MICROSCOPE

Definition:

● Microscope is an optical device used in examining microscopic

structures.
● There’s a lot of applications of microscope in the clinical laboratory
● It is also considered as the most important tools used in the analysis
purposes.

Units of Measurement

1 µm micrometer = 10-6 m = 10-3 mm

1 nm nanometer = 10-9 m = 10-6 mm

1000 nm = 1 µm

1.01 µm = 1 nm

Microscopy: The Instruments

● A simple microscope has only one lens.

● The first simple microscope was first used by Anton Van Leeuwenhoek
● He was the most popular among the microscopies
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● Because of his simple microscope he was able to see a lot of organism

including microscopic helminths and various cell with the aid of fimple
microscope

● In a compound microscope the image from the objective lens is

magnified again by the ocular lens.
● Total magnification = objective lens ´ ocular lens
● It is also known as compound light microscope because it makes used
of light and visible light as it source of illumination.
● “Magnification”- it is the number of times which specimen is enlarge
● The objective lens is also known as “Primary Magnifier”
● The ocular lens is also known as “Secondary Magnifier” that can use to
see the final image of an specimen or substances.
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MAIN PARTS AND ITS DEFINITION

● Stage- it is where the microscopic slides which contain the specimen

placed
● Condenser- it concentrates the lights
● Focusing Knobs- it gives the sharper specimen
● Light- it serves as the source of illumination

TOTAL MAGNIFICATION FOR OBJECTIVE LENSE TYPE

● Scanner magnifies the substances by 4x; it is mainly use for initial

focusing
● LPO- it gives us the general view of the specimen
● HPO and OIL- both detailed the examination
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● Resolution is the ability of the lenses to distinguish two points.

● Resolution is also known as “resolving power”
● A microscope with a resolving power of 0.4 nm can distinguish
between two points ≥ 0.4 nm.
● The ability of lenses to distinguish fine details or objectives
● 0.2 is the limitation without compound microscope
● The viruses as filterable agents identified by other microscope except
compound, it is the electron microscope
● Shorter wavelengths of light provide greater resolution.
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● Refractive index is the light bending ability of a medium.

● The light may bend in air so much that it misses the small
high-magnification lens.
● Immersion oil is used to keep light from bending
● Change the refractive index by staining the specimen
● Staining is the artificial coloring in the specimen

BRIGHTFIELD ILLUMINATION BRIGHT FIELD MICROSCOPE

● Utilized transmitted
light
● Dark objects are
visible against a bright
background.
● Light reflected off
the specimen does not enter
the objective lens

DARKFIELD ILLUMINATION

● Ultilized reflected light with a special condenser

● Light objects are visible against a dark background.
● Light reflected off the specimen enters the objective lens.

PHASE-CONTRAST MICROSCOPY
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● Accentuates diffraction of the light

that passes through a specimen.
● Direct and reflected light rays are
combined at the eye. Increasing contrast
● Binary fission, motility and endospore
● It allows the viewer to examine the
specimen of different degrees of brightness
and contrast.
● It has a lot of applications in clinical
laboratory. Used to demonstrates binary
fission of prokaryotes, movement of motile
microorganism and examination of endospore

There are two sources of lights:

1. Direct light coming from the

illuminator
2. Reflected light

MICROSCOPY: DARK FIELD

● Greater resolution
● Light reaches specimens only from the
side
● Only the specimen itself is illuminated

Microscopy: Phase Contrast

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● May be used to visualize live samples and avoid distortion from cell
stain
● Image contrast is derived from the differential refractive index of cell
structures.
● Detailed examination of internal cellular components
● Identification of cancers and tubar cells

Differential Interference Contrast

● Dual beam optical Microscopy system (light

● Accentuates diffraction of the light that passes through a specimen;
● Uses two beams of light. Adding color
● Better contrast
● Sample principle with phase contrast microscope

CILIATED PROTOZOANS:

1. Macronucleus
2. Micronucleus
3. Vacuoles

Fluorescence Microscopy

● Uses UV light.
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● Fluorescent substances absorb UV light and emit visible light.

● Cells may be stained with fluorescent dyes (fluorochromes).
● Detect antigen and antibodies
● It absorbs light at a shorter wavelength
● Fluorescent dyes has a lot of applications in microbiology and serology

Microscopy: Fluorescence

● Visualization of auto
fluorescent cell structures
(e.g., chlorophyll) or
fluorescent stains
● Can greatly increase the
resolution of cells and cell
structures
● Many functional probes
available

Example for Differential

Fluorescence Stain

Confocal Microscopy

● Uses fluorochromes and a laser light.

● The laser illuminates each plane in a
specimen to produce a 3-D image.
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● Vertical and horizontal cross section are examined

Electron Microscopy

● Uses electrons instead of light.

● The shorter wavelength of electrons gives greater resolution.

Transmission Electron Microscopy

● Ultrathin sections of
(TEM) specimens.
● Light passes through
specimen, then an
electromagnetic lens, to a screen
or film.
● Specimens may be
stained with heavy metal salts.

● 10,000-100,000´; resolution
2.5 nm
● Advantage: We can examine
specimen at different layers

Scanning Electron Microscopy (SEM)

● An electron gun
produces a beam of electrons
that scans the surface of a
whole specimen.
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● Secondary electrons emitted from the specimen produce the image

● 1000-10,000´; resolution 20 nm
● No requirement of ultrathin
preparation of sample
● Surface pictures of microorganism
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Scanning-Probe Microscopy

● Scanning tunneling microscopy uses

a metal probe to scan a specimen.
● Resolution 1/100 of an atom.
● Very useful for DNA analysis

● Atomic force microscopy uses a

metal and diamond probe inserted
into the specimen
● Produces 3-D images.