SCHOOL OF DENTISTRY

General Luna Road, Baguio City Philippines 2600

Telefax No.: (074) 442-3071 Website: www.ubaguio.edu E-mail Address: ub@ubaguio.edu

Module 1:
MICROSCOPY

Two Principles in a microscope

 Having a series of 2 magnifying lenses

o Objective lenses
o Eyepiece/Ocular

1. Magnification

 ability of an optical system to enlarge an image expressed in

magnifying powers (the number of times an image is enlarged).

o Total Magnification = power of the objective x power of the ocular

2. Resolution

 ability of an optical system to gather fine details or to

separate 2 adjacent objects into 2 entities expressed
in resolving powers. (Clarity)

 Note: Human eye resolution - 0.1 mm resolution ‚

→ an image can be seen as 2 separate entities if

there is at least 0.1 mm differential gap between them.

Other terminologies:

1. Numerical aperture

 light gathering ability of the lens system. It is an ability of the

objective not the eyepiece. NA of 1.0 can be only achieved if
we are working in air.
OBJECTIVE MAGNIFYING POWER NUMERICAL COLOR
APERTURE

SCANNING 4X 0.10 RED

LPO 10X 0.25 YELLOW

HPO 40X 0.65 BLUE

OIL IMMERSION 100X 1.25 WHITE

2. Oil Immersion Technique

 Oil has a refractive index near to glass and of the lens.

 Refractive index : The amount of light that is scattered
 RI of air = 1.0 , RI of glass =1.518

3. Parcentrality

 maintaining focus when changing magnifications

4. Parfocality

 keeping the specimen centered within the field of view when

switching between objectives

General Categories of Microscope

1. LIGHT MICROSCOPE

 Uses visible light as the light source

 Bright field microscope

 Dark field microscope

 Phase contrast microscope

 Fluorescence microscope

 Polarizing microscope
2. ELECTRON MICROSCOPE

 utilizes electron beams as the light source

a. Transmission electron microscope

b. Scanning electron microscope

 USES:

o Examination of T. pallidum

o For unstained living organism

o For fluorescent microscope

 DESCRIPTION:

o Light microscope with dark-field apparatus

 Specimen: bright or glows brightly

3. PHASE CONTRAST

 DESCRIPTION:

o an optical microscopy technique that converts

phase shifts in light passing through a transparent
specimen to brightness changes in the image.

 USES:

o For moving, living and unstained cells

o cellular detail that are not visible with a simpler

bright field microscope
3. POLARIZING MICROSCOPE

 USES:

o Improves the quality of birefringent materials (crystals)

 DESCRIPTION:

o The interaction of plane-polarized light with a doubly

refracting (birefringent)

o specimen to produce two individual wave components

(ordinary ray and extraordinary ray) that are polarized

in mutually perpendicular planes.

4. FLUORESCENT MICROSCOPE

 USES:

o Fluorescent dyes stained organisms

 DESCRIPTION:

o Light or energy: extreme or rich in UV radiation

(Tungsten Halogen Lamp)

o Equipped with exciter filter

o Selected light excites the fluorochrome in specimen

o Has barrier filter

- b/n objective and observer’s eyes

5. SCANNING ELECTRON MICROSCOPY (SEM)
Use: For studying the texture, topography and surface

o feature, resolution ~ 10 nm
 Beam of electrons.
 The electrons interact with atoms in the sample, producing
various signals that can be detected and that contain
information about the sample's surface topography and
composition

6. TRANSMISSION ELECTRON MICROSCOPE (TEM)

Use: Lattice imaging, resolution < 0.2 nm

 Beam of electron
 Disadvantage: Silhouette picture
 Source of electrons: magnetic lenses
 Electron micrographs
 Fixed with osmium tetroxide or glutaraldehyde or
paraformaldehyde

Transmission Electron Microscope Scanning Electron Microscope

Aspect (TEM) (SEM)
Operating Uses transmitted electrons to create an Uses backscattered and secondary
Principle internal structure image electrons for surface imaging
Very high resolution (sub-nanometer High resolution (nanometer to tens of
Resolution
level) nanometers)
Specimen
Ultra-thin (50-100 nanometers) Thicker specimens (micrometers)
Thickness
Sample Conductive or coated with a thin
Extremely thin and electron-transparent
Preparation conductive layer
Internal structure, detailed cellular
Image Types Surface morphology, texture, shape
organelles, nanoparticles
Provides information about internal
Image Depth Focuses on the surface topography
structures
Relies on signal generation from
Contrast Relies on differences in electron density
electron interactions
Less likely to damage thicker
Beam Damage Can cause damage to thin specimens
specimens
Cell organelle study, nanomaterial Surface characterization of materials,
Applications
analysis, crystalline lattices biological samples