Conventional, Apodized, and Relief Phase-Contrast Microscopy

Abstract

Non-absorbing colorless specimens (phase objects) can be visualized either by chemical staining (histology) or by the so-called optical contrasting (staining). The latter is achieved by converting optical phase shifts within the specimen, invisible to human eye, to intensity differences in the microscopic image. Out of several available modes of microscopic phase visualization, conventional, apodized, and relief phase contrast are described in detail. A comparison with relief contrast alone, that is, the off-axis (schlieren) illumination mode is presented as well. Images of various phase specimens of biological origin are shown, demonstrating the strong and weak points of each mode. Physiological aspects of image comprehension, facilitated by shading and other visual cues to depth structure are briefly discussed. A phase-contrast microscope equipped with an objective hosting no phase annulus is presented; the latter is located in a pupil projection (optically relayed) plane, in an external attachment unit. This setup enables phase-contrast and, for example, conventional epi-fluorescence and total internal reflection fluorescence (TIRF) images to be acquired with a single objective lens. Such modality is demonstrated in growth cones of neuroblastoma–glioma hybrid mouse–rat cells and touch receptor neurons of Caenorhabditis elegans. Examples of phase-contrast imaging in electron and X-ray (synchrotron radiation) microscopy are also presented.

Glossary

Terminology in phase-contrast microscopy has not been entirely consistent over time. An additional complexity arises when various imaging modes are combined (e.g., relief phase contrast), and a specific terminology is used in electron microscopy.

Amplitude objects

Objects that attenuate light intensity (e.g., opaque granules or stained cells or tissue sections). To visualize them, phase contrast or other ways of ►optical contrasting (staining) is unnecessary. However, such objects typically also induce phase shifts (phase-amplitude objects); cf. ►phase objects.

Anoptral contrast

A variant of negative phase contrast (Reichert) that employed phase plates made of soot featuring smaller reflections than metallic coatings common in other microscopes.

Apodized phase contrast

Phase contrast enhanced by apodization. Neutral density (i.e., non–phase-shifting) filters of annular shape are placed immediately next to the phase annulus. Either two or multiple (graded-transmittance) apodization annuli may be used; only the former option is available on a commercial basis. Apodizing filters are sometimes referred to as modulation filters, the latter being a more general term.

A­type/B­type phase plate

Obsolete terms (coined by American Optical [Spencer] Co) meaning that a light-attenuating (metallic) layer is coated onto the ►conjugate/complementary area of the ►phase plate.

B­minus phase plate

An obsolete term for a ►phase plate yielding ►positive phase contrast (also referred to as ►dark contrast), in which neither the ►conjugate nor complementary area is light-attenuating. Cf. ► Zernike phase contrast.

Conjugate/complementary area

Parts of a ►phase plate. The conjugate area (passing mainly direct or undiffracted light) is optically conjugate with the annular opening in the condenser diaphragm. The complementary area (passing diffracted light only) is the rest of the ►phase plate.

Conventional phase contrast

Non-apodized, non-relief phase contrast in light microscopy. In electron microscopy, phase-contrast observation is relatively new, thus the term “conventional” is irrelevant.

Dark/bright contrast (or “dark/bright phase”)

Older terms for ►positive/negative phase contrast, originally coined by American Optical (Spencer) Co and nowadays used by Nikon. The dark/bright contrast terminology is consistent with sufficiently thin phase-retarding objects such as smaller cells in water. For example, dark-low-low (DLL), dark-low (DL), or dark-medium (DM) denotes a positive phase-contrast objective with a phase annulus of very low, low, or medium attenuation (transmittance ca. 45%, 25%, or 14%, respectively), rendering thin enough phase-retarding objects as less or more dark on bright background. The DL and DM objectives are also available in ►apodized versions (ADL and ADM). An ADH (apodized–dark–high) objective features transmittance of only ca. 6% and the highest phase-visualization sensitivity. Bright-medium (BM) denotes a negative phase-contrast objective with a medium-attenuation phase annulus (transmittance ca. 14%). Objectives currently made by Zeiss are mostly of positive type (marked “Ph1,” “Ph2,” etc.), and only rarely negative (“Ph2­”). A lens with two phase annuli (positive and negative) is also available (“Ph1 Ph2­”). In Olympus’s long-barrel (LB) series objectives (no longer commercially available), the following designations were used: PL/PLL (positive low/low­low) and NM/NH (negative medium/high). Objectives that used to be made by Leitz (“Pv” series) were either of “n” or “h” (positive), or “­h” (negative) type, with transmittance approximately equivalent to that of DL or DM, or BM lenses, respectively. Cf. ►anoptral contrast.

Defocus phase contrast

A term sometimes used in electron microscopy. Its meaning is the same as what is called “imitation phase contrast” in the present chapter, as achieved by slightly defocusing a bright-field image.

Hilbert differential contrast

A term sometimes used to denote a phase-contrast mode in electron microscopy, employing a π­type (λ/2 or 180° phase shift), asymmetric phase plate, suitable for relatively thicker objects (e.g., ultrathin sections or whole ice-embedded bacterial cells). Earlier, it was sometimes referred to as “Difference contrast TEM” (DTEM). Images are rendered in a quasi­3D appearance, similar to the ►relief phase contrast ones in light microscopy.

Hoffman modulation contrast

One of the so-called ►schlieren imaging modes. It employs an asymmetric slit diaphragm in the condenser and a graded-transmittance filter (“modulator”) in the objective. “Nikon Advanced Modulation Contrast” (NAMC) and Olympus’s “Relief Contrast” are its near-identical, double-slit variations; cf. ►relief contrast. Leica’s “Integrated (or Intermediate) Modulation Contrast” (IMC) allows for the modulator to be easily removed from the optical path as it is placed outside the objective (an ►optical-relay setup). A more simple variant, “Emboss Contrast” has been introduced by Nikon.

Optical contrasting (staining)

Visualization of refractive index differences within a specimen. The relevant imaging modalities include dark field (including ►Rheinberg illumination), ►Hoffman modulation contrast (and any other ►schlieren imaging mode, e.g., ►relief contrast), phase contrast, interference contrast, differential interference contrast (DIC) after Smith/Nomarski, and polarization microscopy. A narrower definition of “optical staining” only refers to the use of color filters in the optical path of the microscope, which however does not necessarily enhance the contrast in the images.

Optical path difference (OPD, proportional to object-to-medium phase shift)

Physical thickness (d) of the object (e.g., a cell) multiplied by a refractive index difference (n₁ − n₂) between the medium surrounding it (e.g., a physiological solution) and the object itself. For example, OPD of −λ/6 translates to an object-to-medium phase shift of −60°. Optical thickness (or optical path length) is defined as n₂d in non-absorbing objects. Often used (incorrectly) instead of OPD.

Optical-relay setup

See ►pupil-projection (optical-relay) setup.

Phase objects

Objects that shift only the phase of light, without attenuating its amplitude (e.g., unstained cells or tissue sections). Under a properly adjusted bright-field illumination, such objects are almost invisible. Chemical staining (as in classical histology or fluorescence microscopy) may be used to visualize them. An appropriate optical setup enabling ►optical contrasting (staining) represents a less invasive alternative.

Phase plate (also referred to as “diffraction plate”)

A glass plate at the objective back focal (transform) plane, with a thin coating of dielectric (i.e., phase-shifting) material either at the ►conjugate or complementary area. As the transform plane is often located between tightly packed lenses compounding the objective the coating is deposited directly on one of the lenses in that case. Cf. ►A­type/B­type phase plate.

Positive/negative phase contrast

Imaging modes rendering thin enough phase-retarding (phase-advancing) objects as dark/bright (bright/dark), relative to the background. The phase annulus advances/retards the direct light, typically by 90° (λ/4). At a phase shift of 180° (λ/2) in the phase annulus, the two terms lose their meaning as positive and negative phase contrast yield identical images. Cf. ►anoptral contrast and ►dark/bright contrast.

Pupil-projection (optical-relay) setup

A system in which the objective back focal plane (approx. the exit pupil in low-power objectives) is optically relayed (duplicated) to a more accessible location. A ►phase plate may then be placed in an external, detachable unit rather than in the objective lens itself. This is of advantage when switching, for example, between total internal reflection fluorescence (TIRF) and phase-contrast modes as the need to exchange the objective lens is eliminated. Such systems are offered by Leica (“Integrated [or Intermediate] Phase Contrast,” IPH) and Nikon, and used to be available on Zeiss microscopes.

Relief contrast

One of the so-called ►schlieren imaging modes rendering ►phase objects in a relief-like (quasi­3D) appearance. It employs an asymmetric cut-off diaphragm in the condenser (off­axis illumination). A refined form of relief contrast is referred to as ►Hoffman modulation contrast.

Relief phase contrast

A combination of ►relief contrast and phase contrast, available from Zeiss as “variable relief” (VAREL) contrast. Not to be confused with ►variable phase contrast.

Rheinberg illumination

A refined form of dark-field illumination, in that the background is not dark but colored. Objects illuminated by another (contrasting) color are well visible.

Schlieren imaging

A collective term for ►optical contrasting (staining) modes employing one or two asymmetrical (relief, cutoff) diaphragms in the optical path (e.g., ►Hoffman modulation contrast and ►relief contrast). “Schlieren” (from German, meaning “streaks”) denotes shading patterns in the images which thus attain a relief-like (quasi-3D) appearance.

Variable phase contrast

A system enabling a gradual adjustment of the phase shift and transmittance, with a set of optical elements that emulate the ►phase plate. The most popular device of this kind is called “Polanret” by American Optical (Spencer) Co. This and other similar devices are no longer commercially available. An equivalent in electron microscopy is represented by a system employing an electrostatic (tunable) phase plate. Cf. ►relief phase contrast.

Zernike phase contrast

A term sometimes used to denote a phase-contrast mode in electron microscopy, employing a π/2­type (λ/4 or 90° phase shift), axially symmetrical phase plate with a tiny central opening for unscattered electrons (an equivalent of direct or undiffracted light in light microscopy). It is particularly suitable for very thin objects (e.g., protein particles). An equivalent of ►B-minus phase plate in light microscopy.