MICROSCOPY

DEFINITION OF TERMS:
Resolving power/Resolution Working Distance Magnification Numerical Aperture

Refractive Index

Resolving Power/Resolution
Capacity of the microscope to
separate clearly 2 points

Determined by the shortest wavelength of visible light and maximum numerical aperture

Working Distance Distance between the surface of the lens and the surface of the cover glass or the specimen when it is in sharp focus
Objectives with large NA and great resolving power have short working distanceworking distances

Magnification
Ratio of image size to the actual size Objective Magnification X Ocular Magnification

Numerical Aperture
Measure of the size or angle of the cone of light entering the aperture of the lens

Light-gathering capacity of the microscope

A number that indicates the resolving power of a lens system Derived mathematically from the refractive index(n) of the optical medium (for air, n=1) and the angle of light (u) made by the lens NA= n X sin u The larger the NA, the higher the magnification

Refractive Index
Measure of the light-bending ability of the medium such as glass or air Light bends as it passes from the glass slide into the air; bending is due to the fact that the glass slide and air have different refractive indices Immersion oil has the same refractive index as the glass; immersion oil placed between the glass slide and the OIO lens prevents the refraction or scattering of light rays that would otherwise be lost to the objective lens

The Properties of the Microscope Objectives

property
magnification NA

scanner

LPO

HPO

OIO

4X 0.01
40 mm

10X 0.25
16 mm

40-45X 0.55-0.65
4 mm

90-100X 90-100
1.8-2.0 mm

Approximate focal length

Working distance

17-20 mm

4-8 mm

0.5-0.7 mm

0.1 mm

Approximate resolving 2.3 um 0.9 um 0.35 um 0.18 um power with light of 450 nm Scanner-shortest objective; largest lens; red ring; lowest magnification;

locates various structure LPO- shorter; larger lens; yellow ring; lower magnification; for initial focusing; gives general outline HPO- longer; smaller lens; blue ring; higher magnification; gives details of specimen OIO- longest; small lens; white ring; highest magnification; examines microorganisms

Units of Measure
When measuring microorganisms, we use the metric system; has the advantage of easy conversion by a single factor of 10 The standard unit of length is the meter (m)

Microorganisms and their structural components are measured in even smaller units, such as micrometers and nanometers
Micrometer (um)= 10-6 m Nanometer (nm)= 10-9 m Angstrom (A)= 10-10 m or 0.1 nm

TYPES OF MICROSCOPE
Light or Optical Microscope a. polarizing microscope b. differential interference c. phase-contrast d. fluorescence e. dark-field f. bright-field UV Microscope Electron Microscope a. TEM b. SEM

The Light Microscopes

Uses visible light as a source of illumination Specimen appears against a dark or bright background

Magnification: 2,000X; Resolution: 0.2 um

Brightfield illumination is used for stained smears; most common is the binocular compound microscope Unstained cells are observed using modified compound microscopes

The Nature of Light Waves

How are waves quantified?

Crest and trough Amplitude - maximum excursion from its undisturbed or relaxed position. Waves travel at a speed, v. The number of crests that pass at a specific point in space is called a waves frequency or f, and is recorded in units of Hertz. Period - the time it takes for one complete cycle, measured in seconds. This is known as P. Wavelength - the distance a wave travels during one complete oscillation.
f = 1/P Wavelength() = speed x P or speed/f

MECHANISMS OF LIGHT MICROSCOPE

The principle is based on the wave nature of light rays, and the fact that light rays can be in phase (their peaks and valleys match) or out of phase. If the wave peak of light rays from one source coincides with the wave peak of light rays from another source, the rays interact to produce reinforcement (relative brightness) However, if the wave peak from one light source coincides with the wave through from another light source, the rays interact to produce interference (relative darkness)

Brightfield Microscope imaging of S. aureus

POLARIZING MICROSCOPE
Birefringence : Capacity to change the direction of the axis of light Modification : with two filters POLAROID : between the light source and condenser ANALYZER : at the draw tube Applications : mineral elements ash residues spindle fiber

A POLARIZING MICROSCOPE

the meteorite Tieschitz

needles of urates from gout

PHASE-CONTRAST
Distinguishing features: uses a special condenser containing an annular (ringshaped) diaphragm; the diaphragm allows direct light to pass through the condenser, focusing light on the specimen and a diffraction plate in the objective lens; direct and reflected or diffracted light rays are brought together to produce the image Principle: Combination of in-phase and out-of-phase; different protoplasmic constituents produce phase variations One set of light rays comes directly from the light source; the other set comes from light that is reflected or diffracted from a particular structure in the specimen

When the 2 sets of light rays- direct rays and reflected or diffracted rays- are brought together, they form an image on the ocular lens, containing areas that are relatively light (in phase), through shades of gray, to black (out of phase)
Application : facilitates detailed examination of the internal structures of living specimens

DIFFERENTIAL INTERFERENCE
Like phase contrast, uses differences in refractive indexes to produce images However, uses 2 light beams separated by beamplitting prisms, adding contrasting colors to the specimen Provides a colored 3D image

Differential Interference Contrast Microscope

FLUORESCENCE MICROSCOPE
Takes advantage of fluorescence, the ability to absorb light at a shorter wl (ultraviolet) and emit it at a longer wl (visible) Uses an UV or near-UV source of illumination that causes specimen to emit light or fluoresce; The specimen appears luminescent bright object against a dark background the specimen can either be fluorescing in its natural f orm or stained with fluorochromes e.g. fluorochrome auramine for M. tuberculosis (glows yellow) fluorescein isothiocyanate (FITC) for B. anthracis (appears apple green)

FLUORESCENCE MICROSCOPE contd

Parts: LIGHT SOURCE : Hg vapor lamp
: Quartz iodine lamp

OPTICAL SYSTEM :

2 barrier filters

1 dichroic beamsplitting mirror

Application: for fluorescent-antibody techniques (immunofluorescence) to rapidly detect and identify microbes in tissues or clinical specimens

FLUORESCENCENCE MICROSCOPE

Fluorescence Microscope Configuration

DARKFIELD MICROSCOPE
Uses a special condenser with an OPAQUE DISC that blocks light from entering the objective lens directly Light reflected by the specimen enters the objective lens, and the specimen appears light against a bright background APPLICATION : examines living microorganisms that are invisible in brightfield microscopy, do not stain easily, or are distorted by staining; frequently used to detect T. pallidum in the diagnosis of syphilis

objective lens

stage

condenser lens

DARKFIELD MICROSCOPE CONFIGURATION

red blood cells under dark field microscope

CONFOCAL MICROSCOPE
A computerized microscope that uses laser light to illuminate one plane of a specimen at a time

Images are taken point-by-point and reconstructed with a computer

Application: obtains 2- or 3-dimensional images of cells for biomedical application

CONFOCAL MICROSCOPE CONFIGURATION

SCANNED-PROBE MICROSCOPE
Uses probes to examine surface of a specimen at a very close range without causing modification or damage Application: 1. maps atomic and molecular shapes 2. characterizes magnetic and chemical properties 3. determines temperature

SCANNED-PROBE MICROSCOPE contd Types:

1. Scanning Tunneling Microscope (STM) - uses a thin metal (tungsten) probe - provides incredibly detailed views of molecules such as DNA

- resolution is greater than that of an EM 2. Atomic Force Microscope (AFM) - uses a metal-and-diamond probe - no special preparation is required - produces a 3D image

Detector

SCANNED PROBE MICROSCOPE CONFIGURATION

flagellae of Pseudomonas putida

UV MICROSCOPE
utilizes quartz, fluorite, and other ultraviolettransmitting lenses the image is made visible by photography, fluorescence of special glasses, or television; in a scanning instrument the receptor is a multiplier phototube.

UV MICROSCOPE

ELECTRON MICROSCOPE
Ultrastructure and subcellular structures

UNIQUE FEATURES : e- beams (W filaments) fluorescent screen/TV monitor electromagnetic field

ELECTRON MICROSCOPE
ADVANTAGES: high resolution; high magnification DISADVANTAGES : Requires vacuum enclosed system high voltage mechanical stability different way of specimen preparation well-trained staff

Two Types of ELECTRON MICROSCOPE

filament condenser lens condenser aperture stage objective lens objective aperture

projector lenses