GOOD MORNING….!

PHASE C

ONTRAST

MICROSCOPE

D R R A V I K U M A R V

D E P T O F O R A L P A T H O L O G Y

& M I C R O B I O L O G Y

G D C , K O T T A Y A M .

INTRODUCTION

- The microscope is commonly described as an instrument used for seeing small objects.

- The human eye sees because of two properties of the light entering the eye from the objects seen.

- The eye recognizes only differences in brightness and differences in color.

- Differences in brightness of different objects or their component parts give rise to brightness contrast; differences in color cause color contrast.

- Brightness = Amplitude

- Color = Wavelenght

The improvement of microscopes since their invention about 350 years ago has proceeded along two general lines

First, there has been improvement of the instrument itself in order to make it a better and more convenient image-forming apparatus.

The second approach is to consider the specimen as an essential part of the optical system and, as it were, to build the microscope around the specimen.

Until the invention of the phase microscope no very useful means were available for observing differences in optical path in a specimen.

Optical path is the linear path of light through a transmitting medium multiplied by the index of refraction of the medium.

Refractive index in a microscopic specimen depends upon the specimen's physical and chemical properties.

In optics the refractive index (or index of refraction) n of a substance (optical medium) is a number that describes how light, or any other radiation, propagates through that medium

More fundamentally, n is defined as the factor by which the wavelength and the velocity of the radiation are reduced with respect to their vacuum values

The speed of light in a medium is v = c/n, where c is the speed in vacuum.

In physics, the wavelength of a sinusoidal wave is the spatial period of the wave the distance over which the wave's shape repeats.

It is usually determined by considering the distance between consecutive corresponding points of the same phase, such as crests, troughs, or zero crossings, and is a characteristic of both traveling waves and standing waves, as well as other spatial wave patterns.

Wavelength is commonly designated by theGreek letter lambda (λ). 

Amplitude is the magnitude of change in the oscillating variable with each oscillation within an oscillating system

Peak amplitude

Peak to peak amplitude

Semi amplitude

RMS amplitude

The speed and wavelength of light changes when it passes through media with different refractive indices

AirR.I.=1.0

AirR.I.=1.0

WaterR.I.=1.33

GlassR.I.=1.5

Phase contrast microscopy makes these phase difference visible

The details of many biological and industrial specimens are characterized by differences in refractive index rather than by differences in light absorption. Under an ordinary microscope such details are invisible, unless the aperture of the condenser or objective is made so small that the resolving power suffers a serious deterioration with resultant loss in the observer's ability to interpret what he sees.

Light can be considered a form of wave motion consisting of sinusoidal waves. When a light wave traverses a medium of different optical path the phase of the light wave is altered. This alteration may be visualized as simply a displacement of the wave in its direction of propagation. If a microscopic specimen contains details that differ from each other in optical path, the phase of that portion of the illuminating wave front passing through the detail is changed. If a microscope can delineate change of phase as a change in brightness or color, the eye, photographic plate, or photocell will be able to detect the microscopic areas causing the phase changes.

Constructive interference Destructive interference

We can see amplitude (intensity) differencesTutorial

•Illumination bypasses Specimen > no phase shift

•Illumination passes through thin part of Specimen > small phase retardation

•Illumination passes through thick part of Specimen > larger phase retardation

WALK INTO THE PAST….

Abbe, first reported on the effect of introducing phase changes on light waves within the optical system of the microscope.

(1892),he introduced glass wedges into the rear focal plane of the microscope objective and thereby changed the phase relationships

Later Bratuscheck introduced absorbing strips of soot at the back focal plane of the microscope objective to weaken the brightness of the zero-order spectrum.

These observations were made before 1892 at the time when Abbe was much concerned with establishing the diffraction theory of image formation in the microscope, and they were aimed to substantiate such knowledge. It is certain that they did not find their way into practical applications of the microscope.

A. E. Conrady and J. Rheinberg in 1905, published experimental results showing that a phase reversal exists in different orders of diffraction spectra produced by the light.

It is to Professor F. Zernike, of the University of Groningen, that we are indebted for the first application of phase contrast principles to the microscope and for an explanation of these principles.

Zernike's first published work (1934) on this subject showed the advantage of the phase contrast method over the familiar knife-edge method in testing the quality of optical systems.

In 1935 Zernike discussed the application of the phase contrast method to microscopy, although it is indicated that Zernike realized the importance of the phase contrast method in microscopy as early or earlier than 1932.

However, it was not until 1941 that Kohler and Loos, of the firm of Carl Zeiss, published a paper showing the results of their extensive and convincing experiments with the Zernike method.

This phase contrast technique proved to be such an advancement in microscopy that Zernike was awarded the Nobel prize (physics) in 1953. 

FRITS ZERNIKE

OPTICAL FUNDAMENTALS

Imagine light waves passing through a glass window. 

Although we think of a glass window as smooth,

if we zoomed in we would see tiny imperfections.

Light passing through different parts of the glass window would actually travel through different distances of glass depending on the glass thickness at a particular point, as shown in the diagram 

The peaks and troughs of the waves no longer match up

We say these waves have a difference in 'phase‘

phase differences arise because the light travels through different distances depending on the thickness of the glass at each point.

 a phase contrast microscope is used to transform phase differences into intensity differences, increasing contrast.

Today, two main types of phase contrast are positive and negative. Since the observed particles are usually thin and transparent, these polar contrasts provide strikingly different images.

Positive phase contrast reveals medium to dark gray images on a lighter grey background; these images often have a bright halo along the edge of the sample.

Negative phase contrast is the opposite. The specimen appears lighter with a dark background; they also have a dark halo outlining the image.

Phase Contrast can be performed in two different ways, on upright microscopes and inverted microscopes.

Upright microscopes are the most common type of microscope, designed with the objective lenses positioned above the sample, looking downward and usually have shorter working distances

Inverted microscopes are the most durable and easy to use for cell microscopy and tissue culture work including the diagnosis of tumor cells.

Specialized long-working distance phase contrast optical systems have been developed for inverted microscope employed for tissue culture investigations and sperm cell motility for in vitro-fertilization.

Interaction of Light Waves with Phase Specimens

An incident wavefront present in an illuminating beam of light becomes divided into two components upon passing through a phase specimen

The primary component is an undeviated (or undiffracted; zeroth-order) planar wavefront, commonly referred to as the surround (S) wave, which passes through and around the specimen, but does not interact with it.

 In addition, a deviated or diffracted spherical wavefront (D-wave) is also produced, which becomes scattered over a wide arc (in many directions) that passes through the full aperture of the objective.

After leaving the specimen plane, surround and diffracted light waves enter the objective front lens element and are subsequently focused at the intermediate image plane where they combine through interference to produce a resultant particle wave (often referred to as a P-wave).

Detection of the specimen image depends on the relative intensity differences, and therefore on the amplitudes, of the particle and surround (P and S) waves.

If the amplitudes of the particle and surround waves are significantly different in the intermediate image plane, then the specimen acquires a considerable amount of contrast and is easily visualized in the microscope eyepieces.

Otherwise, the specimen remains transparent and appears as it would under ordinary bright field conditions (in the absence of phase contrast or other contrast-enhancing techniques).

In terms of optical path variations between the specimen and its surrounding medium, the portion of the incident light wavefront that traverses the specimen (D-wave), but does not pass through the surrounding medium (S-wave), is slightly retarded. For arguments in phase contrast microscopy, the role of the specimen in altering the optical path length (in effect, the relative phase shift) of waves passing through is of paramount importance

 the optical path length (OPL) through an object or space is the product of the refractive index (n) and the thickness (t) of the object or intervening medium as described by the relationship:

When light passes from one medium into another, the velocity is altered proportionally to the refractive index differences between the two media.

when a coherent light wave emitted by the focused microscope filament passes through a phase specimen having a specific thickness (t) and refractive index (n), the wave is either increased or decreased in velocity

The difference in location of an emergent wavefront between the specimen and surrounding medium is termed the phase shift 

n(2) is the refractive index of the specimen and n(1) is the refractive index of the surrounding medium. The optical path difference results from the product of two terms: the thickness of the specimen, and its difference in refractive index with the surrounding medium.

For individual cells in tissue culture, the optical path difference is relatively small. A typical cell in monolayer culture has a thickness around 5 micrometers and a refractive index of approximately 1.36.

The cell is surrounded by a nutrient medium having a refractive index of 1.335, which yields an optical path difference of 0.125 micrometer, or about a quarter wavelength (of green light).

Subcellular structures generate much smaller retardations. These small optical path differences produce a linear reduction in intensity with increasing phase shift (the image grows progressively darker) up to a point (depending upon phase plate configuration), after which, the specimen image becomes brighter through reversal of contrast.

In phase contrast microscopy, the intensity of an image is dependent on a variety of factors including absorption at the phase plate, the degree of phase advancement or retardation at the phase plate, and the relative sign of this phase shift.

Oxytricha saprobia in brightfield (left) and with phase contrast illumination (right)

To get a better understanding of how phase contrast illumination works, we study two wave fronts

 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  newly generated spherical wave fronts that are retarded by 90° (λ/4) emanate from 'O

the purple area that contains now "unperturbed" plane waves and spherical wave front

 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).

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. Blue and orange indicate D-wave and S-wave, respectively. The resulting wave (D + S), indicated by yellow, has a reduced amplitude.

PARTS:

Presented in Figure is a cut-away diagram of a modern upright phase contrast microscope, including a schematic illustration of the phase contrast optical train.

Partially coherent illumination is produced by the tungsten-halogen lamp

Light is directed through a collector lens and focused on a specialized annulus (labeled condenser annulus) positioned in the substage condenser front focal plane.

Wavefronts passing through the annulus illuminate the specimen and either pass through undeviated or are diffracted and retarded in phase by structures and phase gradients present in the specimen.

Rays are segregated at the rear focal plane by a phase plate and focused at the intermediate image plane to form the final phase contrast image observed in the eyepieces.

Two specialized accessories are required to convert a brightfield microscope for phase contrast observation.

A specially designed annular diaphragm, which is matched in diameter and optically conjugate to an internal phase plate residing in the objective rear focal plane

The condenser annulus is typically constructed as an opaque flat-black (light absorbing) plate with a transparent annular ring, which is positioned so the specimen can be illuminated by defocused, parallel light wavefronts emanating from the ring.

The microscope condenser images the annular diaphragm at infinity, while the objective produces an image at the rear focal plane

The condenser annulus either replaces or resides close to the adjustable iris diaphragm in the front aperture of the condenser. 

Under conditions of Köhler illumination, surround light waves that do not interact with the specimen are focused as a bright ring in the rear focal plane of the objective

The D waves passes through the diffraction plane at various locations across the entire objective rear aperture.

Thus, the two wavefronts do not overlap to a significant extent, and occupy distinct portions of the objective rear focal plane.

The phase contrast annuli must be specifically matched to a particular objective equipped with a corresponding phase plate

By matching the condenser annulus to the objective phase plate, the microscope can be aligned to superimpose illuminating light rays passed through the annulus onto the objective phase ring to achieve phase contrast illumination.

A majority of the popular universal condenser systems designed for phase contrast microscopy are equipped with three or more removable annular diaphragms, which are available for use with 4x, 10x, 20x, 40x, 60x, and 100x objectives containing the appropriate phase plates.

The PH1 - smallest aperture – 10x and 20 x

The PH2 – 40x and 60x

The PH3 – 100x

The PHL - 4x and 5x, in long working distance condensers 

Condenser annulus inserts are circular aluminum plates having a stamped ring of varying dimensions in the center

 After the stamping operation, annular disk units are anodized and dyed with a flat-black pigment to absorb stray light and ensure that illuminating light rays passing through the annulus follow a defined pathway. 

The central light stop,  is positioned in the center of the plate and secured by three slim tabs spaced at 120-degree intervals.

Modern universal condenser system turrets usually contain between five and eight open slot positions that can be fitted with annular phase contrast plates, DIC Nomarski (Wollaston) prism plates, or darkfield light stop plates.

The phase contrast condenser annular diaphragm plates are inserted into the appropriate position in the turret and secured into place with a spring clip 

Whenever a universal microscope condenser is utilized, be certain to ensure that the diaphragm is opened wider than the diameter of the condenser annulus. In fact, it is a good idea to always open the diaphragm to its widest position during phase contrast observations.

Phase contrast condensers are available in a wide spectrum of optical correction levels, ranging from the basic Abbe design  to highly corrected aplanatic-achromatic systems that feature excellent performance.

 In addition, specialized phase contrast condensers are available with long working distance (LWD) optics & extra long working distance (ELWD).

Single annulus phase condensers were very popular at one time, but have been largely supplanted by the universal turret models. The older condensers require removal and insertion of different phase annuli as the objective is changed

PHASE CONTRAST OBJECTIVES

The most important attribute of objectives designed for phase contrast microscopy is the presence of a specialized phase plate

Phase plates are not interchangeable between objectives and are often permanently etched into one of the internal lens elements

The etched ring is coated with a partially absorbing metallic film that reduces light transmission, and is usually made so that the phase of light passing through will be advanced by one quarter-wavelength relative to light that is transmitted through the rest of the glass.

The inset illustrates the phase plate that is positioned in the rear focal plane

The range of phase contrast objectives available from the major manufacturers covers almost the complete gamut of correction factors, including achromatic, plan achromatic, fluorite, and apochromatic. 

 In addition, phase contrast objectives are manufactured that vary in the neutral density and retardation value of the phase plate to produce a wide spectrum of contrast levels in both positive and negative phase contrast modes.

Presented below is a comparison of digital images recorded using the currently available Nikon

The images are the same viewfield from a fixed and mounted preparation of Zygnema filamentous algae. (a),(b), (c) compare the dark low low, dark low and apodized phase contrast objectives.

The dark and bright medium phase contrast objectives (d) and (e))demonstrate positive and negative phase contrast.

DL (Dark Low - Medium Contrast) - DL objectives produce a dark image outline on a light gray background, and are the typical objectives utilized for all-purpose phase contrast observation.

These objectives are designed to furnish the strongest dark contrast in specimens having a major difference in refractive index from that of the surrounding medium.

The DL phase contrast objective is the most popular style for examination of cells and other semi-transparent living material and is especially suited for photomicrography and digital imaging.

DLL (Dark Low Low - Low Contrast) - Similar in design to the DL objective, the DLL series yields better images in brightfield illumination and is often employed as a "universal" objective in microscope systems that utilize multiple illumination modes such as fluorescence, DIC, brightfield, and darkfield.

The DLL phase contrast objective produces less contrast than the DL objective, but features higher light transmission values, optical correction, and numerical aperture than the standard DL counterpart.

A majority of the DLL phase contrast objectives offered by the manufacturers have fluorite or apochromatic aberration correction levels.

ADL (Apodized Dark Low - Medium Contrast) - Recently introduced, the apodized phase contrast ADL objectives contain a secondary neutral density ring on either side of the central ring in the phase plate.

Addition of the secondary rings assists in reducing unwanted "halo" effects often associated with imaging large particles or specimen features (such as nuclei, whole cells and fibers) in phase contrast microscopy.

Apodized objectives are available in plan achromat optical correction and feature contrast levels similar to the DL objective series.

DM (Dark Medium - High Contrast) - DM objectives produce a dark image outline on a medium gray background.

These objectives are designed to be used for high image contrast with specimens having small phase shifts or refractive differences, such as fine fibers, flagella, cilia, granules, and very small particles.

Usually restricted to higher magnification objectives having large numerical apertures (fluorites and apochromats), DM phase contrast objectives perform well with very thin specimens, but often display a reversal of contrast when thick specimens are imaged.

BM (Bright Medium - High Negative Contrast) - Often referred to as negative phase contrast, BM objectives produce a bright image outline on a medium gray background.

BM objectives are ideal for visual examination of bacterial flagella, fibrin bundles, minute globules, and for blood cell counting.

For easy identification manufacturers  inscribe important specifications  on the outer barrel in green letters

 In addition, phase contrast objectives have inscriptions on the barrel to indicate the objective is designed for phase contrast and also the matching annulus designation.

A typical series of phase contrast objectives are presented.

 As a general rule, when objective numerical aperture and magnification is increased, the phase plate width and diameter both decrease and the condenser annulus size increases

The positive phase plate produces dark contrast and contains a partially absorbing film designed to reduce the amplitude of the surround wavefront.

In addition, this plate contains phase retarding material designed to shift (retard) the phase of the diffracted light by 90 degrees

In  negative phase plate  both materials are placed within the circular phase ring so that the undiffracted surround wavefront becomes the only species affected, and is attenuated and retarded in phase by 90 degrees.

Contrast is modulated in phase objectives by varying the properties of the phase plate, including the absorption of the metallic film (or anti-reflective coatings), the refractive index of the phase retarding material, and the thickness of the phase plate.

Phase plates are produced by vacuum deposition of thin dielectric and metallic films onto a glass plate or directly onto one of the lens surfaces within the microscope objective

 The role of the dielectric film is to shift the phase of light, while the metallic film attenuates undiffracted light intensity. 

 The thickness and refractive indices of the dielectric, metallic, and anti-reflective films, as well as those of the optical cement, are carefully selected to produce the necessary phase shift.

In optical terminology, phase plates that alter the phase of surround light relative to diffracted light by 90 degrees (either positive or negative) are termed quarter wavelength plates because of their effect on the optical path difference

THE PHASE TELESCOPE

If the condenser annulus is not in exact alignment with the fixed phase plate in the objective the contrast effect afforded will be dramatically compromised.

This task can be accomplished using either a phase telescope, which can be inserted into one of the eyepiece observation tubes (in place of a normal eyepiece), or a Bertrand lens built into the microscope binocular (or trinocular) eyepiece tube assembly.

The phase telescope, also commonly referred to as an auxiliary telescope or auxiliary microscope consists of a simple two or three lens telescope

The focal length of the phase telescope ranges from about 150 to 200 millimeters enabling the device to focus on the objective rear focal plane when inserted into eyepiece.

The Bertrand lens is more sophisticated, and acts as a relay lens, transferring an image of the objective focal plane to the microscope intermediate image plane located in the eyepiece aperture diaphragm

 On most microscopes equipped with a Bertrand lens, the lens can be rotated into and out of the optical pathway by means of a small thumbwheel mechanism located beneath the eyepiece tubes

Both the phase telescope and Bertrand lens must be equipped with a mechanism for adjustment of focus, because the location of the objective rear focal plane can vary with magnification 

Focusing is accomplished simply by twisting the eye tube until the objective rear focal plane is sharply focused.

PHASE CONTRAST MICROSCOPE ALIGNMENT ensure that all objectives contain phase plates and are firmly seated

in the nosepiece.

The objectives and annular plates should also be sequentially ordered in their arrangement from lower to higher magnification

Shown below is the phase contrast kit used on the National Optical 160 series microscopes.  

 It consists of 4 objective lenses, a centering telescope and Zernike phase condenser lens.

The long adjustment screws on the phase condenser push in to engage set screws for proper alignment of the phase ring.

There is a thumb wheel on the opposite side used to dial in the proper setting to match the power of the objective lens. 

There is also a "BF" setting on the thumb wheel for brightfield.  This allows you to use the phase objectives as standard brightfield lenses.

Not all phase contrast microscopes are the same but generally they rely on similar techniques to set up the system for optimum results.’

 In the system shown, the phase condenser has five settings that you spin with your thumb ( 10X, 20X, 40X, 100X and BF)  BF is "brightfield", no phase.  

To set up your microscope for phase optics, you first set it at BF and focus on the specimen. 

 Adjust the height of the condenser for optimum image quality.

 Next, set the condenser turret to the phase setting for that particular lens and remove the specimen.

The controls that stick out from both sides on the back are for centering the condenser.

Next, you remove one of the eyepiece lenses and insert the centering telescope in its place.

 The set screw is used to focus the centering telescope. 

 When looking through the telescope, you will see two rings.  

They may or may not be concentric.  By turning centering adjustment screws on the condenser, you align the rings so that they become concentric.  

Once the microscope has been aligned for phase contrast, it will generally hold its centration for a considerable number of objective/annuli changes, but should be checked periodically to ensure proper alignment.

If the microscope starts to slip out of alignment, the images appearing in the eyepieces (or on a computer monitor) will appear increasingly more like those observed with brightfield illumination.

Most of the microscope manufacturers provide a green interference or absorption filter with their auxiliary phase contrast kits to produce monochromatic light having the same wavelength used for the original calibration of the objective phase plates.

As a result, contrast is increased when the filter is inserted into the optical pathway

 A majority of the commercial phase plates are designed to produce phase shifts of a quarter wavelength in the green (550 nanometers) portion of the visible light spectrum.

Theoretically, if white light is utilized instead of monochromatic light, extinction by interference will not be complete for all colors, and contrast will suffer. 

 This limitation is particularly important if achromat objectives, which are corrected for chromatic aberration only in the green region, are utilized for phase contrast observation or image recording.

 However, with the current highly corrected fluorite and apochromatic objectives, the difference in contrast often is negligible, and therefore, insignificant.

How to get good images..?

The key to successful imaging with phase contrast illumination is to properly align the microscope, and to ensure that sufficiently thin specimens are spread evenly within the mounting medium on the microscope slide.

Images made with exceedingly thick specimens often suffer from out-of-focus blur and contrast inversion artifacts that can be difficult to interpret.

If the microscope is utilized for an extended period of time, occasionally check the objective rear focal plane to verify alignment of the condenser annulus with the phase plate in the objective.

INTERPRETATION OF PHASE CONTRAST IMAGES Images produced by phase contrast microscopy are relatively

simple to interpret when the specimen is thin and distributed evenly on the substrate

When using positive phase contrast optics, which is the traditional, images appear darker than the surrounding medium when the refractive index of the specimen exceeds that of the medium.

 Phase contrast optics differentially enhance the contrast near the edges

image density can be utilized as a gauge for approximating relationships between various structures.

 In effect, a series of internal cellular organelles having increasing density, such as vacuoles, cytoplasm, the interphase nucleus, and the nucleolus (or mitotic chromosomes), are typically visualized as progressively darker objects relative to a fixed reference, such as the background.

In order to avoid confusion regarding bright and dark contrast in phase contrast images, the optical path differences occurring within the specimen preparation should be carefully considered

 As discussed above, the optical path difference is derived from the product of the refractive index and the specimen (object) thickness, and is related to the relative phase shift between the specimen and background waves.

For example, a small specimen having a high refractive index can display an identical optical path difference to a larger specimen having a lower refractive index.

The external medium can also be replaced with another having either a higher or lower refractive index to generate changes in specimen image contrast. In fact, the effect on image contrast of refractive index variations in the surrounding medium forms the basis of the technique known as immersion refractometry.

Two very common effects in phase contrast images are the characteristic halo and shade-off contrast patterns in which the observed intensity does not directly correspond to the optical path difference

 In all forms of positive phase contrast, bright phase halos usually surround the boundaries between large specimen features and the medium. Identical halos appear darker than the specimen with negative phase contrast optical systems

Halos occur in phase contrast microscopy because the circular phase-retarding (and neutral density) ring located in the objective phase plate also transmits a small degree of diffracted light from the specimen.

problem is compounded by the fact that the width of the zeroth-order surround wavefront projected onto the phase plate by the condenser annulus is smaller than the actual width of the phase plate ring

The halo effect can also be significantly reduced by utilizing specially designed phase objectives that contain a small ring of neutral density material surrounding the central phase ring material near the objective rear aperture. These objectives are termed apodized phase contrast objective

In practice, halo reduction and an increase in specimen contrast with apodized optical systems can be achieved by the utilization of selective amplitude filters

These amplitude filters consist of neutral density thin films applied to the phase plate surrounding the phase film.

Transmittance increase from 25 to 50 % with their use.

Shade-off is another very common optical artifact in phase contrast microscopy, and is often most easily observed in large, extended phase specimens

The intensity profile of a large, uniformly thick positive phase contrast specimen often gradually increases from the edges to the center, where the light intensity in the central region can approach that of the surrounding medium

This effect is termed shade-off, and is frequently observed when examining extended planar specimens, such as material slabs (glass or mica), replicas, flattened tissue culture cells, and large organelles.

The shade-off phenomenon is also commonly termed the zone-of-action effect, because central zones having uniform thickness in the specimen diffract light differently than the highly refractive zones at edges and boundaries.

Halo and shade-off artifacts depend on both the geometrical and optical properties of the phase plate and the specimen being examined.

 Wider phase plates having reduced transmittance tend to produce higher intensity halos and shade-off, whereas the ring diameter has a smaller influence on these effects

In addition, these effects are heavily influenced by the objective magnification, with lower magnifications producing better images.

ADVANTAGES The capacity to observe living cells and, as such, the ability to examine

cells in a natural state, study bilological processes.

Observing a living organism in its natural state and/or environment can provide far more information than specimens that need to be killed, fixed or stain to view under a microscope

High-contrast, high-resolution images

Ideal for studying and interpreting thin specimens

Ability to combine with other means of observation, such as fluorescence

Modern phase contrast microscopes, with CCD or CMOS computer devices, can capture photo and/or video images

In addition, advances to the phase contrast microscope, especially those that incorporate technology, enable a scientist to hone in on minute internal structures of a particle and can even detect a mere small number of protein molecules.

DISADVANTAGES

Annuli or rings limit the aperture to some extent, which decreases resolution

This method of observation is not ideal for thick organisms or particles

Images may appear grey or green, if white or green lights are used, respectively, resulting in poor photomicrograph

Shade-off and halo effect, referred to a phase artifacts

Shade-off occurs with larger particles, results in a steady reduction of contrast moving from the center of the object toward its edges

Halo effect, where images are often surrounded by bright areas, which obscure details along the perimeter of the specimen

CONCLUSION

The phase contrast microscope opened up an entire world of microscopy, providing incredible definition and clarity of particles never seen before.

These transparent specimens could not be explored because they do not have the capacity to absorb light.

Zernike found a way to manipulate light paths through the use of strategically placed rings and his system is a staple of most modern microscopes.

Although there are a few disadvantages, such as shade-off and halo distortions, phase contrast provides highly detailed, well-contrasted images

The most important breakthrough is the ability to observe living particles in a natural state.