cells or water samples from a pond need the additional LWD feature. Only this construction allows a focusing “through” the sample, inspecting the complete fluid layer. A short look on motorized inverted microscopes gives us an interesting hint: Before changing the objective by rotating the nosepiece, first drive down the revolving nosepiece. This will avoid scratches on the front lens. Parfocality of the objectives in this case is less relevant. 

Following all these short remarks, our message is quite clear: If you like to find the appropriate optics, think about the characteristics of your sample. If you like to create an “illegal” combination of optics and microscope, think about the restrictions of each optical concept. There is no jack of all trades device.

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