MICROSCOPE STAGE

● A device used to examine small objects or the fine ● Square platform with an opening at the center
and detailed structure of larger objects. ● It is where the slide is placed during focusing
● The primary tool of the biologist and knowledge of > KINDS OF GLASS THAT CARRIES OR HOLD THE SPECIMEN-
microscopy is essential to those engaged in (Glass slide: rectangle) (Cover slip: square)
studying cells. STAGE CLIPS
● Paired structures on either side of the stage
ACCORDING TO THEIR FUNCTIONS ● It is used to hold the slide in place
● The parts of the compound microscope are MIRROR RACK
generally divided into: 3 ● Found below the stage and attached to the pillar
1. Mechanical part ● Holds the mirror
2. Illuminating part MIM

3. Magnifying part ILLUMINATING AND MAGNIFYING PARTS

MECHANICAL PARTS —> we manipulate
● The mechanical parts comprise all those structures
that support the whole device and which enable
the operator to use the illuminating and
magnifying parts.
BASE
● May be Y- or U-shaped stand that supports the
microscope
PILLAR
● A short piece of metal attached to one end of the
base. It also supports the microscope
HANDLE/ARM
● Curved metallic part arising from the pillar
● It is used in holding the microscope
INCLINATION SCREW/JOINT
● Found at the junction of the pillar and the handle
● It is used to tilt the microscope
/BODY TUBE
● Cylindrical structure arising vertically from the
handle
● It holds the dust shield and the nosepiece with the
objects at the lower end
DRAW TUBE
● Upper and smaller end of the body tube bearing
eyepiece or ocular
REVOLVING NOSEPIECE
● Circular structure at the lower end of the body
tube and above the stage to which the objectives
are attached
DUST SHIELD
● A thin circular structure at the lower end of the
body tube
● It is used to protect the objectives and the
specimen from dust
ADJUSTMENT SCREWS
● Two pairs of wheels attached to either side of the
body (UPPER: COARSE ADJUSTMENT KNOB/SCREW;
LOWER: FINE ADJUSTMENT KNOB/SCREW)
A. COARSE ADJUSTMENT KNOB
● Bigger wheels used to adjust the lower power
objective in focusing
/ B. FINE ADJUSTMENT KNOB
● Smaller wheels used to delicate focusing in
connection with the high power (HPO) and oil
immersion (OIO) objectives
● It is also used to make the specimen more clear
and vivid
Obj
● Made of glass
● Mostly mirror
● The illuminating and magnifying parts consists
mainly of lenses (employed as a means of
magnification) and glass parts
OCULAR OR EYEPIECE
● Found on the draw tube through which the
operator peeps during actual focusing
MIRROR/ILLUMINATOR
● Found below the stage near the base provided
with concave and plane surfaces
● It is used to collect and directlight to the specimen
● Has 2 sided >CONCAVE FACE>FLAT/CONVEX FACE
● Concave Mirror: Used when there is light available
● Flat/Convex Mirror: Used when viewing specimens
in bright light or natural light
IRIS DIAPHRAGM
● Found below the stage consisting of regularly
arranged circular blades
● Used to regulate a central opening to decrease or
increase the light reflected on the object
CONDENSER
● A lense found immediately beneath the hole of
the stage
● Used to concentrate light rays on the specimen
OBJECTIVES
● Tube-like structures attached to the revolving
nosepiece
A. LOWER POWER OBJECTIVE (LPO)
● Often marked with 10x or 6x
● It is usually shorter than the other two objectives
● It usually forms the general outline or wider portion
of the object
B. HIGH POWER OBJECTIVE (HPO)
● Often marked 45x or 43x
● Longer than LPO
● It forms a bigger image of the object in focus
● Used to examine living microorganisms suspended
in drops of water
● Used to enlarged specimens that are so small
under LPO
C. OIL IMMERSION OBJECTIVE 1101 )
● Often marked with 100x or 90x
● Has the biggest degree of magnification and is
used to examine stained smear preparations of
microorganisms using immersion oil as its medium
● Use of oil reduces the refraction of light to help
create a clear image
.

power Obj .

Low
LPO
-
power
HPO
-
High
Oil
Immersion
-

010 opj .
TOTAL MAGNIFICATION:
● The number tells the operator how many times the ● Makes use of visible light - a wavelength varying
focused specimen is magnified from 380 to 770 nanometers
FORMULA: ● In a compound microscope the image from the
● Total magnification = magnifying power of the objective lens is magnified again by the ocular lens
eyepiece x magnifying power of the objective FORMULA:

:
used Total Magnification = objective lens x ocular lens

EXAMPLE: COMPOUND MICROSCOPE

- Magnifying power of the eyepiece = 10
- magnifying power of the objective used = 100
(OIO)
Therefore:
10 x 100 = 1000x
- The specimen in focus is magnified 1000 times its
actual size using the oil immersion objective
OBSERVING MICROORGANISMS THROUGH A MICROSCOPE
UNITS OF MEASUREMENT
● Meter (m)
● Micrometer (um)
● Nanometer (nm) [smallest measurement such as
virus, prions]
● Microorganisms are measured in micrometers (um)
10^-6 m (is equal to 1 meter), and in nanometer
(nm) 10^-9 m
● 1 um = 10^-6 m = 10^-3 mm
● 1 nm = 10^-9 m = 10 ^-6 mm
● 1000 nm = 1 um
● Uses visible light
● Resolution or resolving power is the ability of the
lenses to distinguish 2 points (fine detail and
structure)
● A microscope with a resolving power of 0.4 nm
can distinguish between two points >/= 0.4 nm
● Resolving the power of a compound light
microscope is 2.0 um; maximum magnification is
2000x nn
● Shorter wavelengths of light provide greater
resolution
● Refractive index iis the light bending ability of a
medium —> oil immersion objective (oio)
● 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
● Immersion oil is used with the oil immersion lens to
reduce light loss between the slide and the lens
TYPES OF COMPOUND MICROSCOPE
Big PI ¥ 1. Brightfield Illumination/Microscope
BD PDF
t
£ 2. Darkfield Illumination/Microscope
DF

● 0.001 um = 1 nm DIC 3. Phase-Contrast Microscope

NOMARSKI 4. Differential Interference Contrast Microscope (DIC)
norm OR -

HISTORICAL NOTE: ski

or Nomarski Microscope
● During the late 1600’s, Anton Van Leeuwenheok 5. Fluorescence Microscope
used the simple microscopes to observe tiny
objects 1. BRIGHTFIELD ILLUMINATION/MICROSCOPE
● Had a maximum magnifying power of about x 300 ● Dark objects are visible against a bright
(300 times) background
● Light reflected off the specimen does not enter the

{
EARLY COMPOUND MICROSCOPE objective lens
Zaccharias Janssen ● Used for stained smear (Gentian violet) -

● Optician in middleburg, Holland, developed the

1st Compound Microscope around 1600 opn.ae, , 2. DARKFIELD ILLUMINATION/MICROSCOPE
● Poor quality and not able to see bacteria DISK
● Useful in viewing unstained specimens
Joseph Jackson Lister (father of Joseph Lister) ● Light objects are visible against a dark background
● Various improvement ● Light reflected off the specimen enters the
objective lens
MICROSCOPE ● Most useful for detecting the presence of
● Refers to an instrument which allows one to view extremely small organisms
manute object, which are normally too sml to be ● Used to view spirochetes
resolved by the naked eye I.
small
> A brightfield microscope can be converted to a darkfield
4 TYPES OF MICROSCOPE microscope by replacing the condenser on a brightfield

]
you TECH
-

1. Simple Light Microscope microscope with a darkfield condenser

2. Compound light Microscope SEES
3. Electron Microscope I /
3. PHASE-CONTRAST MICROSCOPY
HIGH -
TECH
4. Scanned-probe Microscope ● Accentuates diffraction or direct and reflected of
the light that passes through a specimen, around
2 KINDS OF LIGHT MICROSCOPE the specimen
1. Simple Light Microscope: has only one convex lens ● Have a bright ring of light surrounding them
- magnifying glass ● Allows the detailed observation of living organisms
2. Compound Microscope: has 2 lenses; magnifying ● Permits detailed examination of the internal
lens system - ocular lens and objective lens structures of microorganisms such as endospores
 ● Useful in the study of binary fission and motility
SCANNING-PROBE MICROSCOPY
4. DIFFERENTIAL INTERFERENCE CONTRAST ● Scanning tunneling microscopy (STM) uses a metal
→ uses 3D
MICROSCOPY probe to scan a specimen
image
● Called as “NOMARSKI MICROSCOPE ● Resolution 1/100 of an atom
● Accentuates diffraction of the light that passes ● Atomic force microscopy (AFM) uses a metal and
through a specimen; uses 2 beams of light diamond probe inserted into specimen
● Provides a colored, 3-dimensional image of the ● Produces 3D images
object being observed ● Used to study the molecular and atomic shapes of
● It allows detailed observations of living cells organisms
● Determine the variations in temperature inside the
5. FLUORESCENCE MICROSCOPY cell and chemical properties
● Uses UV light
● Fluorescence absorb UV light and emit visible light
● Cells may be stained with fluorescence dyes
(FLUOROCHROMES) such as chlorophyll
● Microorganisms appear as bright objects againts a
dark background
● Fluorescence microscopy is used primarily in a
diagnostic procedure called
“FLUORESCENT-ANTIBODY (FA)” technique

CONFOCAL MICROSCOPY —> well distincted, and clear

specimen
● Uses fluorochromes or fluorescent dye and a laser
light
● The laser illuminates each plane in a specimen to
produce a 3-D image
● Using a computer to process the images,
2-dimensional and 3-dimensional images of cells
can be produced
● Used to study the physiology of cell

ELECTRON MICROSCOPY
● Uses electrons instead of light
● The shorter wavelength of electrons gives greater
resolution
● -
Electromagnets, instead of glass lenses, control
-

focus, illumination and magnification

● Used to observe extremely small infectious agents
such as virus
● Cannot be used to observe living organism

-
because it will die in the use of electromagnets

KINDS OF ELECTRON MICROSCOPE

1. Transmission Electron Microscopy (TEM)
2. Scanning Electron Microscopy (SEM)

1. TRANSMISSION ELECTRON MICROSCOPY

● Ultrathin sections of specimens
● Light passes through specimen, then an
electromagnetic lens, to a screen ir film
● Specimens may be stained with heavy metal salts
● 10,000-100,000x; resolution 2.5 nm (mas mazoom in
compared to scan electron microscope
2. SCANNING ELECTRON MICROSCOPY
● An electron gun produces a beam of electrons
that scans the surface of a whole specimen
● Secondary electrons emitted from the specimen
produce the image
● Enables scientists to observe living cells at
extremely high magnification and resolution under
physiological resolution