Chapter 1.

MICROSCOPY TECHNIQUES
Microbiological Technology in Medicine
Ngoc Hoang Trinh
CONTENTS

WEEK LESSON CONTENTS

Chapter 1. MICROSCOPY TECHNIQUES

1.1 Introduction
1.2 Light microscopy
01
01 1.3 Phase-contrast and dark field microscopy
02
1.4 Confocal laser scanning microscopy
03
1.5 Transmission Electron Microscopy (TEM)
1.6 Scanning Electron Microscopy (SEM)
Chapter 1. MICROSCOPY TECHNIQUES

1.1 Introduction
 Terms

 The word microscope is derived from the Greek words micros (small)
and skopeo (look at)

 The microscope is the microbiologist’s oldest and most fundamental tool

for studying microorganisms

 Microbiology did not exist before the invention of the microscope.

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1.1 Introduction

 History of microscopy

 The first known description of microorganisms is famous book

Micrographia (1665) by Robert Hooke

• The first book devoted to microscopic observations

• Hooke illustrated many microscopic images

including the fruiting structures of molds
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1.2 Introduction

 The first person to see bacteria was the Dutch

draper and amateur microscopist Antoni van
Leeuwenhoek (1632–1723).

• Van Leeuwenhoek constructed extremely simple

microscopes containing a single lens to examine
various natural substances for microorganisms
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1.2 Introduction
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1.1 Introduction
 Applications

 Today, light microscopy is used not only in microbiology, pathology, and

cell biology but also in metallurgy, materials science, computer chip
design, and microsurgical applications.
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1.2 Light microscopy

 In a light microscope the sample is illuminated with visible light.

 Magnification
 describes the capacity of a microscope to enlarge an image. All
microscopes employ lenses that provide magnification.

 Resolution

 is the ability to distinguish two adjacent objects as distinct and separate.

The limit of resolution for a light microscope is about 0.2 μm

• two objects that are closer together than 0.2 μm cannot be resolved as distinct
and separate.
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1.2 Light microscopy

 The modern compound light microscope contains two types of lenses,

objective and ocular, that function in combination to magnify the image.

 Ocular lenses & objective lenses

 Microscopes used in microbiology have ocular lenses that magnify 10–

30X and objective lenses that magnify 10–100X

 The total magnification = the ocular lens’s magnification x the objective

lens’s magnification
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1.2 Light microscopy

 Limit of resolution for most light microscopes

 Magnification of 1000X is required to resolve objects 0.2 μm in diameter,

which is the limit of resolution for most light microscopes

 With 100X objective, an optical grade oil is placed between the

microscope slide and the objective

• Lenses on which oil is used are called oil-immersion lenses.

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1.2 Light microscopy

Chapter 1. MICROSCOPY TECHNIQUES

1.2 Light microscopy

 Improving contrast in light microscopy

 In light microscopy, specimens are visualized because of differences in

contrast that exist between them and their surroundings

 Bacterial cells typically lack contrast, and hence they are difficult to see
well with the bright-field microscope.

 There are several ways to boost contrast

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1.2 Light microscopy

 Improving contrast in light microscopy

 Cells can be stained to improve contrast, and staining is commonly used

to visualize bacteria with bright-field microscopy.

• Dyes can be used to stain cells and increase their contrast

so that they can be more easily seen in the bright-field
microscope

• An important differential-staining procedure used in

microbiology is the Gram stain
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1.2 Light microscopy

 Gram stain

 Bacteria can be divided into two major groups: gram-positive

and gram-negative.

 Which colors appear after Gram-stain?

 Why do bacteria have different colors after Gram-stain?

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1.2 Light microscopy

 Gram stain
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1.3 Phase-contrast and dark field microscopy

 Staining often kills cells and can distort their features. Two forms of light
microscopy improve image contrast of live cells. These are phase-contrast
microscopy and dark-field microscopy

 Phase-contrast microscopy

 based on the principle that cells differ in refractive index (that is, the ability
of a material to alter the speed of light) from their surroundings.
 Light passing through a cell thus differs in phase from light passing
through the surrounding liquid.
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1.3 Phase-contrast and dark field microscopy

 Phase-contrast microscopy

 This subtle difference is amplified by a device in the objective lens of the

phase-contrast microscope called the phase ring, resulting in a dark image
on a light background

Cells visualized by different types of light microscopy. The same field of cells of Saccharomyces cerevisiae
visualized by (a) bright-field microscopy, (b) phase-contrast microscopy, and (c) dark-field microscopy.
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1.3 Phase-contrast and dark field microscopy

 The dark-field microscope

 light does not pass through the specimen. Instead, light is directed from the
sides of the specimen and only light that is scattered when it hits the
specimen can reach the lens. Thus, the specimen appears light on a dark
background

 Dark-field microscopy often has better resolution than light

microscopy

 Dark-field microscopy is a particularly good way to

observe microbial motility, as bundles of flagella
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1.3 Phase-contrast and dark field microscopy

 The dark-field microscope
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1.4 Confocal laser scanning microscopy

 A confocal scanning laser microscope (CSLM) is a computer controlled
microscope that couples a laser to a fluorescence microscope.

 The laser generates a high-contrast, three-dimensional image and allows

the viewer to access several planes of focus in the specimen
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1.4 Confocal laser scanning microscopy

 The laser beam is precisely adjusted such that only a particular layer within
a specimen is in perfect focus at one time.

 Cells struck by the laser fluoresce to generate the image as in fluorescence

microscopy.
 Cells in CSLM preparations can also be stained with fluorescent dyes to
make them more distinct.

 The laser then scans up and down through the layers of the sample,
generating an image for each layer.

 A computer assembles the pictures to compose the many layers into a

single high-resolution, three dimensional image
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1.4 Confocal laser scanning microscopy

 CSLM is particularly useful when thick specimens need to be examined.

Figure Confocal scanning laser microscopy. (a) Confocal image of a microbial biofilm community. The
green, rod-shaped cells are Pseudomonas aeruginosa experimentally introduced into the biofilm. Cells
of different colors are present at different depths in the biofilm. (b) Confocal image of a filamentous
cyanobacterium growing in a soda lake
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1.5 Transmission Electron Microscopy (TEM)

 The transmission electron microscope (TEM) is used to examine cells and

cell structure at very high magnification and resolution.

 The resolving power of a TEM is much greater than that of the light
microscope, even allowing one to view structures at the molecular level

 Whereas the resolving power of a light microscope is about 0.2 micrometer,

the resolving power of a TEM is about 0.2 nanometer, a thousandfold
improvement.
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1.5 Transmission Electron Microscopy (TEM)

 The transmission electron microscope (TEM) is used to examine cells and
cell structure at very high magnification and resolution.

Figure electron micrographs. (a) Micrograph of a thin section of a dividing bacterial cell, taken by transmission
electron microscopy (TEM). (b)TEM of negatively stained molecules of hemoglobin.
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1.5 Transmission Electron Microscopy (TEM)

 If only the external features of an organism are to be observed, thin sections
are unnecessary. Intact cells or cell components can be observed directly in
the TEM by a technique called negative staining

TEM at College of Agriculture and Life Sciences, Seoul National University

TEM image indicated cell morphology of strain Paraburkholderia flava LD6T
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1.6 Scanning Electron Microscopy (SEM)

 For optimal three-dimensional imaging of cells, a scanning electron
microscope (SEM) is used.

 In scanning electron microscopy, the specimen is coated with a thin film of a

heavy metal, typically gold.

 An electron beam then scans back and forth

across the specimen.

 Electrons scattered from the metal coating

are collected and projected on a monitor to
produce an image
Chapter 1. MICROSCOPY TECHNIQUES

1.6 Scanning Electron Microscopy (SEM)

 A wide range of magnifications can be obtained with the SEM, from as low
as 15:up to about 100,000:, but only the surface of an object is typically
visualized

 Electron micrographs taken by either TEM or

SEM are black and-white images.