Materials Characterization

MT21005

Various Modes of Optical Microscopy

Topics Covered

 Reflected and transmitted light Microscopy

 Bright field Illumination
 Dark field Illumination
 Phase contrast Microscopy
 Polarised light Microscopy
 Differential Interference Microscopy
 Fluorescence Microscopy
Anatomy of a
modern OM
 Reflected light microscopy
• Reflected light microscopy is often referred to as
incident light, epi-illumination, or metallurgical
microscopy.
• The optical pathway for reflected light begins with
illuminating rays originating in the lamp housing
for reflected light.
• This light next passes through the collector lens
and into the vertical illuminator where it is
controlled by the aperture and field diaphragms.
• After passing through the vertical illuminator, the
light is then reflected by a beam-splitter (a half
mirror or elliptically shaped first-surface mirror) • In kohler illumination, the collector lenses and
through the objective to illuminate the specimen. various type of field diaphragm are present.
• Light reflected from the surface of the specimen • Collector lens basically forms a magnified image of
re- enters the objective and passes into the the filament which is now used as a virtual source.
binocular head where it is directed either to the • The purpose of all of these different lenses is to
eyepieces or to a port for photomicrography. make the light the way you want whether a parallel
ray of light or not.
• Bright field is the method of choice for imaging
specimens that remain opaque even when
ground to a thickness of 30 microns.
• The range of specimens falling into this
category includes most metals, ores, ceramics,
many polymers, semiconductors (unprocessed
silicon, wafers, and integrated circuits), slag,
coal, plastics, paint, paper, wood, leather, glass
inclusions, and a wide variety of specialized
materials.
• Because light is unable to pass through these
specimens, it must be directed onto the surface
and eventually returned to the microscope
objective by either specular or diffused
reflection.
 Transmitted light Microscopy
• Transmitted light microscopy is associated with
the light passing from the source to the opposite
side of the lens.
• This method is used to distinguish the
morphological characteristics and optical
proprieties of the observed material.
• The light rays are organized and conducted
through the instrument so the light can be
polarized and redirected.
• The light from the condenser will fill the plane of
the objective and a ray of light will be projected
to lighten the field.
• The condenser is able to control the angle of the
illumination which permits the right balance of
resolution and contrast in the microscope.
• The light passes through the sample and it will go
to the objective where the image will be
magnified.
 Bright-field Illumination
• Bright-field illumination relies upon changes in light absorption, refractive
index, or color for generating contrast.
• As light passes through the specimen, regions that alter the direction, speed,
and/or spectrum of the wavefronts generate optical disparities (contrast) when
the rays are gathered and focused by the objective.
• Resolution in a bright-field system depends on both the objective and
condenser numerical apertures, and an immersion medium is required on both
sides of the specimen (for numerical aperture combinations exceeding a value
of 2.3).
• Digital cameras provide the wide dynamic range and spatial resolution required
to capture the information present in a brightfield image.
• Background subtraction algorithms, using averaged frames taken with no
specimen in the optical path, increases contrast dramatically.
• Angle of illumination defines the type of image formation. Direct beam is used
in bright-field image formation.
• Till now whatever image formation in optical microscope we saw is brightfield
image.
 Enhancing contrast in optical microscope
• In transmitted light microscopy, the specimen quality does not always
lend itself to easy observation and image recording with excellent
contrast as in simple bright-field imaging mode.
• Investigations dealing with inherently low-contrast specimens
(unstained bacteria, thin tissue slices, and adherent live cells), rely on
specialized contrast-enhancing techniques for imaging of virtually
transparent samples.
• In the course of examining unstained specimens, poor light
absorption by the specimen results in extremely small variations in
the intensity distribution difference between the specimen and the
background.
• When the background is bright, the human eye requires local
intensity fluctuations of at least 10-20% to be able to recognize • Generally, image from the dark-field
specimen details. imaging mode is suffered from poor
(𝐼 𝑠 − 𝐼(𝑏) × 100) contrast.
• Contrast (%) 𝐶=
𝐼(𝑏) • Therefore, it is important to have
small background intensity to
where 𝑰(𝒃) is the intensity of the background and 𝑰(𝒔) is the specimen achieve better contrast in dark field
intensity. imaging mode.
 Dark-field microscopy
• Darkfield microscopy is a specialized illumination technique
that capitalizes on oblique illumination to enhance contrast
in specimens that are not imaged well under normal
brightfield illumination conditions.
• The condenser directs a cone of light onto the specimen at
high azimuths.
• Light passing through the specimen is diffracted, reflected,
and/or refracted by optical discontinuities enabling these
faint rays to enter the objective.
• Zeroth-order wavefronts do not directly enter the objective • Only first order beam (diffracted beam) is
front lens element. passes through the objective lense due to
oblique illumination.
• First order beam is forming the background and
other higher order beam interfere with it and
gives contrast. So total background intensity
goes down and contrast enhances.
• Only by using light stop, it changes from bright
field to dark field imaging mode. No any other
difference.
 Example of contrast enhancement in DF
• The specimen can be visualized as a
bright object on an otherwise black
background.
• Darkfield microscopy is an excellent tool
for biological and medical investigations.
It can be effectively used at high
magnifications to image living bacteria,
or at low magnifications to view and
image cells, tissues, and whole mounts.
• Recently, a renewed interest in
transmitted darkfield microscopy has
arisen due to its advantages when used
in combination with fluorescence
microscopy. OM images of the green algae

• In dark field, the direct beam is completely avoided which removes the complete background.
Therefore, contrast is enhanced much better.
Phase Contrast Microscopy
 Phase Contrast Microscopy
• First described by the Frits Zernike in 1934, phase Human Blood cell
contrast earned its discoverer the Nobel Prize for
physics in 1953 while revolutionizing basic
biomedical research on living cells.
• Phase contrast employs an optical mechanism to
translate minute variations in phase into
corresponding changes in amplitude, which can be
visualized as differences in image contrast.
• The technique is ideal for thin unstained specimens
(such as culture cells on glass), which are
approximately 5 to 10 μm thick above the nucleus,
but less than 1 μm thick at the periphery. • Mostly this microscopy is done in transmitted
• Such specimens barely exhibit any light absorption mode with direct beam.
in the visible portion of the spectrum and the • Such a thin specimen can not produce the
human eye cannot detect them in bright-field and better contrast either in bright or dark field
dark-field illumination. mode imaging.
• Very small differences exist between the refractive
indices of the cells and their surrounding aqueous
solutions and within the cells between the cytoplasm
and the cell nucleus.
• Phase contrast makes these tiny differences in
refractive indices visible by the use of optical
manipulation; for example, it translates them into
differences in intensity that can be visually observed
and recorded.
• The optical effect employed in this case consists of a
shift of phase relationships between light wavefronts
traveling through different portions of the specimen.
• The higher the refractive index of a medium, the smaller
the speed or velocity of light in the medium.
• During their journey through cell nuclei, cytoplasm, or • Due to difference in thickness and
water, the light waves are shifted (retarded) by small refractive index, the speed of the light is
degrees, since these media have slightly different different and this difference in speed of
refractive indices. light produces shift in the phase.
• Before their entry into the specimen, the waves
are still in phase (wavefronts beneath the
coverslip), but this is no longer the case when they
have passed through the various materials having
different refractive indices.
• A light wave that has passed through a cell nucleus
lags behind the light waves that only had to pass
through water. The amount of lag is called a phase
shift.
• The amount of the phase shift depends on what
media (refractive index) the waves have passed
through on their paths, and how long the paths
were through these media.
• The human eye cannot see these phase shifts in
the microscope image, it is only able to distinguish • Therefore, we have to finally transform this
between different intensities and colors. phase shift into amplitude/intensity
difference.
• Therefore, the phase contrast technique uses optical
tricks to translate phase shifts into grey values.
• All of the wavefronts are ultimately recombined to form
the intermediate image by the objective lens.
• The wavefronts that have all been retarded to varying
degrees by details in the specimen are superimposed in
the intermediate image plane where they amplify or
attenuate each other (depending on the phase), forming
the final phase contrast image.
• These interference processes in the intermediate image
create bright and dark areas of the various structures
having refractive index (and/or thickness) variations in
the specimen.
 Example of phase contrast image
• Background • Depending
intensity is upon phase
very high shift of
due to direct different
light, result region, the
in bright amplitude of
image but direct beam
with poor is getting
contrast. modified.
That’s why
the contrast
is enhanced.

• This is not the problem of resolution; it is the problem of contrast.

• Other kind of features also visible within single cell. So not only you are enhancing the
contrast between background and the cells but contrast within cells is also enhanced. That is
why phase contrast microscopy works so good for this kind of biological system.
 Requirement for phase contrast OM
• The microscope must be equipped with a specialized condenser containing an annulus or a series of annuli
matched to a set of objectives containing phase rings (designed as Ph) in the rear focal plane.
• The technique is not useful for thick specimens (such as plant and animal tissue sections) because shifts in
phase occur in regions removed from the focal plane that distort image detail.
• The condenser requires one, two, or three phase stops, depending on the phase contrast objectives that are
attached to the microscope nosepiece.
• The required ring diameter increases with the numerical aperture (high apertures require the maximum
diameter (Ph3), for example 0.9 in air or 1.3 with oil immersion).

• An annular ring at the

condenser side to produces
the light rays with the same
phase.
• A phase ring at objective
side to recombine all the
beams which are coming
with different different
phases.
 Halo and Shade-Off artefacts in phase contrast OM
• Phase contrast is an excellent method to
increase contrast when viewing or imaging
living cells in culture, but typically results in
halos surrounding the outlines of edge
features.
• Halos arise because of the configurational
parameters of the phase contrast microscope
optical system.
• 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.
• The presence and severity of halos in phase
contrast is dependent on the refractive index
difference between the specimen and the
surrounding medium.
Polarized light microscopy
 Polarized light microscopy
• Polarized light microscopy is a contrast-enhancing
technique that dramatically improves the quality
of an image acquired with birefringent materials
when compared to other techniques.
• To observe specimens that are visible primarily
due to their optically anisotropic character
(birefringent).
• The microscope must be equipped with a
polarizer, positioned in the light path somewhere
before the specimen, and an analyzer (a second
polarizer), placed in the optical pathway between
the objective rear aperture and the observation
tubes or camera port.

• Polarized light microscopes possess a high degree of sensitivity and can be used for both qualitative and
quantitative studies for a wide range of anisotropic specimens.
• Qualitative polarizing microscopy is primarily used by mineralogists, geologists, and chemists.
 Polarization of light
• Polarized light is most commonly produced by
absorption of light having a set of specific vibration
directions in a dichroic medium.
• Light is an electromagnetic wave, it has electric field
vectors. When the electric field vectors of light are
restricted to a single plane by filtration, then the
light is said to be polarized with respect to the
direction of propagation and all waves vibrate in the
same plane.
• Linearly polarized light waves are generated by
polarizers that filter out a privileged plane from the
statistical confusion of vibrational directions
prevailing in natural light.
• When illuminated, many specimens turn the vibration direction of the polarized light out of the plane
produced by the polarizer.
• Materials having birefringent properties rotate the plane of light polarization, appearing bright on a dark
background.
 Application of polarizer
• The hazard of driving, or performing other daily activities, with a large amount of glare in one's eyes has
resulted in the development of polarized sunglasses.
• The lenses of such sunglasses contain polarizing filters that are oriented vertically with respect to the frames.
• The electric field vectors of the blue light waves are oriented in the same direction as the polarizing lenses
and, therefore, are passed through.
• In contrast,the red light wave represents glare, which is parallel to the surface of the highway.
• Since the red wave is perpendicular to the filters in the lenses, it is successfully blocked by the lenses.
 Working principle
• Polarized light microscopy is conducted by viewing the specimen between crossed polarizing elements
inserted into the optical train before the condenser and after the objective.

• There are two polarizing

elements in a polarizing
microscope. These elements
can be rotated through 360o.
• The first polarizer is placed beneath the
specimen stage.

• Another polarizer (analyzer) is placed above

the objective and can be moved in and out
of the light path as needed.

• When both the polarizer and analyzer are

inserted into the optical path, their vibration
azimuths are positioned at right angles to each
other.

• In this configuration, the polarizer and analyzer

are crossed, with no light passing through
the optical system and a dark background
present in the eyepieces.
 Material requirement
• Polarized light microscopy is capable of providing information on absorption
color and optical path differences between materials of differing refractive Light refraction by water

indices, in a manner similar to brightfield illumination, but the technique can

also distinguish between isotropic and anisotropic substances.
• Isotropic materials, which include a variety of gases, liquids, unstressed glasses
and cubic crystals, demonstrate the same optical properties when probed in all
directions. These materials have only one refractive index and no restriction on
the vibration direction of light passing through them.
• In contrast, anisotropic materials, which include 90% percent of all solid
substances, have optical properties that vary with the orientation of incident
light with the crystallographic axes.
• They demonstrate a range of refractive indices depending both on the
propagation direction of light through the substance and on the vibrational
plane coordinates. More importantly, anisotropic materials act as beam-
splitters and divide light rays into two orthogonal components.
• The technique of polarizing microscopy exploits the interference of the split light
rays, as they are re-united along the same optical path to extract information
about anisotropic materials.
 Polarizer

Birefringence White light

Analyzer

Retardation
• Birefringence is optical property of a material
having a refractive index that depends on the
polarization and propagation direction of light.
• The two orthogonal components of light (ordinary
Plane
and extraordinary waves) travel at different speeds
polarized light
through the specimen and, as a result, observe
Two components
different refractive indices, a phenomenon known resulting from
as birefringence. birefringence

• The faster wavefront emerges first from the

specimen to generate an optical path difference
with the slower wavefront.
• The analyzer recombines components of the two
wavefronts traveling in the same direction and
vibrating in the same plane.
• This polarizing element ensures that the two
wavefronts have the same amplitude at the time of
recombination for maximum contrast.
 Lambda plate
• Polarized light microscope optical train also include an auxiliary element known as lambda or retardation plate
that is used for quantitative analysis.
• In polarized light, this lambda plate converts contrast into colors.
• As in phase contrast, optical path differences give rise to colors, although this time with polarized light and
birefringent material in the specimen.
• The path differences leads to an extinction of certain wavelengths in the light through interference.
• Only certain colors remain from the white light and create beautiful, colored pictures.

Polarized light microscopy image of Aplite

 Phase identification by reflected POM
• Polarizing microscopy offers an immense amount of information about the composition and
three- dimensional structure of a range of samples.
• Valuable in manufacturing and research, polarizing microscopy is relatively inexpensive and an
accessible research and quality control tool that can provide information unavailable with any other
technique.

YBa2Cu307-x superconductor material: (a) Tetragonal phase and

(b) Orthorhombic phase with multiple twinning (100 X).
Differential Interference Contrast (DIC) Microscopy
 Differential Interference Contrast
• Differential interference contrast (DIC) microscopy,
also known as Nomarski interference contrast
(NIC) or Nomarski microscopy, is an optical
microscopy technique used to enhance the
contrast in unstained, transparent/opaque
materials.
• The technique was developed by Polish physicist
Georges Nomarski in 1952.
• DIC works on the principle of interferometry to gain
information about the optical path length of the
material, to see otherwise invisible features.
• Images so produced using DIC have a pseudo 3D-
effect.

• Optical path difference is determined by product of refractive index difference (between the specimen
and its surrounding medium) and the thickness (geometrical distance) traversed by a light beam between
two points on the optical path.
 Differential Interference Contrast
• DIC introduces contrast to images of specimens which
have little or no contrast when viewed using brightfield
microscopy.
• DIC requires plane-polarized light and additional light-
shearing (Nomarski) prisms to exaggerate minute
differences in specimen thickness gradients and
refractive index.
• Lipid bilayers, for example, produce excellent contrast
in DIC because of the difference in refractive index
between aqueous and lipid phases of the cell.
• This image is similar to that obtained by phase
contrast microscopy but without the bright diffraction
halo.

Biological specimen imaged in transmitted DIC

microscopy
• DIC works by separating a polarized light source into two orthogonally polarized mutually coherent parts
which are spatially displaced (sheared) at the sample plane, and recombined before observation.
• The interference of the two parts at recombination is sensitive to their optical path difference (i.e. the
product of refractive index and geometric path length).
• Polarized light is required for the technique: Unpolarised light enters the microscope and is polarized at 45°.
• The polarized light enters the first Nomarski-modified Wollaston prism and is separated into two rays
polarized at 90° to each other, the sampling and reference rays.
• The two rays are focused by the condenser for passage through the sample. These two rays are focused
so they will pass through two adjacent points in the sample, ~0.2 μm apart.
• The sample is effectively illuminated by two coherent light sources, one with 0° polarisation and the
other with 90° polarisation.
• The rays travel through adjacent areas of the sample; the separation is normally of the order of the
resolution of the microscope.
• They will experience different optical path lengths where the areas differ in refractive index or thickness.
This causes a change in phase of one ray relative to the other due to the delay experienced by the wave
in the more optically dense material.
• The passage of many pairs of rays through pairs of adjacent points in the sample (and their absorbance,
refraction and scattering by the sample) means an image of the sample will now be carried by both the
0° and 90° polarised light.
• These, if looked at individually, would be bright field images of the sample, slightly offset from each other.
• The light also carries information about the image invisible to the human eye, the phase of the light.
• The rays travel through the objective lens are focused for the second Nomarski- modified Wollaston prism.
• The second prism recombines the two rays into one polarised at 135°.
• The combination of the rays leads to interference, brightening or darkening the image at that point according
to the optical path difference.
• This prism overlays the two bright field images and aligns their polarisations so they can interfere.
• However, the images do not quite line up because of the offset in illumination.
Image formation in DIC mode

• Image is generated from two identical bright field images

being overlaid slightly offset from each other (typically ~0.2
μm), and the subsequent interference due to phase
difference converting changes in phase (and so optical path
length) to a visible change in darkness.
 Depth perception in DIC mode
Wollaston Prism Orientation Wollaston Prism Orientation Wollaston Prism Orientation

Dark

Light

• The DIC image has the appearance of a three-dimensional object under very oblique illumination,
causing strong light and dark shadows on the corresponding faces.
• The direction of apparent illumination is defined by the orientation of the Wollaston prisms.
• This interference may be either constructive or destructive, giving rise to the characteristic
appearance of three dimensions.
 Comparison between polarized light and DIC modes

Ti-6Al-4V alloy with tint etching: slightly uncrossed Ti-6Al-4V alloy with tint etching: DIC mode
(45–129) polarized light
 Comparison between phase contrast and DIC modes
• The halo and shade-off artifacts that plague the
phase contrast optical system are largely absent in
differential interference contrast images.
• In positive phase contrast, specimen features with a
higher refractive index than the surrounding medium
are encircled with a bright fringe (halo) that often
obscures edge detail.
• Although halos can be suppressed to a limited
degree by configurational modifications to the
objective phase plate design, they cannot be
eliminated entirely.
• In some cases, differential interference contrast
images suffer from excessive brightness along edges
having very steep optical gradients, but this effect
usually occurs when bias retardation values are too
low, and can be circumvented by proper adjustment
of the Nomarski prism position.
Fluorescence Microscopy
 Fluorescence
• Fluorescence is the emission of light by a substance that
has absorbed light or other electromagnetic radiation.
• One radiative mechanism by which excited electrons
may relax is a light-emitting transition from the lowest
excited state (S1) to ground state (S0) in a fast (10-9 to 10-
6 sec) process.

• The energy difference is dissipated by emitting a

photon.
• Similar to excitation, emission may generally relax to a
variety of vibrational levels of the ground state (S0),
resulting in a bandwidth of possible wavelengths of
the emitted photon. Willemite in natural and UV light
• Since the electron shed some of the original
excitation energy by vibrational relaxation, the
emitted photon will be of lower energy and thus of
longer wavelength.
ℎ. 𝑐
𝐸 = ℎ. 𝑓 =
λ
 Phosphorescence
• Unlike fluorescence, a phosphorescent material does
not immediately re-emit the radiation it absorbs.
• The slower time scales of the re-emission are
associated with "forbidden" energy state transitions in
quantum mechanics.
• As these transitions occur very slowly in certain
materials, absorbed radiation is re-emitted at a lower
intensity for up to several hours after the original
excitation.
• Phosphorescent material can store the absorbed light
energy for some time and release light later, resulting in
an afterglow that persists after the light has been
switched off.
• Depending on the material, this afterglow can last
anywhere from a few seconds to hours.
 Fluorescence vs. Phosphorescence
• Both fluorescence and phosphorescence are based on the ability of a substance to absorb light and emit light
of a longer wavelength and therefore lower energy. The main difference is the time in which it takes to do so.
• In fluorescence, the emission is basically immediate and therefore generally only visible, if the light source is
continuously on (such as UV lights).
• More energy is dissipated by non-radiative processes during phosphorescent relaxation than in fluorescence.
• The energy difference between the absorbed and emitted photon is bigger and the wavelength shift more
pronounced.
 Principle of fluorescence
microscopy
• The fluorescence process is governed by three
important events, all of which occur on timescales
that are separated by several orders of magnitude.
• Excitation of a susceptible molecule by an incoming
photon happens in femtoseconds (10-15 seconds),
while vibrational relaxation of excited state
electrons to the lowest energy level is much slower
and can be measured in picoseconds (10-12 seconds).
• The final process, emission of a longer wavelength
photon and return of the molecule to the ground
state, occurs in the relatively long time period of
nanoseconds (10-9 seconds).
• A fluorescence microscope uses fluorescence and
phosphorescence instead of, or in addition to,
scattering, reflection, and attenuation or absorption.
 Components of fluorescence microscope
• The specimen is illuminated with light of a specific
wavelength (or wavelengths) which is absorbed by the
fluorophores, causing them to emit light of longer
wavelengths (i.e., of a different color than the absorbed light).
• Illumination light is separated from the much weaker emitted
fluorescence through the use of a spectral emission filter.
• Typical components of a fluorescence microscope
– Light source (Xenon arc lamp or mercury-vapor lamp
are common; more advanced forms are high-
power LEDs and lasers)
– Excitation filter
– Dichroic mirror (or dichroic beam-splitter),
– Emission filter
• Because of the tremendously sensitive emission profiles,
spatial resolution, and high specificity of fluorescence
investigations, the technique is an important tool in genetics
and cell biology.
• The filters and the dichroic beam-splitter are chosen to match the spectral excitation and
emission characteristics of the fluorophore used to label the specimen.
• The distribution of a single fluorophore (color) is imaged at a time. Multi-color images of several
types of fluorophores must be composed by combining several single-color images.
• Because only a narrow bandwidth of light is reflected by the dichromatic mirror, illumination
wavelengths shorter than 445nm and longer than 515nm that manage to pass through the excitation
filter are also transmitted through the dichromatic mirror.
• Not all of the light having wavelengths above 510 or below 450 nm is transmitted through the mirror. A
small percentage of this light is reflected by the mirror through the objective and onto the specimen.
How fluorescent microscope works?
• Distribution of single fluorophore (color) is imaged at
a time.
• Multi-color images of several types of fluorophores
must be composed by combining several single-
color images.
• The images are captured sequentially using a digital
CCD camera, then overlaid to give a complete
image.
• Fluorescent imaging of the three components in a
dividing human cancerous cell.
• DNA is stained blue, a protein called INCENP is
green, and the microtubules are red.
• Each fluorophore is imaged separately using a
different combination of excitation and emission
filters.
 Specimen preparation
• In order for a the specimen to be suitable for fluorescence microscopy, it must
be fluorescent.
• There are several methods of creating a fluorescent sample; the main
techniques are labelling with fluorescent stains or, in the case of biological
samples, expression of a fluorescent protein.
• In the life sciences, fluorescence microscopy is a powerful tool which allows
the specific and sensitive staining of a specimen in order to detect the
distribution of proteins or other molecules of interest.
• There is a diverse range of techniques for fluorescent staining of biological
samples. Some of these are small molecules which are intrinsically fluorescent
and bind a biological molecule of interest.
• Others are drugs, toxins, or peptides which bind specific cellular structures and
have been derivatised with a fluorescent reporter.
• A sample of herring sperm stained with SYBR green in a cuvette illuminated by
blue light in an epifluorescence microscope.
• The SYBR green in the sample binds to the herring sperm DNA and, once
bound, fluoresces giving off green light when illuminated by blue light.
 Examples of fluorescent microscopy images

Yeast cell membrane visualized by some

Endothelial cells: Nuclei are stained blue I,
membrane proteins fused with fluorescent
microtubules are marked green and actin
markers. Imposition of light from both of
filaments are labeled red
markers results in yellow color.
Bright field illumination, sample Dark field illumination, sample Phase contrast illumination, sample
contrast comes from absorbance of contrast comes from light scattered contrast comes from interference of
light in the sample. by the sample. different path lengths of light
through the sample.

Cross-polarized light illumination,

sample contrast comes from rotation
of polarized light through the sample.