Richard Rupert T.

Vicencio, RMT
DMMC Institute of Health Sciences
▪ is an instrument used to see objects that are too small to be seen by
the naked eye.

Microscopy - is the science of investigating small objects and structures

using such an instrument.
1. Lens system
2. Illumination system
3. Body consisting of a base, body tube, and nosepiece

1. Lens system
- Primary components of the lens system are the oculars, the
objectives, and the coarse- and fine adjustment knobs
2. Illumination system
- contains the light source, condenser, and field and iris
diaphragms.
Objects to be examined are placed on a platform, referred to as the
mechanical stage.
1. Light Microscopy
1. Brightfield
2. Darkfield
3. Phase Contrast
4. Polarizing
5. DIC
6. Fluorescence microscope
7. Confocal laser scanning

2. Electron Microscopy
1. Scanning
2. Transmission
▪ Microscopes used in clinical practice are light microscopes.
▪ consists of two lens systems (combination of lenses) to magnify the image.
▪ A compound light microscope with a single eye-piece is called monocular; one with
two eye-pieces is said to be binocular.
▪ A compound light microscope is the most common microscope used in microbiology

▪ Electron Microscope - These microscopes provide a higher magnification and are

used for observing extremely small microorganisms such as viruses.
1. Brightfield
- commonly used type of microscope.
- in which objects appear DARK against a LIGHT background, is most
frequently used in the clinical laboratory
- employs the basic microscope previously described with a light source
emitting light in the visible wavelength range.
- It is mainly used with stained preparations.
- Differential staining may be used depending on the properties of different
structures and organisms.
1. Brightfield
Simple design
- Light directed at specimen
is absorbed to form image
- Unstained specimens have
poor contrast
- Stained specimens show
excellent contrast
- Ideal for stained bacteria,
cells, tissues
- good resolution
- Bright background, dark
specimen
- tungsten or halogen light
source
1. Brightfield Microscope
1. Brightfield Microscope
▪ consists of a two-lens system combined with a light source.
▪ The FIRST LENS system is located in the OBJECTIVE and is adjusted
to be near the specimen.
▪ The SECOND LENS system, the OCULAR LENS, is located in the
EYEPIECE.
▪ The path of light passes through the specimen up to the eyepiece.
1. Brightfield Microscope
KEY COMPONENTS
1. Magnification
2. Resolving Power
3. Limit Resolution
4. Numerical Aperture (N.A)
5. Focal Length
KEY COMPONENTS
1. Magnification
• the ability of the microscope to render fine
detail of an object visible.
▪ Ocular Lenses
▪ Objective Lenses
o Scanner
o Low Power Objective (LPO)
o High Power Objective (HPO)
o Oil Immersion Objective (OIO)
KEY COMPONENTS
1. Magnification
• Ocular Lenses
▪ magnifies 10x
• Objective Lenses
▪ Scanner = 4x
▪ LPO = 10x
▪ HPO = 40x
▪ OIO = 100x
KEY COMPONENTS
1. Magnification

Objectives Objective Ocular Lens Total

Lens Magnificatio Magnificatio
Magnificatio n n
n
Scanner 4x 10x 40x
LPO 10x 10x 100x
HPO 40x 10x 400x
OIO 100x 10x 1000x
KEY
COMPONENTS
1. Magnification
▪ Immersion oil (cedarwood oil)
is used to keep light from
bending.
KEY COMPONENTS
2. Resolving Power
• Ability to distinguish two
adjacent points
▪ Depends on the wavelength of light
and numerical aperture
KEY COMPONENTS
KEY COMPONENTS
2. Resolving Power
• Depends on the wavelength of light and
numerical aperture
• Numerical aperture – Expression relating the
measurement of light that is delivered to the
specimen by the condenser and gathered by
the objective.
• Wavelength of light
KEY COMPONENTS
2. Resolving Power
½ of the wavelength of light used
numerical aperture
KEY COMPONENTS
3. Limit Resolution
• The smallest object that can be
seen distinctively, as
accomplished by:
▪ shortest wavelength of visible light
▪ maximal numerical aperture
KEY COMPONENTS
3. Limit Resolution
KEY COMPONENTS
4. Numerical Aperture
(N.A)
• Expression relating the
measurement of light that is
delivered to the specimen by the
condenser and gathered by the
objective.
• Indication of the light gathering
power of lens.
KEY COMPONENTS
4. Numerical Aperture (N.A)
KEY COMPONENTS
5. Focal Length
• The distance between the
end of objectives and the
object in focus.
Key Term
❑ Par Focal
➢If one objective is in focus
and when a switch is made
to another objective the
focus will still be there
2. Darkfield Microscope
▪ is a technique used in the clinical laboratory to enhance visualization of
specimens that cannot be seen easily viewed with a bright-field
microscope.
▪ the field of view is DARK and the organisms are ILLUMINATED. A
special condenser is used which causes light to reflect from the
specimen at an angle
▪ is used for observing bacteria such as treponemes (which cause
syphilis) and leptospires (which cause leptospirosis).
▪ often used for unstained specimens.
2. Darkfield
Microscope
- A method from the 19th
century
- Bright specimen, dark
background
- Light not scattered by
the specimen bypasses
the objective, therefore
making the “field” dark.
- Can see very small
objects but resolution
is variable
- High contrast, good for
unstained, live, and
motile specimens
2. Darkfield Microscope
3. Phase Contrast
▪ allows the examination of LIVE UNSTAINED organisms.
▪ These alter the phase relationships of the light passing through the object and that
passing around it.
▪ special condensers and objectives are used.
▪ Converts differences in refractive index in a specimen to differences in image
brightness.
▪ The central portion of the light source is blocked, creating a ring of light from the
condenser that illuminates the specimen.
3. Phase
Contrast
- technology from
1940’s
- provides high
contrast, good
resolution
- good for bacteria,
flagella, cilia,
organelles such as
mitochondria
- good for unstained or
live mounts
- phase halos
(artifacts) occur
3. Phase Contrast
Microscopy
4. Polarizing Microscope
▪ Detects specimens that are birefringent (have the characteristic of double refraction,
i.e. the velocity of light refracted by a substance is not the same in all directions)

▪ The specimen is placed between two polarizers crossed at 90 o to each other (one in
condenser and one in objective).

▪ use of polarized light aids in the identification of crystals and lipids.

▪ Both substances have the ability to rotate the path of the unidirectional polarized light
beam to produce characteristic colors in crystals and Maltese cross formation in
lipids
4. Polarizing
Microscope
- bright image, dark
background
- used for substances
with highly organized
molecular structure,
such as crystals,
minerals
- can be quantitative
4. Polarizing Microscope
4. Polarizing Microscope
▪ Two polarizing filters must be installed in a crossed configuration
▪ The FIRST FILTER, the polarizing filter, is placed in the condenser filter holder
▪ The SECOND filter, the analyzer, is placed in the head between the objectives and
the ocular.
5. Differential Interference
Contrast Microscopy
▪ provides a three-dimensional image showing very fine structural detail by splitting
the light ray so that the beams pass through different areas of the specimen.
▪ The advantage of interference-contrast microscopy is that an object appears bright
against a dark background but without the diffraction halo associated with phase-
contrast microscopy
6. Fluorescence microscope
▪ specimens are stained with fluorochromes/ fluorochrome complexes.
▪ Light of high energy or short wavelengths (from halogen lamps or mercury vapour
lamps) is then used to excite molecules within the specimen or dye molecules
attached to it.
▪ It is used to detect BACTERIA and VIRUSES within cells and tissues through a
technique called immunofluorescence.
▪ Auramine differential staining for acid-fast bacilli is one application of the technique;
▪ rapid diagnostic kits have been developed using fluorescent antibodies for identifying
many pathogens.
5. Differential
Interference
Contrast
Microscopy
- produces 3-D images
- excellent resolution,
high NA, high contrast
- good for unstained
specimens, live
mounts; can see
membranes within cells
- detects changes in
refractive index of
specimen Hoffman
modulation contrast
microscopy (HMCM)
5. Differential Interference
Contrast Microscopy
▪ Two types of interference-contrast microscopy are available:

1. Modulation contrast (Hoffman)

2. Differential-interference contrast (Nomarski
6. Fluorescence microscope
▪ The excitation filter selects the excitation wavelength of light from a light source.
▪ The emission filter selects a specific wavelength of emitted light from the specimen
to become visible.
- uses uv light source =
mercury or xenon arc lamp.
- high contrast, high resolution
image
- special fluorescent dyes used
to locate “molecules” in a
specimen
- black background, bright-
stained specimen
- no condenser required, light
comes from above (“epi”)
specimen
- multiple fluorescent probes
available
- detects small quantities,
molecules; can use antibody
staining techniques
6. Fluorescence microscope
7. Confocal laser scanning
▪ A laser is focused at a plane in the specimen and scans the specimen in a horizontal
(XY) plane.

▪ Only light from the plane of focus reaches the detector. The scanned image (an optical
section) is digitally recorded.

▪ Images from consecutive focal planes can be recorded, and composite or 3-D images
can be digitally created.
7. Confocal
laser
scanning
- krypton/argon laser
- high resolution,
sharp image
- high sensitivity
- Can be used in
reflectance or
fluorescence mode
- eliminates
background
interference
7. Confocal laser scanning
 2. Electron Microscopy

1. Scanning Electron Microscope

2. Transmission Electron Microscope
1. Scanning Electron
Microscope
▪ Fixed, dehydrated specimens are mounted on stubs and surface-
coated with gold, palladium or rhodium.
▪ The specimen is placed in a vacuum and an electron beam scans back
and forth over it.
▪ Electrons that bounce off the metal-coated specimen surface are
collected, converted to a digital image and displayed on a TV-like
monitor.
1. Scanning Electron
Microscope
▪ Electron beam is focused using a magnetic field
▪ SEM provides a 3-D image
▪ Gives information about external topography of specimen
▪ Much higher resolution and magnification than possible in LM
1. Scanning Electron
Microscope
2. Transmission Electron
Microscope
▪ Fixed, dehydrated specimens are embedded in a resin, hardened, sectioned, stained
with heavy metals such as uranium and lead, and inserted into the electron column in
the microscope

▪ The electron beam is absorbed or deflected by the heavy metal stains and shadows
are cast onto film or a phosphorescent plate (image is a shadow) at the bottom of the
column.
▪ 2-D image
▪ reveals internal cell structure
▪ high resolution, high magnification
▪ electron beam is focused by magnetic field
2. Transmission Electron
Microscopes
Focusing Specimen
1. Always start with the SCANNING OBJECTIVE.
2. Focus using the COARSE ADJUSTMENT KNOB to bring the
object into focus.
3. Use only the FINE ADJUSTMENT KNOB when using the OIO.
4. If the specimen is too light or too dark, try adjusting the iris
diaphragm and the light intensity (illuminator) knob.
5. Keep both eyes open to reduce eye strain