Phase contrast microscope

D R R AV I K U M A R V
D E P T O F O R A L PAT H O L O G Y
& MICROBIOLOGY
G D C , K O T TAYA 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

Air Glass Water Air
R.I.=1.0 R.I.=1.5 R.I.=1.33 R.I.=1.0

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

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
Tutorial
We can see amplitude (intensity) differences

•Illumination bypasses
Specimen > no phase shift

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

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.

 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.

Phase contrast microscope

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