Beyond Brightfield: “Forgotten” Microscopic Modalities

Abstract

“Forgotten” microscopic modalities, devices, and accessories derived from or complementary to brightfield microscopy are briefly surveyed. These include off­axis illumination, schlieren contrast, Abbe diffraction apparatus, Rheinberg illumination, darkfield and phase-contrast microscopy combined, incident-illumination microscopy, camera lucida, and comparison microscopy. Examples of their use are shown. While most of them are no longer or only rarely available or used, they are still important for proper understanding of image formation, contrast generation, and data interpretation in microscopy. In some cases, they are superior to their more modern counterparts.

Appendices

Appendix 1 (Hands-on Demonstration)

Diffraction and Resolution

Inspect a diatom specimen⁵ using an objective fitted with a built-in iris diaphragm (e.g., Nikon ×100/0.50 − 1.30) at different settings (i.e., different effective numerical aperture, NA). In this way, it is possible to artificially reduce the objective’s resolving power to the extent (NA = 0.40 in our example) that the finest details in the diatom image completely disappear (Fig. 3B).

Alternatively, use an ordinary objective (having no iris diaphragm) jointly with a diffraction funnel fitted under the ocular head of an upright microscope. Insert S2 slider (with a built-in iris diaphragm) into the funnel (Figs. 4 and 5).

Inspect the objective back focal plane using a centering (“phase”) telescope or Bertrand lens⁶ to monitor the diffraction maxima of different orders while closing and opening the diaphragm in (or above) the objective lens. Typically, 0th and 1st order maxima will be visible (the former representing direct or undiffracted light).

Note that the diffraction patterns may be clearly observed only with the condenser aperture diaphragm closed as much as possible (down to a “pinhole”).

Illuminate the diatom specimen with light of different wavelengths. Blue light (shorter wavelength, ca. 450 nm) is more likely to resolve fine details than red light, ca. 700 nm) as the resolving power is inversely proportional to NA. For this effect to be sufficiently prominent, the objective (or S2 slider) iris controlling the effective NA needs to be set to an appropriate value (0.80 in the example shown in Fig. 3C, D), as checked by viewing the diffraction patterns in the objective back focal plane.

Be aware that different diatom species feature structures of different periodicities.

Appendix 2 (Exercise)

Setting Köhler Illumination

(minor adaptation of a text originally written by Dr. Lisa Cameron, Light Microscopy Core Facility, Duke University, Durham, NC, USA)

Following is a step-by-step protocol for Köhler illumination with transmitted light. An interactive version is also available at the online Microscopy U(niversity)⁷ and elsewhere⁸. For information on focusing the light source, please see the “Focusing the light source” section further below.

First, open all diaphragms. Raise the condenser to its highest point (on an upright microscope). Put a well-stained specimen on the stage, and inspect it with a low power (10×) objective.

Focus the objective lens to obtain a sharp image of the specimen by using the coarse and fine focus controls. This first step sets the correct relationship between the specimen and objective lens.

On a binocular microscope, each user may need to adjust the eyepieces for their own eyes for optimal focus. At least one eyepiece will have an adjustment collar. Use one eye to look down the microscope and focus on some detail in the specimen while keeping the eye which uses the eyepiece with adjustable collar closed. Then switch and use the other eye. Turn the adjustable eyepiece collar to focus the same detail in the image as sharp as before. This procedure is referred to as diopter adjustment and is recommended every time a microscope is used, for the sake of microscopist’s visual comfort. Setting Köhler illumination is possible without it if using one eye only.

Focus the condenser by first closing the illuminated field diaphragm and then adjust the height of the condenser with the condenser focus knob until a sharp image of the field diaphragm is seen superimposed on the image of the specimen (Fig. 1C). Make sure that the condenser diaphragm is wide open. This adjustment sets the correct relationship between the condenser lens and the specimen. If the microscope has an Abbe condenser, this image will likely have a fringe of color around the field aperture.

Center the condenser lens. To do this, make the image of the field diaphragm concentric with the field of view (Fig. 1D) using the condenser centering screws. This adjustment makes the optical axis of the condenser lens coincide with that of the microscope as defined by the field diaphragm and the objective lens.

Adjust the area of the field that is illuminated. Open the field diaphragm until its image is just outside the field of view; readjust the condenser centering if necessary (as you open it). This ensures that illumination falls only on the area of specimen within the field of view, and that the diameter of the primary image is only a little larger than the field-limiting diaphragm as seen by the eyepiece. This prevents light from falling on the internal walls of the microscope to be scattered to produce hot spots and haze, reducing contrast in the final image.

Adjust the aperture diaphragm (illuminating aperture) in the condenser. To do this, remove the eyepiece, or turn the Bertrand lens into position if available—look down the microscope tube from ca. 100 mm above the tube, and observe the back focal plane of the objective, the disc of light at the base of the tube. More conveniently, use a centering (“phase”) telescope in place of the eyepiece, in the same way as during adjustment of the annular diaphragm for phase-contrast imaging. Close the aperture diaphragm until the image of the iris is approximately 70–80% of the viewing field (the aperture of the objective). Replace the eyepiece (or remove the Bertrand lens). The working (effective) aperture of the condenser is now slightly smaller than the aperture of the objective lens. Do not close the diaphragm too far; this will cause a serious deterioration in the quality of the image.

Adjust the brightness of illumination using the control on the lamp power supply, or by inserting neutral density filter(s). These are usually found along the base of the microscope between the lamp and the field diaphragm. The microscope optical adjustments or diaphragms should not be used to control brightness. This will adversely affect the quality of the image. For instance, if the condenser diaphragm is closed too much, the image will appear too contrasty, as refractile structures will be highlighted too much due to diffraction effects; and with it wide open, there will be glare due to stray light (internal reflections). The resolution is poor in both. In a microscope with absolutely no internal reflections the setting is optimal when the effective numerical aperture of the condenser (adjustable by its diaphragm) matches the NA of objective in use. As such microscopes do not exist in reality the abovementioned setting of ca. 70–80% is recommended. Image contrast is slightly improved yet the diffraction artifacts thus introduced are minimal.

For a higher power objective:

Rotate the nosepiece to the 40× dry objective. Owing to parfocality of objective design, the 40× objective should be almost in focus after aligning the microscope for 10×; it was not the case in very old microscopes.

As before, focus and center the image of the field diaphragm using the condenser focus knob and the condenser centering knobs. The aperture of the field diaphragm will need to be readjusted.

Remove an eyepiece (ideally replace it with the centering [“phase”] telescope), or use the Bertrand lens to observe the back focal plane of the objective. Notice that the area illuminated for the low power objective is much smaller than the diameter of the back aperture of the 40× objective.

Adjust the condenser diaphragm so that the effective NA of the condenser is about the same as the objective NA.

For a high-power oil immersion objective:

Rotate the nosepiece so that a high-power oil immersion objective is near-vertical. Just before it is clicked into place, stop and add oil to the coverslip, as close as possible to the optical axis (light beam coming from condenser prealigned at smaller magnifications, see above). Be sure the oil droplet does not have any bubbles. Use immersion oil provided by the microscope manufacturer, as there are some slight differences. Ideally, the refractive index of the oil, coverslip, and objective lenses should be the same.

Click the oil immersion objective into place. The space between the front lens of the objective and the coverslip should now be filled with oil.

Remove an eyepiece (ideally replace it with the centering [“phase”] telescope) or use the Bertrand lens to view the back aperture of the objective. Open the condenser diaphragm to almost fill the objective aperture.

Replace the eyepiece (or remove the Bertrand lens) and observe the specimen. Adjust the field diaphragm until the edge just matches the field of view. Strictly speaking, the condenser should again be readjusted as above (focus and centering). However, switching from ×40 to ×100 objective will rarely misalign the condenser beyond tolerable limit.

Focusing the light source:

Remove the diffuser from the lamp housing or along the base of the microscope stand, if possible, in order to see bulb and filament. Lamp illumination should fill most of the front aperture of the condenser. Put a sheet of lens paper on the specimen stage to help visualize the area of illumination. Focus light on the lens paper by moving the lamp-focusing knob. Then remove the eyepiece (ideally replace it with the centering [“phase”] telescope) or insert the Bertrand lens to view the back focal plane of the objective. Be sure the lamp filament is centered and focused in the plane of the condenser diaphragm. Adjust the collector lens on the lamp housing.

N.B.: Many student microscopes and more recently released modern research ones do not have illumination bulb adjustments, but are designed to deliver even illumination.