Microscopy

Mariejim Diane P. Lee, RMT, MSMT

OUTLINE:

Types of Microscopes
Parts of a Compound Microscope
Focusing
Terminologies
Care for the Microscope

SEEING IS STILL BELIEVING.

Chromosomes from a glioblastosma
sample using spectral karyotyping

Microscope from the Gk words:

Mikros small
Skopein to see or look
To see objects that are too
small for our naked eye

How does it work?

Magnification
Resolution
Contrast

Light Microscopes

Bright field
Dark field
Phase Contrast
Polarizing

DIC
Fluorescent
Confocal Laser
Scanning

Electron Microscopes
Transmission Electron Microscope
(TEM)

Scanning Electron Microscope

(SEM)

Bright field Microscopy

Most common
Background bright
Image dark
Ideal: bacteria, cells
and tissues

Dark field Microscopy

Background dark
Image bright
Ideal: unstained, live and
motile specimens
Only oblique light strikes
the specimen

Phase Contrast Microscopy

Special condensers that
throw light out of phase,
passing through the object at
different speeds
Ideal: unstained and live
mounts
Bacteria, flagella, cilia,
organelles (mitochondria)
Only oblique light strikes the
specimen

Polarization Microscopy
Background dark
Image bright
Ideal: birefringent (double
refraction), highly
organized molecular
structure substances
2 polarizers

DIC Microscopy
Differential interference
contrast
Resembles PCM but more
sensitive (3D)
Ideal: unstained and live
mounts
Membranes within cells
Polarizing lenses

Fluorescence Microscopy
UV as light source
UV hits object excites the
electron various shades of
colors

Background dark
Image bright
High resolution
Immunofluorescence

Confocal Microscopy
Uses a laser light
Image placed on a digital
computer screen for analyzing
Sharp image, high sensitivity
Eliminates background
interference

Electron Microscope

Beam of electrons
Higher magnification
Greater resolving power
Finer detail; viruses
Electrons travel in a vacuum
Image is created on a monitor

TEM
2D view
Thins/ultrathin slices of
microorganisms
Coated before viewing
Densely coated part deflect
the electron beam
Both dark and light areas
show up

SEM
Detailed structure
3D image
Lower magnification than
TEM
Specimen is coated (gold
or palladium); black and
white pictures

Parts of a Compound Microscope

Structural Parts
Head/Body: contains the
optical parts
Base: support; contains
the illuminator
Arm: connector between
the head and the base

Parts of a Microscope

Parts of a Compound Microscope

Mechanical Parts
Base

V or U-shaped structure for support

Pillar

Vertical extension of the base to which the arm is attached

Arm

Curved basic part; handle; support for the optical parts

Inclination Joints

Movable parts which facilitate tilting of the microscope

Stage

Platform where you put the slide with specimen; provided

with a central aperture that allows light to pass

Stage clips

Pair of metals holding the slide in place

Body tube

Hollow cylinder; housing the lenses; objective to eyepiece

Draw tube

Smaller cylinder holding the eyepiece

Dust shield

Fixed plate (body tube); protects the objectives

Revolving nosepiece

Rotary head holding the objectives; shifting

Adjustment knobs

Adjust the objectives

Parts of a Compound Microscope

Illuminating Parts
Mirror

2-faced; one is plain or flat and concave;

reflects light; frame is held by mirror rack or
fork
Plain: bright light; Concave: low intensity
natural/artificial source
Abbe condenser Under the stage; fixed or provided with
adjustment for lowering/raising; illuminate,
condense or concentrate the reflected light
Iris diaphragm
Plate that is supported with numerous
apertures of varying sizes to regulate the
amount of light that enters the condenser

Parts of a Compound Microscope

Magnifying Parts
Eyepiece or ocular

Detachable tube on top of the draw tube; dark pointer

Objectives
Scanner

Shortest; widest lens opening; lowest magnification;

observe wider field of object

LPO

Shortest tube with large lens opening; 10x; general outline

of the object; locate various parts of the specimen

HPO

Longer tube with smaller lens opening; 40-45x; details of

the specimen

OIO

Longest tube with a very small lens opening; 90-100x;

cedar wood oil; lowered until touches the oil to prevent
refraction of light

Body Tube

The body tube holds the objective lenses and the

ocular lens at the proper distance

Diagram

Nose Piece
The Nose Piece holds the objective lenses and can be
turned to increase the magnification

Diagram

Objective Lenses
The Objective Lenses increase magnification (usually
from 10x to 40x)

Diagram

Stage Clips
These 2 clips hold the slide/specimen in place on the
stage.

Diagram

Diaphragm
The Diaphragm controls the amount of light on the
slide/specimen

Turn to let more light in or to

make dimmer.

Light Source
Projects light upwards through the diaphragm, the
specimen and the lenses
Some have lights, others have mirrors where you
must move the mirror to reflect light

Diagram

Ocular Lens/Eyepiece
Magnifies the specimen image

Diagram

Arm
Used to support the microscope when carried. Holds
the body tube, nose piece and objective lenses

Diagram

Stage
Supports the slide/specimen

Diagram

Coarse Adjustment Knob

Moves the stage up and down (quickly) for focusing
your image

Diagram

Fine Adjustment Knob

This knob moves the stage SLIGHTLY to sharpen the
image

Diagram

Base
Supports the microscope

Diagram

Standard Objective Nomenclature

Parts of a Compound Microscope

Terminologies in Microscopy
Magnification
Ratio of the apparent size of an
object as seen thru the
microscope and the actual size
of the object
Focusing
Adjustment of the distance
between the objective lens &
the specimen; clear image
Virtual or floating image
Image seen in microscopic field

Terminologies in Microscopy
Microscopic field
Bright circular illuminated area as seen
through the eyepiece
Working distance
Between the objective lens and the top
of the cover glass
Higher magnification, the shorter is the
working distance (OIO)
Angular aperture
Angle/cone of light rays capable of
entering the front lens of the objective
from a point in the object

Terminologies in Microscopy
Depth of focus
Thickness of the object
that may be seen at one
time under focus; it
increases with a decrease
in the NA and
magnification

Numerical aperture
Measurement of the
ability of the condenser
and objective lens to
gather light; higher
magnification of the
objective, large the NA

Terminologies in Microscopy
Resolving power
Ability of the lens to clearly
separate or distinguish 2
points/images; determined
by the shortest wavelength
of visible light and max. NA
Parfocal
Objectives & eyepiece
where no change in focus
has to be made during
shifting of objectives
Refraction
Bending of light rays away
from the objective lens
when light passes from the
glass slide to the air

Care for the Microscope

Care for the Microscope

Check for loose or missing parts. Report immediately to
your instructor.
Never touch the lenses with your finger.
Never allow liquids to come in contact with any part of
the microscope.
Do not tilt unless necessary.
Do no force the lever of the diaphragm to full limit.

Care for the Microscope

Observe the following guidelines before you keep the
microscopes inside the cabinet:
1. The body tube is lowered; objective does not
touch the condenser.
2. Upright position with LPO/scanner in place
3. Stage clips are loosely set and in forward
direction
4. Diaphragm is wide open
5. Mirror is in vertical position and is facing forward