PHASE CONTRAST

MICROSCOPY
MS. SHABABANO SIDDIQUE
ASSISTANT PROFESSOR
DEPARTMENT OF PHARMACEUTICAL
CHEMISTRY
SSR COLLEGE OF PHARMACY

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Introduction
■ The microscope is defined as an instrument used for seeing small
objects.
■ The human eye see only because of property of the light entering the
eye from objects seen.
■ The eye has the ability to recognize only the difference in brightness
and difference in color.
■ Difference in brightness of different objects or their component parts
give rise to brightness contract and difference in color causes color
contrast.
■ Brightness contract involve amplitude and color contract involve
wavelength

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■ Improvement in microscope can be done in two general ways.
■ First, there has been improvement of the instrument itself in order to
make it a better and more convenient image forming apparatus.
■ Second approach is to consider the specimen as an essential part of the
optical system and as it were to build microscope around the specimen.
■ Until the invention of the phase microscope no very useful means were
available for observing the difference 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 microscope specimen depends upon the specimens
physical and chemical properties.

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■ In optics, the refractive index (also known as refraction
index or index of refraction) of a material is a dimensionless
number that describes how fast light travels through the
material. It is defined as

■ where c is the speed of light in vacuum and v is the phase

velocity of light in the medium.
■ Increasing refractive index corresponds to decreasing
speed of light in the material.
■ The refractive index determines how much the path of light
is bent, or refracted, when entering a material. This is
described by Snell's law of refraction, n1 sinθ1 = n2 sinθ2,
where θ1 and θ2 are the angles of incidence and refraction,
respectively, of a ray crossing the interface between two
media with refractive indices n1 and n2.

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PHASE CONTRAST MICROSCOPY MAKES
THESE PHASE DIFFERENCE VISIBLE

■The speed and

wavelength of
the light
changes when it
passes through
media with
different
refractive
indices

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Introduction to Phase Contrast
Microscopy
■ Phase contrast microscopy, first described in 1934
by Dutch physicist Frits Zernike, is a contrast-
enhancing optical technique that can be utilized
to produce high-contrast images of transparent
specimens, such as living cells (usually in culture),
microorganisms, thin tissue slices, lithographic
patterns, fibers, latex dispersions, glass
fragments, and subcellular particles (including
nuclei and other organelles).

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■ Partially coherent illumination produced by the tungsten-halogen lamp
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.
■ Undeviated and diffracted light collected by the objective is
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.

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The Phase Contrast
Microscope
■ The most important concept
the segregation of surround
underlying the design of a phase contrast microscope is
and diffracted wavefronts emerging from the specimen,
which are projected onto different locations in the objective rear focal plane
(the diffraction plane at the objective rear aperture).
■ In addition, the amplitude of the surround (undeviated) light must be reduced and the
phase advanced or retarded (by a quarter wavelength) in order to maximize
differences in intensity between the specimen and background in the image plane.
■ The mechanism for generating relative phase retardation is a two-step process, with
the diffracted waves being retarded in phase by a quarter wavelength at the
specimen, while the surround waves are advanced (or retarded) in phase by a phase
plate positioned in or very near the objective rear focal plane.
■ Only 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, is placed in the condenser front focal plane.

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■ The condenser annulus is typically constructed as an opaque flat-
black (light absorbing) plate with a transparent annular ring, which is
positioned in the front focal plane (aperture) of the condenser 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 (where
a conjugate phase plate is positioned, as discussed below).
■ The condenser annulus either replaces or resides close to the
adjustable iris diaphragm in the front aperture of the condenser.
■ Whenever we are conducting phase contrast experiments utilizing a
condenser with both a phase annulus and an aperture diaphragm,
check to ensure that the iris diaphragm is opened wider than the
periphery of the phase annulus.
■ Phase contrast is also insensitive to polarization and birefringence
effects, which is a major advantage when examining living cells
growing in plastic tissue culture vessels.

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■ 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 diffraction plane).
■ Under these conditions, the objective rear focal plane is conjugate to the
condenser front aperture plane, so non-diffracted (zeroth order) light
waves form a bright image of the condenser annulus at the rear aperture
of the objective (superimposed over the image of the phase plate).
■ The spherical wavefront that is diffracted by the specimen (the D waves)
passes through the diffraction plane at various locations across the
entire objective rear aperture.
■ The distribution (amount and location) of diffracted light depends on the
number, size, and refractive index differential of light-scattering objects
in the specimen.
■ For most specimens, only a small portion of the incident light waves are
diffracted, and a majority of the light passes through undeviated to
eventually illuminate the entire image plane.

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■ In contrast, the surround planar wavefront occupies a smaller
proportion of the objective rear aperture, which corresponds to the
conjugate of the condenser annulus.
■ Thus, the two wavefronts do not overlap to a significant extent, and
occupy distinct portions of the objective rear focal plane.
■ Because the direct, zeroth order light and diffracted light are spatially
separated in the diffraction plane, the phase of either wave
component (surround, S or diffracted, D) can be selectively
manipulated without interfering with the other.

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■ In general, specimens having a higher refractive index than the
surrounding medium appear dark on a neutral gray background, while
those specimens that have a lower refractive index than the bathing
medium appear brighter than the gray background.
■ This is not always the case, however, because specialized phase
contrast objectives having higher neutral densities coupled to lower
retardation values (one-eight of a wavelength or less) can produce
contrast inversion in thicker specimens.
■ The net result is that regions with very high optical path differences
begin to appear bright.

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■ In order to modify the phase and amplitude of the spatially separated
surround and diffracted wavefronts in phase contrast optical systems, a
number of phase plate configurations have been introduced.
■ Because the phase plate is positioned in or very near the objective rear
focal plane (the diffraction plane) all light passing through the
microscope must travel through this component.
■ The portion of the phase plate upon which the condenser annulus is
focused is termed the conjugate area, while the remaining regions are
collectively referred to as the complementary area.
■ The conjugate area contains the material responsible for altering the
phase of the surround (undiffracted) light by either plus or minus 90-
degrees with respect to that of the diffracted wavefronts.
■ In general, the phase plate conjugate area is wider (by about 25 percent)
than the region defined by the image of the condenser annulus in order
to minimize the amount of surround light that spreads into the
complementary area.

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Applications of Phase
contrast Microscopy
■ To produce high-contrast images of transparent specimens, such as
– living cells (usually in culture),
– microorganisms,
– thin tissue slices,
– lithographic patterns,
– fibers,
– latex dispersions,
– glass fragments, and
– subcellular particles (including nuclei and other organelles).
■ Applications of phase-contrast microscopy in biological research are
numerous.

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Thank you

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