Phase contrast microscopy is an optical microscopy illumination

Lighting

Lighting or illumination is the deliberate application of light to achieve some practical or aesthetic effect. Lighting includes the use of both artificial light sources such as lamps and light fixtures, as well as natural illumination by capturing daylight...

 technique of great importance to biologists in which small (invisible to the human eye) phase shifts in the light passing through a transparent specimen are converted into (visible) amplitude

Amplitude

Amplitude is the magnitude of change in the oscillating variable with each oscillation within an oscillating system. For example, sound waves in air are oscillations in atmospheric pressure and their amplitudes are proportional to the change in pressure during one oscillation...

 or contrast

Contrast (vision)

Contrast is the difference in visual properties that makes an object distinguishable from other objects and the background. In visual perception of the real world, contrast is determined by the difference in the color and brightness of the object and other objects within the same field of view...

 changes in the image.

A phase contrast microscope

Microscope

A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...

 does not require staining to view the slide. This type of microscope made it possible to study the cell cycle

Cell cycle

The cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication . In cells without a nucleus , the cell cycle occurs via a process termed binary fission...

.

When light travels through a medium other than vacuum

Vacuum

In everyday usage, vacuum is a volume of space that is essentially empty of matter, such that its gaseous pressure is much less than atmospheric pressure. The word comes from the Latin term for "empty". A perfect vacuum would be one with no particles in it at all, which is impossible to achieve in...

, interaction with this medium causes its amplitude

Amplitude

Amplitude is the magnitude of change in the oscillating variable with each oscillation within an oscillating system. For example, sound waves in air are oscillations in atmospheric pressure and their amplitudes are proportional to the change in pressure during one oscillation...

 and phase

Phase (waves)

Phase in waves is the fraction of a wave cycle which has elapsed relative to an arbitrary point.-Formula:The phase of an oscillation or wave refers to a sinusoidal function such as the following:...

 to change in a way which depends on properties of the medium. Changes in amplitude arise from absorption of light, which is often wavelength dependent and may give rise to colours. The human eye measures only the energy of light arriving on the retina, so changes in phase are not easily observed under optimal bright field illumination, yet often these changes in phase carry much important information.

The same situation applies in a typical microscope with "Kohler" bright-field illumination, i.e., although the phase variations introduced by the sample are preserved by the instrument (at least within the instrumental limits of imaging perfection) this information is lost in the process öf image recording, which measures only light intensity. In order to make phase variations observable, it is necessary to combine the light passing through the sample with a reference so that the resulting interference reveals the phase structure of the sample.

This problem was first appreciated by Frits Zernike

Frits Zernike

Frits Zernike was a Dutch physicist and winner of the Nobel prize for physics in 1953 for his invention of the phase contrast microscope, an instrument that permits the study of internal cell structure without the need to stain and thus kill the cells....

 during his study of diffraction gratings

Diffraction grating

In optics, a diffraction grating is an optical component with a periodic structure, which splits and diffracts light into several beams travelling in different directions. The directions of these beams depend on the spacing of the grating and the wavelength of the light so that the grating acts as...

. During the course of his work he realised that it is necessary both to achieve interference with a reference beam, and (for maximizing the contrast achieved with the technique) to introduce a phase shift to this reference beam so that the no-phase-change condition gives rise to completely destructive interference.

He later realized that the same technique can be applied to optical microscopy. The necessary phase shift is introduced by rings etched accurately onto glass plates so that they introduce the required phase shift when inserted into the optical path of the microscope. When in use, this technique allows the phase of the light passing through the object under study to be inferred from the intensity of the image produced by the microscope. This methodology is known as the phase-contrast technique.

In optical microscopy many biological objects such as cell components in protozoans, bacteria and sperm tails are fully transparent unless stained. (Staining is a difficult and time-consuming procedure which can destroy or alter the specimen sructure). The difference in densities and composition within the imaged objects however often give rise to changes in the phase of light passing through them, hence they are sometimes called "phase objects". Using the phase-contrast technique makes these structures visible and allows their study in living specimens.

This phase contrast technique proved to be such an advancement in microscopy that Zernike was awarded the Nobel prize

Nobel Prize

The Nobel Prizes are annual international awards bestowed by Scandinavian committees in recognition of cultural and scientific advances. The will of the Swedish chemist Alfred Nobel, the inventor of dynamite, established the prizes in 1895...

 (physics) in 1953.

Background

The technique was invented by Frits Zernike

Frits Zernike

Frits Zernike was a Dutch physicist and winner of the Nobel prize for physics in 1953 for his invention of the phase contrast microscope, an instrument that permits the study of internal cell structure without the need to stain and thus kill the cells....

 in the 1930s for which he received the Nobel prize

Nobel Prize

The Nobel Prizes are annual international awards bestowed by Scandinavian committees in recognition of cultural and scientific advances. The will of the Swedish chemist Alfred Nobel, the inventor of dynamite, established the prizes in 1895...

 in physics

Physics

Physics is a natural science that involves the study of matter and its motion through spacetime, along with related concepts such as energy and force. More broadly, it is the general analysis of nature, conducted in order to understand how the universe behaves.Physics is one of the oldest academic...

 in 1953. Phase-contrast is an imaging mode available on most advanced light microscopes and is most commonly used to provide contrast of transparent specimens such as living cells or small organisms.

Description

A practical implementation of phase-contrast illumination consists of a phase ring (located in an aperture

Aperture

In optics, an aperture is a hole or an opening through which light travels. More specifically, the aperture of an optical system is the opening that determines the cone angle of a bundle of rays that come to a focus in the image plane. The aperture determines how collimated the admitted rays are,...

 plane located somewhere behind the front lens element of the objective) and a matching annular

Annulus (mathematics)

In mathematics, an annulus is a ring-shaped geometric figure, or more generally, a term used to name a ring-shaped object. Or, it is the area between two concentric circles...

 ring, which is located in the conjugate primary aperture

Aperture

In optics, an aperture is a hole or an opening through which light travels. More specifically, the aperture of an optical system is the opening that determines the cone angle of a bundle of rays that come to a focus in the image plane. The aperture determines how collimated the admitted rays are,...

 plane (location of the condenser's aperture).

Two selected light rays, which are emitted from one point inside the lamp's filament, are focused by the field lens exactly inside the opening of the condenser annular ring. Since this location is precisely in the front focal plane of the condenser, the two light rays are then refracted in such way that they exit the condenser as parallel

Parallel (geometry)

Parallelism is a term in geometry and in everyday life that refers to a property in Euclidean space of two or more lines or planes, or a combination of these. The assumed existence and properties of parallel lines are the basis of Euclid's parallel postulate. Two lines in a plane that do not...

 rays. Assuming that the two rays in question are neither refracted nor diffracted in the specimen plane (location of microscope slide), they enter the objective as parallel rays. Since all parallel rays are focused in the back focal plane of the objective, the back focal plane is a conjugate aperture plane to the condenser's front focal plane (also location of the condenser annulus). To complete the phase setup, a phase plate is positioned inside the back focal plane in annulus.

Only through correctly centering the two elements can phase contrast illumination be established. A phase centering telescope that temporarily replaces one of the oculars can be used, first to focus the phase element plane and then center the annular illumination ring with the corresponding ring of the phase plate. In some microscopes models, an in-built Bertrand lens serves the same purpose as the telescope.

An interesting variant in phase contrast design was once implemented (by the microscope maker C. Baker, London) in which the conventional annular form of the two elements was replaced by a cross-shaped transmission slit in the substage and corresponding cross-shaped phase plates in the conjugate plane in the objectives. The advantage claimed here was that only a single slit aperture was needed for all phase objective magnifications. Recentring and rotational alignment of the cross by means of the telescope was nevertheless needed for each change in magnification.

Image appearance

Phase contrast images have a characteristic grey background with light and dark features found across the sample. Light and dark fringes appear around regions with a change in optical density, for example the boundary between water and a cell. This normally manifests itself as a bright halo around a dark object.

Technical details

To understand how phase contrast illumination works, we study two wave fronts (see the figure to the right). This figure simplifies a few things. First, the condenser annulus is just a small aperture located in the center (see the plane labeled '1') and the phase plate is also just covering a small aperture (located in the plane labeled '3'). Second, the optical system is greatly simplified by showing only two single lenses to represent all optical elements.

The plane labeled '1' is the front focal plane of the condenser. The light emanating from the small aperture 'S' is captured by the condenser and emerges as light with only parallel wavefronts from the condenser. When these plane waves (parallel wave fronts) hit the phase object 'O' (located in the object plane labeled '2'), some of this light is diffracted (and/or refracted) while moving through the specimen. Assuming that the specimen does not significantly alter the amplitudes of the incoming wavefronts but mainly changes phase relations with respect to the "unperturbed" wavefronts, newly generated spherical wave fronts that are retarded by 90° (λ/4) emanate from 'O' (see the purple area that contains now "unperturbed" plane waves and spherical wave fronts). It is important to note that there are now two types of waves, the surround wave or S-wave and the diffracted wave or D-wave, which have a relative phase-shift of 90° (λ/4). - The objective focuses the D-wave inside the primary image plane (labeled '4'), while it focuses the S-wave inside the back focal plane (labeled '3'). The location of the phase plate 'P' has now a profound impact on the S-wave while leaving most of the D-wave "unharmed". In what is known as positive phase contrast optics, the phase plate 'P' reduces the amplitude of all light rays traveling through the phase annulus (mainly S-waves) by 70 to 90% and advances the phase by yet another 90° (λ/4). However, the phase plate leaves most of the D-waves "untouched". Hence the recombination of these two waves (D + S) in the primary image plane (labeled '4') results in a significant amplitude change at all locations where there is a now destructive interference due to a 180° (λ/2) phase shifted D-wave. The net phase shift of 180° (λ/2) results directly from the 90° (λ/4) retardation of the D-wave due to the phase object and the 90° (λ/4) phase advancement of the S-wave due to the phase plate. Without the phase plate, there would be no significant destructive interference that greatly enhances contrast. With phase contrast illumination "invisible" phase variations are hence translated into visible amplitude variations. The destructive interference is illustrated in the figure to the left. Blue and orange indicate D-wave and S-wave, respectively. The resulting wave (D + S), indicated by yellow, has a reduced amplitude.

Implementing phase contrast function using a 4F correlator

We can see how the phase contrast principle works by considering the figure below, which shows a 4F correlator (see Fourier optics

Fourier optics

Fourier optics is the study of classical optics using Fourier transforms and can be seen as the dual of the Huygens-Fresnel principle. In the latter case, the wave is regarded as a superposition of expanding spherical waves which radiate outward from actual current sources via a Green's function...

) that implements the phase contrast function (in this figure, magnification is unity, so it cannot really be called a microscope in the usual sense).

With reference to this figure, we assume a plane wave incident from the left and a phase transmittance function of the form:


The term, in the exponent is known as the phase (see phase (waves)

Phase (waves)

Phase in waves is the fraction of a wave cycle which has elapsed relative to an arbitrary point.-Formula:The phase of an oscillation or wave refers to a sinusoidal function such as the following:...

) of the transmittance function. If this "phase object" is thin, so that then,


Film (or detectors) respond to variations in amplitude, not phase. The transmittance function above will have very small variations in amplitude, since the two terms in the transmittance function are in phase quadrature. For maximum contrast, we will prefer to have these two terms in-phase (not in quadrature phase), so that variations in directly impact the amplitude of the transmittance function. We accomplish this by selectively multiplying one term in the equation above by a factor of j, thus bringing the two terms in-phase.

We can accomplish this using the 4F correlator in the following way. We assume a plane wave field incident on the "input plane" of the correlator (on the far left in the diagram). The Fourier transform

Fourier transform

In mathematics, Fourier analysis is a subject area which grew from the study of Fourier series. The subject began with the study of the way general functions may be represented by sums of simpler trigonometric functions...

 (FT) of the phase transmittance function


is formed in the back focal plane of the first lens as


where is the Point spread function

Point spread function

The point spread function describes the response of an imaging system to a point source or point object. A more general term for the PSF is a system's impulse response, the PSF being the impulse response of a focused optical system. The PSF in many contexts can be thought of as the extended blob...

(PSF) of the lens. The PSF is basically just a small dot in the FT plane (the back focal plane of the first lens), whereas the function will be more spread out. Since the PSF is localized to a small region about the optic axis (the horizontal axis) in the FT plane, we may place a small, quarter-wavelength thick dot there. This dot will impart a quarter wavelength phase shift to the term, while leaving the term relatively unaffected.

So, behind the dot, the field has the form:


and both terms are now in-phase. We now FT this field distribution using the second lens, to produce the following field in the "output plane" (the rightmost plane) of the 4F correlator system:


where we now neglect the factor of j common to both terms. Now the phase function, directly modulates transmittance amplitude, making for better contrast in the images.

External links

• The Phase Contrast Microscope
• Phase Contrast Illumination
• Molecular Expressions: Phase Contrast Microscopy
• Microscopy University: Principles of Phase Contrast Microscopy
• Microscopy University: Principles of Phase Contrast Microscopy: Phase contrast microscopy