Microscopy : Fundamentals

Since a microscope is a fixed-working-distance viewfinder, the distance object/frontal lens of a given objective is fixed when the focus is realized. This distance, called 'Working Distance^[1]', usually varies from several millimeters for low magnification objectives (\(×10\)) to several tens of millimeters for higher magnification objectives (\(×100\), immersion). A large working distance facilitates the observation by limiting the risk of collision between the preparation and the objective, and also allows observing specific objects surrounded with raised patterns, such as integrated circuits with their connection slots. However, it is very difficult (but possible at very high cost) to conciliate a large working distance with a high numerical aperture. Thus, manufacturers usually offer, in addition to a standard series of objectives, specific objectives with high working distance for users interested by this property¹. Recent standard objectives of low working distance usually have a mount with a spring to limit the unfortunate consequences of a collision objective/preparation.

In addition, following a dimensional standard imposing a precise distance (usually \(45~mm\)) between the object plane and the objective support (see Fig. 6 of the subsection "Tube lens and other dimensional standard of a microscope"), the focus must normally be preserved when interchanging objectives. One should however realize that the microscope depth of focus is usually very small, since it is on the order of \(\lambda / (NA_{obj} )^2\) where \(NA_{obj}\) is the object-side numerical aperture of the objective (axial focusing error – see the basic optical course). This usually leads to an exceptionally low tolerance on the objective mechanical mount and on the nosepiece. The effective quality with which the focus can be preserved while interchanging objective is called parfocality.

A microscopist often needs to keep recordings of his or her observations. Photomicrography is a great way of achieving this filing. It consists in making a real image of the object under observation on a photographic film, or nowadays more often on a CCD or CMOS camera. For practical reasons (and sometimes for image quality reasons, as the field may not be flat enough) it is not possible on current microscopes to place the camera in the intermediate image plane; in addition, we advise against modifying the eyepiece focus to obtain an real image at the output of the eyepiece. The solution is rather to replace the standard eyepiece with another optical system called projection eyepiece, which is specifically designed to give on the camera an image of required quality (See reference [ ^[2]] ). In any case, one should make sure to match the camera resolution with the image resolution in order to obtain optimum results (see the third case study). To facilitate the image acquisition, it is possible to equip the microscope with a trinocular head, allowing binocular viewing by the observer simultaneously with image recording (of the full image or a part of it) on the (video) camera.