Conventional Light Microscopy

This figure illustrates the refraction of light as it passes from air to water (A) or water to air (B). Air is shown with refractive index n₁, and water with n₂. In general, light passing from a low- to high-density medium (A) bends towards an imaginary line drawn perpendicular to the interface. Conversely, light bends away from such a line as it passes from a high- to low-density medium (B).

When prisms are stacked as in this illustration, the compound effect is to refract light in such a way that it converges at a single point, the focal point (f ). This is due to the increasingly acute angles of each prism as one moves away from the center of the lens. This is the principle behind a simple biconvex lens (a lens that curves “outward” on both surfaces).

This line drawing illustrates the fundamental design of a compound microscope. The object is placed at less than 2 × f₁ in front of the objective lens, so that its image is magnified (as in Fig. 9.1.6B). This real image is projected onto a plane that lies inside the focal distance f₂ for the ocular lens. Consequently, as light from this real image passes through the ocular lens, its light diverges (as in Fig. 9.1.6D). Your eye collects this divergent light and creates a real image on the retina. This produces the effect of a virtual image.

The microscope stage often has two sets of coordinates (x and y) that can be used to record the location of a specific object on a microscope slide. This figure illustrates the appearance of the x coordinate designating a location at 112.6. See text for details.

Establishing Köhler illumination. This illustrates the two major steps in establishing Köhler illumination. First, panel (A) shows how (after focusing the microscope on an object), one should focus the substage condenser using its own focusing knob in order to bring the image of the collector lens into focus. This can be done in one of three ways. (1) If the microscope has a field diaphragm, simply close the field diaphragm down and focus on the edges of the diaphragm polygon. Next, open the diaphragm back up until its image expands just beyond the field of view. (2) If the microscope doesn't have a field diaphragm, drop a clear plastic disk onto the illuminator marked with an “X,” and focus on that, or (3) bring the ground glass of the diffuser into focus and then slightly defocus (the ground glass image can be too subtle for some to catch). Panel (B) shows the step (see step 7 under Logistics of Köhler Illumination) in which one adjusts the condenser diaphragm to the 75% image seen in Figure 9.1.10B.

A cartoon of the diffraction pattern of light as it passes through a tiny aperture or is diffracted around some tiny microscopic object. The central light disk is referred to as the Airy disk and contains ~84% of the light. It is referred to as the zero-order light. The concentric rings of light represent the first-order, second-order diffracted light, etc., and are of decreasing intensity. All microscopic images can be modeled as a collection of such diffraction patterns and the composite interference pattern that occurs through their interaction.

Dark-field microscopy. Panel (A) shows the light-path from a typical bright-field microscope. Most of the image is created by direct, undeviated light. Diffracted light is collected, but its signal is swamped by the undeviated light. Panel (B) shows the effect of placing a dark-field stop into the center of the light path between the illuminator and specimen. Only tangential light passes through the specimen pane. Having no specimen with which to interact, this tangential light misses the objective lens completely and the image is black. (C) When an object passes into the path of this hollow cone of light, light is diffracted, and that diffracted light will be collected by the objective lens. The result is a bright image against a dark background. (See Fig. 9.1.16B for an example).

Phase-contrast microscopy. The upper figure illustrates the basic design of the phase-contrast microscope. A phase annulus lies just below the condenser lens, illuminating the specimen with a hollow cone of light, similar to the dark-field model, yet different. The hollow cone of direct (undeviated) light has a more acute angle, so that its light is collected by the objective lens and contributes to formation of the image. Within the objective lens, there is a phase plate through which the direct, undeviated cone of light is transmitted. In the model illustrated, this phase plate has a raised annulus of glass that is coated with a neutral-density filter to dim the undeviated light. The height of this raised ring is such that light passing through it is phase-delayed by ¼ (see lower diagram). Deviated light, i.e., light that has passed through a diffraction object in the specimen, passes through the unraised center of the phase plate. When light is diffracted, it is also phase delayed by ¼ . Hence, in this design, the deviated light and the undeviated light are brought back in phase with one another, and will interfere positively to create regions of brightness in the image. Since diffraction is the result of light interfering with edges and tiny particles, these are the objects that become highlighted in the final image. Other models of phase microscopes etch the glass of the phase plate, reducing its effective light path so that the direct, undeviated light is advanced ¼ with respect to the deviated (diffracted) light. The net effect is that deviated light is now ½ delayed with respect to undeviated light, and the deviated and undeviated light interfere negatively, producing areas of shadow around edges and particles.