Sample Preparation Techniques in Light

Microscopy | Microbiology
One of the most attractive features of light microscopy is the ease with which most
specimens can be prepared for examination. Preparation often involves nothing
more than mounting a small piece of the specimen in a suitable liquid on a glass
slide and covering it with a glass coverslip.

The slide is then positioned on the specimen stage of the microscope and examined
through the ocular lens, or with a camera. Magnification can be changed simply by
rotating a turret that holds different objective lenses.

Objective lenses capable of high magnification (40 x and greater) generally require
the application of a small drop of immersion oil to the coverslip for optimal viewing.
Specimens that are pathogenic or potentially pathogenic are often killed chemically
prior to viewing by the application of a small amount of fixative (such as an aqueous
buffered aldehyde solution) to the specimen suspension.

Technique # 1. Specimen Processing – Fixation:

Often the first step in preparing the specimen is primary fixation, generally in a
buffered aldehyde fixative. Fixation kills the cells, stabilizes their chemical
components, and hardens the specimen in anticipation of further processing and
sectioning. One way to fix a specimen is simply to immerse it in the fixative solution.

An alternative approach for animal tissues is to pass the fixative through the blood
stream of the animal before removing the organs. This technique, called perfusion,
may help reduce artifacts, false or inaccurate representations of the specimen that
result from chemical treatment or handling of the cells or tissues.
Technique # 2. Embedding and Sectioning:
In most cases, the next step is to prepare thin sections of the fixed specimen. For this
purpose, the specimen is embedded in a medium that will hold it rigidly in position
while sections are cut. The usual choice of embedding medium is paraffin wax. Since
paraffin is insoluble in water, any water in the specimen must first be removed (by
dehydration in alcohol, usually) and replaced by an organic solvent such as xylene,
in which paraffin is soluble.

The processed tissue is then placed in warm, liquefied paraffin and allowed to
harden. Dehydration is less critical if the specimen is embedded in a water-soluble
medium instead of in paraffin. Specimens may also be embedded in epoxy plastic
resin.
Next, the embedded specimen is sliced into thin sections, usually a few (1-10)
micrometers thick. Sections are cut with a microtome, an instrument that operates
somewhat like a meat slicer.

The paraffin or plastic block containing the specimen is mounted on the arm of the
microtome, which advances the block by small increments toward a metal or glass
blade. As successive sections are cut, they usually adhere to one another forming a
ribbon of thin sections. These sections can be mounted on a glass slide, stained (if
desired), and protected with a coverslip.

Technique # 3. Staining:
The purpose of staining is to give distinctive colour characteristics to different kinds
of cellular components. The general approach is to mount the fixed tissue on a
microscope slide and then treat it with any of a variety of dyes and stains that have
been adapted for this purpose. Sometimes the tissue is treated with a single stain,
but more often a series of stains is used, each with an affinity for a different kind of
cellular component.

Technique # 4. Autoradiography:
Autoradiography is a technique that uses photographic film to determine where
within a cell a specific radioactively labeled compound is at the time the cell is fixed
and sectioned for microscopy. Autoradiography can therefore be used to localize
cellular processes to specific structures and to provide information about cell
function.

In essence, the process involves incubating cells, organisms, or tissue with a

radioactively labeled compound, fixing the specimen, sectioning it in the
conventional way, and mounting the fixed sample on a microscope slide.
The slide is then covered with a thin layer of photographic emulsion and placed in a
sealed box for the desired length of time, often for several days or even weeks.
During this time, the radioactivity in the sample exposes the emulsion directly above
it, providing a photographic record of precisely where in the cell the radioactive
compound is located.

Thereafter, the slide containing the specimen and the emulsion is developed in much
the same way as conventional black-and-white film. It is then ready for examination
under the microscope. Autoradiography can be applied to both light microscopy and
electron microscopy. In either case, it provides valuable information about the
localization of specific molecules, structures, or processes within the cell.

Sample Preparation for Fluorescence Microscopy

Fluorescence Microscopy allows specimens to be studied with high sensitivity and specificity through the use of
light. It works on the principle that energy emitted by certain types of materials can be detected as light, if irradiated
with the light of a specific wavelength. A fluorescent chemical compound called a fluorophore absorbs the light of
the specific wavelength that illuminates the specimen and emits light of longer wavelengths.

This emitted light is then separated from the original, brighter excitation light through a filter. In order for
this process to work the sample must be prepared so that it is fluorescent. Samples can be either live
cells or dead fixed cells that maintain their cellular structure, with cell preparation differing between the
two.

Preparing the sample with immunofluorescence

A technique used for samples of fixed cells is immunofluorescence which utilizes the specific binding
abilities of antibodies to its antigen. A specific antibody, called a primary antibody, will bind to the
particular protein being identified. There are two variants of immunofluorescence:

1. Direct immunofluorescence- (in which the primary antibody is directly bound to a fluorophore).
 2. Indirect immunofluorescence- (in which the primary antibody binds to a fluorescent secondary antibody,
which emits light and determines the location of the protein).

Direct immunofluorescence is faster than the indirect version of the method as the indirect washing and
incubation steps are not required. However, the indirect method provides more flexibility as the
fluorescent secondary antibody can be combined with different primary antibodies. Immunofluorescence
cannot be used in live-cell imaging and is restricted through the need to find antibodies that recognize the
protein of interest.

Fixation of the sample has to occur first so that the sample appears as close as possible to a living
sample with no loss of proteins, lipids or other molecules. Cross-linking fixation is a technique in which
aldehyde groups cross link molecules within the cell, though this can hinder antigen binding. Organic
solvents such as methanol or acetone can also be used to fix proteins to their cellular contexts. Though
the structure of the cell is maintained, some solvents can destroy the epitope of an antigen. The method
of fixation has to be specific to the antibody chosen, where a balance between good preservation and
binding ability is found.

Preparing the sample with fluorescent proteins - Green Fluorescent Proteins

For live cells, proteins can be genetically modified to carry a fluorescent protein reporter. Green
Fluorescent Proteins (GFPs) from the jellyfish Aequorea Victoria are used as a fluorescent tag to
visualize proteins within cells. Further engineering has developed several mutant Fluorescent Proteins
(FPs) that are bright, stable and chromatically diverse.

The spectral variety of these FPs means that the simultaneous visualization of several proteins in a cell
can occur. This technique has been applied to live cell imaging techniques. The GFP gene is
incorporated into the region of DNA that codes for the proteins being targeted. As this is controlled by the
same regulatory sequence, when the gene is expressed, GFP is produced at the same time as the
tagged proteins.
Preparing the sample with fluorescent proteins - self labeling enzymes

Another method of labeling proteins is through self-labeling enzymes called Halo tags or SNAP/CLIP
tags. The self-labelling enzymes can covalently attach a fluorescent ligand to its amino acid residue and
are a similar size to FPs. The proteins expressed are not innately fluorescent and only fluoresce when
exposed to the fluorescent ligand. In comparison to labelling with FPs, this technique has the advantage
of restricting the labeling for certain times and areas, allowing for sequential labeling. This technique has
been applied to pulse-chase experiments in which a cellular process is examined as it occurs over time.
Using self-labeling enzymes also allows protein populations to be tracked across a period of time.

Hoffman Modulation Contrast Basics

The Hoffman Modulation Contrast system is designed to increase visibility and contrast in unstained and living
material by detecting optical gradients (or slopes) and converting them into variations of light intensity. This
technique was invented by Dr. Robert Hoffman in 1975, and employs several accessories that have been
adapted to a number of commercial microscopes.
The basic microscope configuration for Hoffman Modulation Contrast is illustrated in Figure 1. An optical
amplitude spatial filter, termed a "modulator" by Hoffman, is inserted on the back focal plane of an achromat
or planachromat objective (although higher correction can also be used). Light intensity passing through this
system varies above and below an average value, which by definition, is then said to be modulated. Objectives
useful for modulation contrast can cover the entire magnification range of 10x to 100x. The modulator has
three zones that are depicted in Figure 2: a small, dark zone near the periphery of the back focal plane which
transmits only one percent of light (areas marked "D" in Figure 2); a narrow gray zone which transmits 15
percent (areas marked "G" in Figure 2); and the remaining clear or transparent zone, covering most of the
territory at the back of the objective, which transmits 100 percent of the light (areas marked "B" in Figure 2).
Unlike the phase plate in phase contrast microscopy, the Hoffman modulator is designed not to alter the phase
of light passing through any of the zones. When viewed under modulation contrast optics, transparent objects
that are essentially invisible in ordinary brightfield microscopy take on an apparent three-dimensional
appearance dictated by phase gradients. The modulator does not introduce changes in the phase relationship
of light passing through different portions of the modulator, but influences the principal zeroth order maxima.
Higher order diffraction maxima are unaffected. Measurements using a Michelson interferometer confirm that
the phase changes of light passed through a Hoffman-style modulator varies (if any) by a factor of less than
λ/20.
Below the stage, a condenser with rotating turret is utilized to hold the remaining components of the Hoffman
Modulation Contrast system. The turret condenser has a brightfield opening with an aperture iris diaphragm for
regular brightfield microscopy and for alignment and establishing proper conditions of Köhler illumination for
the microscope. At each of the other turret openings, there is an off-center slit that is partially covered with a
small rectangular polarizer. The size of the slit/polarizer combination is different for each objective of different
magnification; hence the need for a turret arrangement.

The Hoffman design is such that the slits are located at the front focal plane of the condenser as illustrated in
Figures 1 and 3. When light passes through the off-axis slit, it is imaged at the back focal plane of the objective
(also termed the Fourier plane) where the modulator has been installed. The front focal plane of the condenser
containing the off-axis slit plate is optically conjugate to the modulator in objective back focal plane. Image
intensity is proportional to the first derivative of the optical density in the specimen, and is controlled by the
zeroth order of the phase gradient diffraction pattern.
The principles of modulation contrast provide for at least two basic modulator-slit plate configurations that are
illustrated in Figures 2 and 3. Drawings of the modulator plates shown in Figure 2 are exaggerated and
increased in size for the purposes of this discussion. The arrangement on the left in Figures 2 and 3 (Figure
2(a) and Figure 3(a)) is a symmetrical system where both the modulator gray stripe and the slit are placed in
the optical axis (center) of the microscope. Resolution in this system is limited to:
 Resolution = λ/NA

where NA is the numerical aperture of the objective and λ equals the wavelength average of the imaging light
source. The dark (one percent transmittance) and light or transparent (100 percent transmittance) zones are
identical in size, while the gray (15 percent transmittance) zone is in the form of a narrow stripe that is 10
percent of the diameter of the exit pupil of the objective. The other arrangement (Figures 2 (b) and 3(b)) is
asymmetrical or offset, where the dark area of the modulator lies outside the exit pupil of the objective. The
resolution in this system is much improved and approaches:

Resolution = λ/2(NA)

where values for NA and λ are the same as described above. It is obvious that resolution in the offset system
(Figure 3(b)) is almost twice as good as in the central (Figure 3(a)) system. The transparent (clear) zone in the
offset system fills almost 90 percent of the diameter of the objective exit pupil with the gray and dark zones
filling the other 10 percent.

Below the condenser, a circular polarizer is placed on the light exit port of the microscope (note that both
polarizers are below the specimen). The rotation of this polarizer can control the effective width of the slit
opening. For example, a "crossing" of both polarizers at 90 degrees to each other results in "narrowing" the slit
so that its image falls within the gray area of the modulator, as illustrated in Figure 3. The part of the slit
controlled by the polarizer registers on the bright area of the modulator. As the polarizer is rotated, contrast can
be varied for best effect. A very narrow slit produces images that are very high in contrast with a moderate
degree of coherence. Optical section imaging is also optimized when the slit is adjusted to its narrowest
position. When the circular polarizer is oriented with its vibration direction parallel to that of the polarizer in the
slit, the effective slit width is at a maximum. This reduces overall image contrast and coherence, but yields
much better images of thicker objects where large differences in refractive index exist.

Early designs of the modulation contrast system did not utilize the slit polarizer or the circular polarizer on the
microscope light port, and relied on a single-sized slit as illustrated in Figure 4 for a symmetrical configuration.
In this figure, light from the source passes through a fixed-aperture slit (termed a "slit-plate" in the figure) and
then through a specimen containing phase gradients. These gradients deflect light, according to the direction of
the gradient, into either the clear or dark zones of the symmetrical modulator that is positioned in the rear focal
plane of the objective. The resulting image displays a simple contrast gradient that is dictated by the
positioning and slope of phase gradients in the sample.
In modern advanced modulation contrast systems, both the modulator and the slit are offset from the optical
axis of the microscope. This arrangement permits fuller use of the numerical aperture of the objective and
results in good resolution and details. Shapes and details are rendered in shadowed, pseudo three-
dimensional appearance. These appear brighter on one side, gray in the central portion, and darker on the
other side, against a gray background. The modulator converts optical phase gradients in details (steepness,
slope, rate of change in refractive index, or thickness in specimen details) into changes in intensity of various
areas of the image at the plane of the eyepiece diaphragm. Resulting images have an apparent three-
dimensional appearance with directional sensitivity to optical gradients.

Opposite gradients result in deflection of the slit image to either the very dark part of the modulator or the bright
section of the modulator, as illustrated in Figure 5. In this figure, a hypothetical specimen containing both
positive and negative phase (thickness) gradients and a flat (non-gradient) area is imaged using modulation
contrast optical components. The negative gradient depicted in Figure 5(a) deflects light into the dark area of
the modulator, where it is attenuated to approximately one percent of its former value. Likewise in Figure 5(c),
light deflected by a positive gradient into the clear area of the modulator is not attenuated, and 100 percent of
this light is transmitted into the intermediate image plane. Any non-gradient part of the specimen (Figure 5(b))
and also the background (surround) register on the gray part of the modulator, where about 15 percent of the
light is transmitted into the intermediate image plane. The result is that the intensity of the image area from one
side of the gradient is dark. Intensity from the opposite side of the gradient yields a bright image area, and the
non-gradient areas appear gray on the image, as does the background.
The contrast (related to variations in intensity) of the dark and bright areas against the gray gives a shadowed
pseudo-relief effect. This is typical of modulation contrast imaging. Rotation of the polarizer alters the contrast
achieved and the orientation of the specimen on the stage (with respect to the polarizer and offset slit) may
dramatically improve or degrade contrast.
Since the modulator affects the image of the slit according to how the specimen's details shift the image of the
slit (and thus results in altering light intensities), it is described as an amplitude filter. Hoffman and others have
demonstrated that phase gradients in the specimen, like spatial frequencies, are distributed over the entire exit
pupil of the objective. The optical transmission intensity distribution of the modulator will provide satisfactory
images of a wide variety of objects that produce phase gradients including: all types of cells and tissues (both
live, stained, and unstained), and surface details of crystals, transparent polymers, glasses and other similar
materials. Reflected light modulation contrast microscopy is also useful for imaging grain boundaries in opaque
and metallurgical specimens and surface details of complex integrated circuits and other electronic materials.

There are numerous advantages as well as limitations to modulation contrast. Some of the advantages include
fuller use of the numerical aperture of the objective yielding excellent resolution of details along with good
specimen contrast and visibility. Although many standard modulation contrast objectives are achromats or
planachromats, it is also possible to use objectives with a higher degree of correction for optical aberration, as
discussed above. Many major microscope manufacturers now offer modulation contrast objectives in fluorite-
correction grades, and apochromats can be obtained by special order. Older objectives can often be retrofitted
with a modulator made by Modulation Optics, Inc., the company founded by Dr. Robert Hoffman specifically
to build aftermarket and custom systems.

In addition to the advantages of using higher numerical apertures with modulation contrast, it is also possible to
do "optical sectioning" with this technique. Sectioning allows the microscopist to focus on a single thin plane
of the specimen without interference from confusing images arising in areas above or below the plane that is
being focused on. The depth of a specimen is measured in a direction parallel to the optical axis of the
microscope. Focusing the image establishes the correct specimen-to-image distance, allowing interference of
the diffracted waves to occur at a pre-determined plane (the image plane) positioned at a fixed distance from
the eyepiece. This enables diffracting objects that occur at different depth levels in the specimen to be viewed
separately, provided there is sufficient contrast. The entire depth of a specimen can be optically sectioned by
sequentially focusing on each succeeding plane. In this system, depth of field is defined as the distance from
one level to the next where imaging of distinct detail occurs, and is controlled by the numerical aperture of the
objective. Higher numerical aperture objectives exhibit very shallow depths of field and the opposite holds for
objectives of lower numerical aperture. The overall capability of an objective to isolate and focus on a specific
optical section diminishes as the optical homogeneity of the specimen decreases.

Images appear shadowed or pseudo three-dimensional, enhancing visibility because of differences in contrast
on either side of a detail. There are no halos exhibited in the image, unlike the images produced with phase
contrast optics. Modulation contrast converts phase gradient information into amplitude differences that are
very different from the phase relationship variations (and optical path differences) produced by a phase
contrast microscope. Use of the dark and gray zones in the modulator produce images that contain varying
shades of gray and are devoid of color. It is possible to introduce color into modulation contrast images by
producing modulators that have the gray and dark zones substituted for colored zones of equal transmittance
values. In this case, resulting images from the phase gradient are rendered in colors with similar gradients
having the same tint. Currently, we are unaware of any commercial source of modulation filters that contain
colored zones.
Hoffman Modulation Contrast

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Achromats or planachromats are the most widely utilized objectives for modulation contrast microscopy
because they can yield good images since color is not involved. Using these objectives with a green filter
(placed under the polarizer) will further improve the image because achromats are spherically corrected for
green light. Objectives of higher correction, including fluorites and apochromats, can also be used for
modulation contrast microscopy, but the added expense is often not worth the improvement in image quality,
except at very high magnifications.

The cost of the modulation contrast accessories is considerably below that of differential interference contrast
(DIC) equipment. Although both techniques require turret condensers with components matched to each
objective, DIC-equipped microscopes also contain a polarizer below the condenser and an analyzer that is
placed before the intermediate image plane in the optical path (above the objectives). The presence of a
crossed polarization system, necessary for DIC microscopy, diminishes its effectiveness with samples that
react to polarized light.

Birefringent objects (rock thin sections, crystals, bone, etc.), that can confuse images in DIC, can be examined
since the specimen is not situated between the two polarizers. Further, specimens can be contained in plastic
or glass vessels without deterioration of the image because of polarization effects since such vessels are also
above both polarizers, not between them. This allows the Hoffman system to be far more useful than DIC in the
examination and photomicrography of cell, tissue, and organ culture performed in plastic containers.

When the condenser is set at the brightfield position, objectives with a modulator installed can also be used for
regular brightfield work. Because the modulator is off-axis, little deterioration of the image results. Objectives
(but not the slit-plate condenser) equipped with a modulator can also be used for fluorescence and darkfield
work, but these objectives should be avoided when attempting DIC microscopy. The modulation contrast
system has been used very successfully with polarized light microscopy to enhance the detection of both
optical gradients and birefringency in the specimen. In this application, a non-polarized slit is used and the
polarized light configuration should be parallel polarizers (although crossed polarizers will also produce good
results, albeit with diminished illumination). An objective modified for modulation contrast is very useful using
ordinary polarized illumination without a slit plate in the condenser.

There are also several disadvantages and limitations of the Hoffman Modulation Contrast system. Images must
be viewed with caution because different observers can "see" a "hill" in the image as a "valley" or vice versa as
the pseudo three-dimensional image is observed through the eyepiece. The system is most sensitive to
gradients perpendicular to the length of the slit, resulting in the requirement for some degree of skill in
orientation of the specimen for best effect.

The cost of modification of each objective and the condenser openings must be added to the basic cost of
these accessories themselves. Complex, high numerical aperture, multi-element objectives are difficult or too
expensive to modify. In recent years, Robert Hoffman's company, Modulation Optics of Greenvale, New York
(a wholly owned subsidiary of Slant Fin Corporation), has been producing modified objectives and condensers.
Modulation Optics specializes in modifying objectives produced by the leading microscope manufacturers.
Some objectives are easily modified, while some are difficult or impossible to modify for the modulation optics
specification. However, any objective can be used with one of the company's intermediate tube systems,
including a broad range that spans macro camera lenses to 100x microscope objectives. Also included in this
category are objectives designed to image specimens either dry or with immersion media (oil, water, and
glycerin), single and multiple wavelength objectives, reflected and transmitted light objectives, and objectives
corrected for either infinity or a finite tube length. Modulation Optics designs and manufactures their own
condenser systems to satisfy a wide range of numerical aperture and working distance combinations.

Non-absorbing specimens are not rendered in color, with the exception of those observed using a special
modulator containing semi-transparent colored filters in place of the gray and dark zones. Specimens that
naturally absorb specific wavelengths or lightly stained are rendered in color as well as those observed with the
combination of modulation contrast and fluorescence or with the combination of modulation contrast and
polarized light.
Configuration of a microscope for Hoffman Modulation Contrast is straight-forward and basic steps are outlined
below:

Hoffman Modulation Contrast in Transmitted Light

 Attach the pertinent modulation contrast-enabled objectives to the nosepiece of the microscope, and
install the turret condenser containing the appropriate slit plates. If the microscope is equipped for
differential interference contrast (DIC) or polarizing microscopy, remove all polarizers, retardation
plates, and Wollaston or Nomarski prisms from the optical path.

 Place a stained specimen (preferably a tissue thin section) on the stage and, using the 10x objective
(with a modulator installed), align the microscope for proper Köhler illumination, outlined in our
section on Anatomy of the Microscope. The modulation contrast slit plate should be removed from
the condenser for this operation. If the turret condenser has a position for brightfield illumination with
an aperture diaphragm, rotate the turret to select this condenser.

 View the modulator plate in the back focal plane of the objective, using a Bertrand lens (common on
polarized light microscopes), a phase telescope, or by simply removing an eyepiece and peering
down the eye tube. Make certain that the sample is removed from the optical path or that it is moved
to a clear area on the microscope slide.
 Select the slit aperture plate that corresponds to the 10x objective by moving the appropriate
condenser (from the turret) into the optical path. There should be a set of adjustment screws or a
lever that allows rotation and translation of the illuminating slit plate within the condenser.

 Place the circular polarizing filter on the microscope light port beneath the condenser. Rotate this filter
while observing the slit image through the Bertrand lens (or phase telescope) and observe that the
angle of rotation influences the amount of light (brightness) passing through the polarizer portion of
the slit.

 Translate the image of the slit so that the opened portion lacking a polarizer is superimposed over the
gray region of the modulator plate as illustrated in Figure 3(b). The portion of the slit containing the
polarizing material should be imaged in the clear portion of the modulator just to the right of the gray
region. Rotate the circular polarizing filter and observe how the region of the slit containing the
polarizing material appears and disappears. When the vibration plane of the circular polarizer is
aligned perpendicular to the vibration plane of polarizer in the slit, the slit size if minimized and
maximum contrast is obtained. This maneuver can be practiced using the interactive Java tutorial
below:
Modulation Contrast Slit Alignment

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 Remove the Bertrand lens or phase telescope and replace the eyepiece in the eye tube of the
microscope. Place a specimen candidate on the stage and focus with the 10x objective.

 Readjust the condenser position by refocusing the field diaphragm to achieve a sharp focus. Open the
field iris diaphragm until it is just outside the field of view.

 The image is now ready for observation or photomicrography with modulation contrast. Rotate the
specimen and/or the circular polarizer at the base of the microscope to achieve optimum contrast.
These settings will vary from specimen to specimen.
  Repeat the above steps each time a different magnification is selected for viewing the specimen in
modulation contrast.

Once again, we find that manipulation of light at the front focal plane of the condenser (by means of an offset
slit) and manipulation of light at the back focal plane of the objective (offset modulator) can have a significant
effect upon the image presented in the eyepieces.

(OR)

Hoffman modulation contrast microscopy

Hoffman modulation contrast microscopy (HMC microscopy) is an optical microscopy technique for enhancing the
contrast in unstained biological specimens. The technique was invented by Robert Hoffman in 1975.[2] Like differential
interference contrast microscopy (DIC microscopy), contrast is increased by using components in the light path which
convert phase gradients in the specimen into differences in light intensity that are rendered in an image that appears
three-dimensional. The 3D appearance may be misleading, as a feature which appears to cast a shadow may not
necessarily have a distinct physical geometry corresponding to the shadow. The technique is particularly suitable for
optical sectioning at lower magnifications.[3][4]
An example of the use of HMC illumination is in in-vitro fertilisation, where under brightfield illumination the near-
transparent oocyte is hard to see clearly.
HMC systems typically consist of a condenser with a slit aperture, an objective with a slit aperture, and a polariser which
is fitted between the condenser and the illumination source and is used to control the degree of contrast. The principle of
HMC is used by a number of microscope manufacturers who have introduced their own variants of the technique, for
example, ZEISS improved Hoffman Modulation Contrast (iHMC), Nikon Advanced Modulation Contrast (NAMC),
Olympus Relief Contrast (RC) and Leica's integrated Hoffman Modulation Contrast (iMC)