University of Guyana/DMT 3108/Lecture 6 Light Microscopy/ 2017-18

DMT 3108 General & Environmental Microbiology

Lecture 6 Principles of Light Microscopy

Measurement
Bacteria
0.2m 10m, typically 1-2m in diameter
Chlamydiae ---- approx 0.25 m
Mycoplasmas ---- 0.15-0.3m dia
Protozoa - 10-100m; some as large as 1000m
Viruses - 20-250nm

BRIGHTFIELD MICROSCOPE
Ordinary microscope= BF microscope
Dark image against a brighter background
Simple microscope single biconcave lens
Compound microscope
2 separate lens system
Achieves greater magnification
Microscope has an optical role (magnification) and an illumination role (renders
specimens properly visible)

MICROSCOPE

Arm

Nosepiece

Base

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University of Guyana/DMT 3108/Lecture 6 Light Microscopy/ 2017-18

PARTS OF THE BINOCULAR MICROSCOPE & THEIR USES

Consists of a sturdy metal body or stand composed of a base and an arm to which the
remaining parts are attached
Light source is located at the base
Mirror or electric illuminator

Two focusing knobs located on the arm and can move either the stage or nosepiece to
focus the image
Coarse and fine adjustment knobs
Stage positioned about halfway up the arm and holds microscope slides by either simple
clips or a mechanical stage clip
Mechanical stage allows the microscopist to move the slide around smoothly by
use of stage control knobs

Substage condenser mounted within or beneath the stage

Focuses a cone of light on to the slide
Position fixed in simpler microscopes but can be adjusted vertically in more
advanced models
As the magnification of the objective lens increases, more light is needed
The iris diaphragm (usually located in the condenser), regulates the amount of light
reaching the specimen

Upper Part of the arm holds a revolving nosepiece to which one or more eyepieces or
ocular lenses are attached
Also attached to the nosepiece ---- objective lens (usually 4)
Objective lens ---- varying magnifying power
Objective lens forms an enlarged real image --- not seen by the microscopist
The ocular lens magnifies the real image yielding a virtual image which is seen by the
eye
Total magnification
Eyepiece X Objective

RESOLUTION/RESOLVING POWER
The most important part of the microscope is the objective, which must produce a clear
image, not just a magnified one
Resolution/RP
Ability of a lens to separate or distinguish between small objects that are close
together (as distinct and separate)
RP important indicates the size of the smallest object that can be seen clearly
RP =
2 X NA
Numerical Aperture refers to the size of the cone of light that will enter the objective lens
and the medium in which the lens is suspended; usually air
halfway point between the limits of visible light. Set at 550nm

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University of Guyana/DMT 3108/Lecture 6 Light Microscopy/ 2017-18

Calculate the RP for X10 objective lens

Calculate the RP for the X100 objective lens

WORKING DISTANCE
Distance from the front lens element of the objective to the closest surface of the
coverslip when the specimen is in sharp focus
For objectives designed to be used without coverslips,
the working distance is determined by the linear
measurement of the objective front lens to the specimen
surface
Working distance decreases as the magnification and
numerical aperture both increase

USE OF IMMERSION OIL

Immersion oil increases resolution not magnification

Oil needed b/c light bends abruptly as it leaves the glass slide and enters the air

Both LP and HP objectives are wide enough to capture sufficient light

Oil immersion is so narrow that most light rays bend away and miss objective
Oil has the same RI as glass --- 1.5; RI for air is 1.0
Light does not bend as much when moving from glass to oil