Deparment of Biology

Faculty of Mathemathics and Natural Sciences

Brawijaya University
Malang
Modern microscopists have developed a wide spectrum of useful techniques designed to
aid in contrast enhancement and provide better observation and photomicrography of
specimens .

When imaging specimens in the optical microscope, differences in intensity and/or color
create image contrast, which allows individual features and details of the specimen to
become visible.
Microscopy Techniques
Whats Contrast?
Difference in light intensity between the image and the adjacent
background relative to the overall background intensity
HOW to Produce Contrast ?
absorption of light brightness reflectance
birefringence
light scattering
diffraction
fluorescence
color variations
BRIGHT FIELD MICROSCOPY
Most commonly used microscopy imaging technique is bright field microscopy, where
light is either passed through or reflected off a specimen
Biologists and histologists have used counter staining for over one hundred years; and
this helps to differentiate the various tissues and organelles that can be found in a variety
of subjects that would otherwise be rendered invisible

Drawbacks
The cells are usually killed and therefore cannot be studied whilst moving around their
natural habitat
When to use bright field microscopy
viewing stained or naturally pigmented specimens such as stained prepared slides of
tissue sections
Used when there is enough contrast in the subject matter or artificial staining techniques
are employed .

BRIGHT FIELD
Main uses:
Viewing stained specimens
Pathological exams
Blood tests
Wafer inspections
Liquid crystal board inspections
PHASE CONTRAST MICROSCOPY
Optical phenomena of diffraction and interference are used to add light/dark contrast to
a transparent specimen for imaging. There is no need to stain the specimen as in
brightfield microscopy, so live specimens can be used.
enhances contrasts of transparent and colorless objects by influencing the optical path
of light

Drawbacks
A disadvantage of this method is the appearance of light halos around some objects
("halo-effect").
When to use Phase Contrast microscopy
Phase contrast is preferable to bright field microscopy when high magnifications (400x,
1000x) are needed and the specimen is colorless or the details so fine that color does not
show up well. Cilia and flagella, for example, are nearly invisible in bright field but show
up in sharp contrast in phase contrast
For the procedure itself a special condenser with a ring-shaped mask and an additional
"phase-ring" that is fixed within the back focal plane of the objective is needed
PHASE CONTRAST
Main uses:
Imaging cultured cells
Imaging blood or living cells
DIFFERENTIAL INTERFERENCE CONTRAST MICROSCOPY (D.I.C)
Transforms minute differences in refraction indexes of light passing through an
unstained specimen, or optical path differences from the specimen surface shape, into a
monochromatic shadow-cast image enabling observation.
3D-pseudo effect that it gives and also, unlike phase contrast there are no halos around
the subject
Drawbacks
DIC utilizes optical path differences within the specimen (i.e.: product of refractive index
and geometric path length) to generate contrast the three-dimensional appearance may
not represent reality
Birefringent specimens such as those found in crystals may not be suitable because of
their effect upon polarized light. Similarly, specimen carriers, such as culture vessels,
Petri dishes, etc., made of plastic may not be suitable
When to use DIC?
As with phase contrast microscopy, DIC microscopy may be used with living specimens.
However, it is better suited to thicker specimens.

The DIC set-up consists of:
A Polarizer,a Wollaston prism the Object, Wave train, Objective, Wollaston prism,
Analyzer, and Eyepiece.
DIC
Main uses:
Imaging fibrous structure of nerve
Imaging mitotic spindles
Imaging cellular nucleic structures or
other thick unstained specimens
DARK FIELD MICROSCOPY
A special condenser lens is used to illuminate the specimen diagonally, then observe
light scattering off it. The field of view is darker than bright field microscopy because
illumination light does not enter the objective lens.
Oblique illumination is used to increase the visibility of specimens
Useful in revealing very fine detail especially bacteria

Drawbacks
Dark field is only black and white and is missing the information from the shading ability
of phase contrast
For serious dark field work, one needs to use a dedicated darkfield condenser.
When to use Dark field microscopy
Phase contrast is preferable to bright field microscopy when high magnifications (400x,
1000x) are needed and the specimen is colorless or the details so fine that color does not
show up well. Cilia and flagella, for example, are nearly invisible in bright field but show
up in sharp contrast in phase contrast
All of us are quite familiar with the appearance and visibility of stars on a dark night,
this despite their enormous distances from the Earth. Stars can be readily observed at
night primarily because of the stark contrast between their faint light and the black sky.
PHASE CONTRAST
Main uses:
Microbiological imaging
Blood tests
Detecting microscopic scratches or
irregularities
FLUORESCENCE MICROSCOPY
Specimen is excited with a specific wavelength of light, then fluorescent emissions are
observed.
Drawbacks
When to use Fluorescence microscopy
Used to study specimens, which can be made to fluoresce.
Certain material emits energy detectable as visible light when irradiated with the light of
a specific wavelength. The sample can either be fluorescing in its natural form like
chlorophyll and some minerals, or treated with fluorescing chemicals.


Photo bleaching can significantly cause measurement error
Sample you want to study is itself the light source.
FLUORESCENCE
Main uses:
Imaging and quantification
Assaying antigens in antigen/antibody reactions
Imaging and quantification of intracellular DNA
Analysis of chromosomal abnormalities

Principle of Fluorescence
1. Energy is absorbed by the atom which becomes excited.
2. The electron jumps to a higher energy level.
3. Soon, the electron drops back to the ground state, emitting
a photon (or a packet of light) - the atom is fluorescing
POLARIZING MICROSCOPY
This technique uses the phenomenon of polarization to add contrast and color to
specimen images.
Designed to observe and photograph specimens that are visible primarily due to their
optically anisotropic character.
When this beam passes through certain specimens the plane of the waves is "rotated".
In some cases the extent of rotation varies with wave length, or "colour" ("birefringence)
second filter, referred to as the analyser, prior to viewing
When a birefringent specimen is viewed under these conditions, the rotated light can
pass through the analyser
Drawbacks
When to use Polarizing microscopy
Polarized light is a contrast-enhancing technique that improves the quality of the image
obtained with birefringent materials when compared to other techniques

Proper alignment of the various optical and mechanical components is a critical step that
must be conducted prior to undertaking quantitative analysis between crossed polarizers
alone, or in combination with retardation plates and compensators
Microscope must be equipped with both 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.
POLARIZING
Main uses:
Analysis of optical properties of rocks, ores
Polarization analysis of fine structures within
living organisms and cytoskeletons
Gout testing

Incident light is
polarized, passes
through the sample and
crossed polar analyzer
to an image of a brightly
colored (interference
colored) image of the
pigment crystallite.
Each of these methods is best suited for different uses. Research class
microscopes allow these techniques to be utilized merely by switching
attachments
Modern microscopists and optical engineers have developed a
wide spectrum of useful techniques designed to aid in contrast
enhancement, provide better observation, and assist in the
collection of photomicrographs and digital images of a wide
variety of specimens.
Why consider digital imaging?
(1) Easily transmitted to a wide number of users
(7) Easily annotated with
appropriate software for
inclusion in
presentations or
archives
(6) Digital image capture from
the start saves time and effort
(5) Little or no photographic
expertise is needed
(2) No ongoing costs -
there are no film or
processing fees.
(3) Almost instantaneous
(4) A decision can be immediately taken
on whether the picture is satisfactory
A-D Converters
(eg. CCD Camera,
Frame Grabber Card..
Etc.)
ADSL
ISDN
Etc.
INTERNET
TELEMICROSCOPY APPLICATION
MIC-D
E-Learning Digital Microscope
WWW Community - Website
Portability- in and out of classroom
Limitless general applications


Educational tools Students
Hobbyist
Teaching
Presentations
Sharing
The Olympus MIC-D Digital Microscope
Anatomy of the MIC-D Digital Microscope
- Anatomical Overview
- Brightfield Illumination
- Oblique Illumination
- Darkfield Illumination
- Polarized Illumination
- Reflected Illumination
(B) Digital Image Capture and Processing

New era in optical microscopy education
MIC-D inverted digital microscope
Designed specifically for a wide spectrum
of applications.
A palette of contrast enhancing techniques
Anatomy of the MIC-D Digital Microscope

Anatomical Overview
Unique design that incorporates a CMOS
electronic digital imaging sensor as a substitute
for the traditional eyepieces
inverted illumination system that broadcasts
light from above the specimen
Lamp house and condenser unit that can be
rotated over a 135-degree angle
Six basic mechanical control elements available
to the operator

1. Microscope can be powered on and off with a small switch
2. Single focus knob
3. Gliding stage
4. Diffusion filter (or screen)
5. Rotating Arm
6. Zoom Lens


Anatomy of the MIC-D Digital Microscope

Brightfield Illumination
One of the most commonly utilized observation modes in optical microscopy
Ideal for fixed, stained specimens or other types of samples having
high natural absorption of visible light


6(a) is a stained thin section of tooth enamel formation
6(b) illustrates a common flea
6(c) is a stained thin section of pine staminate
cone tissue
6(d) is from a cephalopod
Almost every compound and stereo microscope is
capable of producing brightfield images at a variety
of magnifications, and many new designs, such as
the Olympus MIC-D microscope, provide digital
imaging capabilities
Anatomy of the MIC-D Digital Microscope

Oblique Illumination
Made possible by off-axis translation of the illuminator head and
condenser assembly
Enhance contrast in specimens that would otherwise remain invisible (or nearly so)
in brightfield illumination.


3(a) is a brightfield image of a crab megalops
3(b) resulting oblique illumination clearly reveals structural
detail
3(c) are wing scales from the Common Nawab
3(d) the butterfly wing scales have a quasi three-dimensional
appearance that clearly reveals the edges and some
surface detail not present in the brightfield image
This method lights the specimen from an oblique
direction for clear observation of contours and
details that are hard to observe with transmitted
light observation.


Anatomy of the MIC-D Digital Microscope

Darkfield Illumination
When the rotation arm of the MIC-D digital microscope is positioned at a
distance greater than 15 degrees from the central (optical) axis
Produces results similar to those observed in true darkfield illumination.


5(a) is a young starfish specimen that has been stained with hematoxylin and prepared as a whole
mount
5(b) A multiply stained shepherd's purse
5(c) exhibits spectacular color and reveals fine specimen detail when viewed under both brightfield
and darkfield illumination
Excellent tool for both biological and
medical investigations.
Effectively used at high magnifications to
photograph living bacteria, or at low
magnifications to view and photograph
cells, tissues, and whole mounts
E.G.: Observe and record data about
fresh and salt-water organisms such as
algae and plankton.


Anatomy of the MIC-D Digital Microscope

Polarized Light Illumination
Designed to observe and record images of specimens that are visible
primarily due to their optically anisotropic character
Specimens have polarizable intramolecular bonds that interact with
polarized light in a direction-sensitive manner to produce phase
retardations that are monitored
6(a) reveals one of the many cholesteric liquid crystalline textures exhibited
6(b) is a digital image of a human bone thin section captured by the MIC-D microscope in oblique
polarized light mode
6(c) is a polystyrene membrane


The MIC-D microscope can be
equipped with a polarizer and analyzer
combination in order to view and capture
digital images from birefringent anisotropic
specimens.
Anatomy of the MIC-D Digital Microscope

Reflected Light Illumination
optical microscopy can be largely divided into two main categories:
transmitted light microscopy and reflected light microscopy
Transmitted light microscopy is utilized for specimens that are
relatively thin and semi-transparent, enabling a significant amount
of light to pass through.
Reflected light microscopy is for specimens that remain opaque even when ground to
a thickness of 30 micrometers or less.


The oblique illumination
angle employed by the
MIC-D digital microscope
in reflected light mode
affords a shadow-cast
effect that enhances
overall specimen
appearance and
increases contrast
(B) Digital Image Capture and Processing




The Olympus MIC-D inverted
digital microscope captures
images with a complementary
metal oxide semiconductor
(CMOS) image sensor housed in
the base and coupled to a host
computer for acquisition,
cataloging and processing of
digital images
(B) Digital Image Capture and Processing




(B) Digital Image Capture and Processing




(B) Digital Image Capture and Processing




(B) Digital Image Capture and Processing




(B) Digital Image Capture and Processing




(B) Digital Image Capture and Processing




MIC-D IT!
DP12
Digital Microscope camera DP12
For Clinical, Laboratory and Basic Research applications
From Classroom to the laboratory
More dedicated use

The Olympus DP12 Microscope Digital Camera
The new compact solution for
professional digital imaging in microscopy
What do you get?
Camera
Body
Computer
Workstation
(DP12-BSW)
Hand
Switch



3.34 million pixels high resolution image
High speed transfer of image data (USB)
3.5 inch LCD monitor for focusing
Uncompressed TIFF format selectable
Easy operation by hand switch
Analog output to external monitor
Standard Configuration
Features
High-resolution 3.34 million pixel imaging

1/1.8 inch interlaced scan CCD with 3.34M pixel resolution - ensures highly precise digital images.
Very fine structures of the specimen are clearly imaged.
The maximum image resolution is 2048 x 1536 pixels.
SmartMedia cards used with DPOF-compliant printers will easily produce superb printed images

Easy Focusing

3.5"LCD monitor : real-time display of large, images for faster, accurate focusing and framing.
Focusing indicator and a 2 x electronic zoom function, sharp focusing is easy even at low
magnifications.
The LCD monitor's tilting function allows image observation at the ideal angle.

Features
Direct scale bar imprint

Calibrated scale bar can be superimposed on the image - horizontal or vertical orientation.
Scale bar can be saved and printed with the image.
Multi-functional, compact control unit

Compact and robust stand alone camera fo use with or without a PC.
The camera head is equipped with a universal C-mount thread The 200,000 pixel 3.5
LCD Monitor is integrated in the control pad forming a compact unit
The tiltable LCD monitor also function as a protective lid for the control unit.
The camera is operated entirely from the control unit.
Users do not need to touch the camera head or the microscope for exposure.
Features
Software for image download and camera control

Equipped with a high-speed USB interface.
Images can easily be downloaded directly from the camera control unit to the PC.
The SmartMedia card is removable, allowing images to be easily transferred to any PC using
optional USB, FlashPath or PCMCIA SmartMedia reader
Easy adjustment for optimum image quality

Three different white balance modes are available for optimal color representation
Exposure metering is selectable between 1% spot and 30% avarage metering.
Users can choose automatic or manual exposure modes.
Large LCD monitor for on-screen image selection

Date, time, shutter speed and file name are displayed and stored together with the microscope image.
Acquire images can be displayed (up to 16 at once) on the screen.
Images can be protected on the SmartMedia card or assigned for subsequent printing.
Image data is retained on the SmartMedia card when the power is turned off.
The Ultimate Image Analysis Solution
OLYMPUS MICRO IMAGE
Fluorescence imaging
Quality assurance
Materials imaging
And various other scientific,
medical, and industrial
applications
Features extensive enhancement and measurement tools and allows you to write application-
specific macros and plug-ins.

Key Benefits?

Over 20 years of development, evolution, and user feedback, OMI provides a full range of utilities
OMIs user-friendly environment
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learning from your images.

Compress lengthy
operations into a single
keystroke
or mouse click with OMIs
Auto-Pro programming
language

Further extend the functionality
of OMI with the following
integrated plug-in modules
Scope-Pro
automated microscope control
AFA
advanced fluorescence acquisition
3D Constructor
three-dimensional reconstruction and measurement.

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imaging community
Work With a Proven Solution
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