Microscopy

Microscopy

• Microscopy is a technical field that involves the use of Microscopical

components such as microscopes or microscope objectives to obtain
greater detail of examined samples.
• Microscope is used to view objects or specimens that are too small to be
seen with just the human eye.
• Definition-A microscope is a high precision optical instrument that uses a
lens or a combination of lenses to produce highly magnified images of
small specimens or objects especially when they are too small to be seen
by the naked (unaided) eye.
• A light source is used (either by mirrors or lamps) to make it easier to see
the subject matter.
 Peering Into the Invisible World (1 of 5)

Figure 2.9 (a) Antonie van Leeuwenhoek (1632–1723) is credited as being

the first person to observe microbes, including bacteria, which he called
“animalcules” and “wee little beasties.” (b) Even though van Leeuwenhoek’s
microscopes were simple microscopes (as seen in this replica), they were
more powerful and provided better resolution than the compound
microscopes of his day. (c) Though more famous for developing the
telescope, Galileo Galilei (1564–1642) was also one of the pioneers of
microscopy. (credit b: modification of work by “Wellcome Images”/Wikimedia
Commons)
 Peering Into the Invisible World (2 of 5)

• While van Leeuwenhoek is

credited with the discovery of
microorganisms, others before
him had contributed to the
development of the microscope.

• These included eyeglass makers

in the Netherlands in the late
1500s (Hans and Zacharias
Janssen), as well as the Italian
astronomer Galileo Galilei, who
used a compound microscope
to examine insect parts.

Figure 2.11 shows a portrait

of Zacharias Janssen.
 Peering Into the Invisible World (3 of 5)

• Whereas van Leeuwenhoek used a

simple microscope, in which light is
passed through just one lens, Galileo’s
compound microscope was more
sophisticated, passing light through two
sets of lenses.
 Peering Into the Invisible World (4 of 5)

• Van Leeuwenhoek’s contemporary, the

Englishman Robert Hooke (1635–1703),
also made important contributions to
microscopy.

• Robert Hooke was the first to observe the

structures that we now know as cells.
Peering Into the Invisible World (5 of 5)

Figure 2.10 Robert Hooke used his (a) compound microscope to

view (b) cork cells. Both of these engravings are from his seminal
work Micrographia, published in 1665.
 TERMS AND DEFINITIONS
Principle
Microscopy is to get a magnified image, in which structures may be resolved
which could not be resolved with the help of an unaided eye.
Magnification
•It is the ratio of the size of an object seen under microscope to the actual size
observed with unaided eye.
•The total magnification of microscope is calculated by multiplying the
magnifying power of the objective lens by that of eye piece.
Resolving power
•It is the ability to differentiate two close points as separate.
•The resolving power of human eye is 0.25 mm
•The light microscope can separate dots that are 0.25µm apart.
•The electron microscope can separate dots that are 0.5nm apart.
 TERMS AND DEFINITIONS
Limit of resolution
•It is the minimum distance between two points to identify them separately.
•It is calculated by Abbé equation.

Working distance
•It is the distance between the objective and the objective slide.
•The working distance decreases with increasing magnification.
 TERMS AND DEFINITIONS
Numerical aperture(NA)
The numerical aperture of a lens is the ratio of the diameter of the lens to its focal
length.

NA can be decreased by decreasing the amount of light that passes through a lens.

Diameter of the lens

 Instruments of Microscopy
• The early pioneers of microscopy opened a window into
the invisible world of microorganisms.

• In 1830, Joseph Jackson Lister created an essentially

modern light microscope.

• The 20th century saw the development of microscopes

that leveraged nonvisible light.

• These advances led to major improvements in

magnification, resolution, and contrast.
 Light Microscopy
• Many types of microscopes fall under the
category of light microscopes, which use light
to visualize images.

• Light microscopes include:

– Brightfield microscopes
– Darkfield microscopes
– Phase-contrast microscopes
– Fluorescence microscopes
– Confocal scanning laser microscopes
 Brightfield Microscopes (1 of 12)

• The brightfield microscope is a compound

microscope with two or more lenses that
produce a dark image on a bright
background.

• Some brightfield microscopes are monocular

(having a single eyepiece), though most
newer brightfield microscopes are binocular
(having two eyepieces).
 Brightfield Microscopes (2 of 12)

• Each eyepiece contains a lens called an

ocular lens, which typically magnify
images 10 times (10x).

• At the other end of the body tube are a set

of objective lenses on a rotating
nosepiece. The magnification of these
objective lenses typically ranges from 4x
to 100x.
 Brightfield Microscopes (3 of 12)

• The ocular and objective lenses work

together to create a magnified image.

• The total magnification is the product of

the ocular magnification times the
objective magnification:

ocular magnification x objective magnification

 Brightfield Microscopes (4 of 12)

Figure 2.12 Components of a typical brightfield microscope.

 Brightfield Microscopes (5 of 12)

• The item being viewed is called a specimen. The specimen is

placed on a glass slide, which is then clipped into place on the
stage (a platform) of the microscope.

• Once the slide is secured, the specimen on the slide is positioned

over the light using the x-y mechanical stage knobs.

• These knobs move the slide on the surface of the stage, but do not
raise or lower the stage.
 Brightfield Microscopes (6 of 12)

• Once the specimen is centered over the light,

the stage position can be raised or lowered to
focus the image.

• The coarse focusing knob is used for large-

scale movements with 4x and 10x objective
lenses.

• The fine focusing knob is used for small-

scale movements, especially with 40x and
100x objective lenses.
 Brightfield Microscopes (7 of 12)

• The illuminator provides intense lighting

typically due to a high-intensity bulb below
the stage.

• Light from the illuminator passes up

through the condenser lens (located
below the stage), which focuses all of the
light rays on the specimen to maximize
illumination.
 Brightfield Microscopes (8 of 12)

• If less-than-maximal light levels are needed,

the amount of light striking the specimen can
be easily adjusted by opening or closing a
diaphragm between the condenser and the
specimen.

• In some cases, brightness can also be

adjusted using the rheostat, a dimmer switch
that controls the intensity of the illuminator.
 Brightfield Microscopes (9 of 12)

• In a brightfield microscope, different colors

can behave differently as they interact with
chromophores (pigments that absorb and
reflect particular wavelengths of light) in
parts of the specimen.

• Chromophores are artificially added to the

specimen using stains, which serve to
increase contrast and resolution.
 Brightfield Microscopes (10 of 12)

• At very high magnifications, resolution

may be compromised when light passes
through the small amount of air between
the specimen and the lens.

• This is due to the large difference between

the refractive indices of air and glass. The
air scatters the light rays before they can
be focused by the lens.
 Brightfield Microscopes (11 of 12)

• A drop of oil can be used to fill the space

between the specimen and an oil immersion
lens, a special lens designed to be used with
immersion oils.

• Since the oil has a refractive index very

similar to that of glass, it increases the
maximum angle at which light leaving the
specimen can strike the lens.

• This increases the resolution of the image.

 Brightfield Microscopes (12 of 12)

Figure 2.13 (a) Oil immersion lenses like this one are used to improve
resolution. (b) Because immersion oil and glass have very similar refractive
indices, there is a minimal amount of refraction before the light reaches the
lens. Without immersion oil, light scatters as it passes through the air above
the slide, degrading the resolution of the image.
 Darkfield Microscopes (1 of 5)

• A darkfield microscope is a brightfield

microscope that has a small but significant
medication to the condenser.

• A small, opaque disk (about 1 cm in

diameter), which is placed between the
illuminator and the condenser lens, blocks
most of the light from the illuminator as it
passes through the condenser on its way to
the objective lens.
 Darkfield Microscopes (2 of 5)

• The only light that reaches the objective is light that has
been refracted or reflected by structures in the
specimen.

• The resulting image typically shows bright objects on a

dark background.
 Darkfield Microscopes (3 of 5)

Figure 2.14 An opaque light stop inserted into a brightfield

microscope is used to produce a darkfield image. The light
stop blocks light traveling directly from the illuminator to the
objective lens, allowing only light reflected or refracted off the
specimen to reach the eye.
 Darkfield Microscopes (4 of 5)

• Darkfield microscopy can often create

high-contrast, high-resolution images of
specimens without the use of stains. This
is particularly useful for viewing live
specimens that might be killed or
otherwise compromised by the stains.
 Darkfield Microscopes (5 of 5)

Figure 2.15 Use of a darkfield microscope allows us to view

living, unstained samples of the spirochete Treponema
pallidum. Similar to a photographic negative, the spirochetes
appear bright against a dark background. (credit: Centers for
Disease Control and Prevention)
 Phase-Contrast Microscopes (1 of 6)

• Phase-contrast microscopes use

refraction and interference caused by
structures in a specimen to create high-
contrast, high-resolution images without
staining.

• This microscope creates an image by

altering the wavelengths of light rays
passing through the specimen.
 Phase-Contrast Microscopes (2 of 6)

• To create altered wavelength paths, an

annular stop is used in the condenser.

• The annular stop produces a hollow cone

of light that is focused on the specimen
before reaching the objective lens.

• The objective contains a phase plate

containing a phase ring.
 Phase-Contrast Microscopes (3 of 6)

• As a result, light traveling directly from the

illuminator passes through the phase ring while
light refracted or reflected by the specimen passes
through the plate.

• This causes waves traveling through the ring to be

about one-half of a wavelength out of phase with
those passing through the plate.

• Ultimately, structures that differ in features such as

refractive index will differ in levels of darkness.
 Phase-Contrast Microscopes (4 of 6)

Figure 2.16 This diagram of a phase-contrast microscope illustrates phase

differences between light passing through the object and background.
These differences are produced by passing the rays through different parts
of a phase plate. The light rays are superimposed in the image plane,
producing contrast due to their interference.
 Phase-Contrast Microscopes (5 of 6)

• Phase-contrast microscopy is often used to observe live

specimens.

• Certain structures, such as organelles in eukaryotic cells

and endospores in prokaryotic cells, are especially well
visualized with phase-contrast microscopy.
 Phase-Contrast Microscopes (6 of 6)

Figure 2.17 This figure compares a brightfield image (left) with a

phase-contrast image (right) of the same unstained simple squamous
epithelial cells. The cells are in the center and bottom right of each
photograph (the irregular item above the cells is acellular debris).
Notice that the unstained cells in the brightfield image are almost
invisible against the background, whereas the cells in the phase-
contrast image appear to glow against the background, revealing far
more detail.
 Fluorescence Microscopes (1 of 8)

• A fluorescence microscope uses fluorescent chromophores called

fluorochromes, which are capable of absorbing energy from a light
source and then emitting this energy as visible light.

• Fluorochromes include naturally fluorescent substances (such as

chlorophylls) as well as fluorescent stains that are added to the
specimen to create contrast.
 Fluorescence Microscopes (2 of 8)

• The microscope transmits an excitation light, generally

a form of EMR with a short wavelength toward the
specimen.

• The chromophores absorb the excitation light and emit

visible light with longer wavelengths.

• The excitation light is then filtered out (in part because

ultraviolet light is harmful to the eyes) so that only
visible light passes through the ocular lens.

• This produces an image of the specimen in bright

colors against a dark background.
 Fluorescence Microscopes (3 of 8)

• Fluorescence microscopes are especially

useful in clinical microbiology.

• They can be used to identify pathogens, to

find particular species within an
environment, or to find the locations of
particular molecules and structures within
a cell.
 Fluorescence Microscopes (4 of 8)

• One of the most important applications of

fluorescence microscopy is a technique
called immunofluorescence, which is used
to identify certain disease-causing microbes
by observing whether antibodies bind to
them.

• There are two approaches to

immunofluorescence:
– Direct immunofluorescence assay (DFA)
– Indirect immunofluorescence assay (IFA)
 Fluorescence Microscopes (5 of 8)

• In direct immunofluorescence assay (DFA),

specific antibodies are stained with a
fluorochrome.

• If the specimen contains the targeted pathogen,

one can observe the antibodies binding to the
pathogen under the fluorescent microscope.

• This is called a primary antibody stain because the

stained antibodies attach directly to the pathogen.
 Fluorescence Microscopes (6 of 8)

• In indirect immunofluorescence (IFA),

secondary antibodies are stained with a
fluorochrome.

• Secondary antibodies do not attach

directly to the pathogen, but they do bind
to primary antibodies.
 Fluorescence Microscopes (7 of 8)

• When the unstained primary antibodies

bind to the pathogen, the fluorescent
secondary antibodies can be observed
binding to the primary antibodies. Thus,
the secondary antibodies are attached
indirectly to the pathogen.

• IFA increases the number of fluorescent

antibodies attached to the specimen.
Fluorescence Microscopes (8 of 8)

Figure 2.19 (a) A direct immunofluorescent

stain is used to visualize Neisseria
gonorrhoeae, the bacterium that causes
gonorrhea. (b) An indirect
immunofluorescent stain is used to
visualize larvae of Schistosoma mansoni, a
parasitic worm that causes schistosomiasis,
an intestinal disease common in the tropics.
(c) In direct immunofluorescence, the stain
is absorbed by a primary antibody, which
binds to the antigen. In indirect
immunofluorescence, the stain is absorbed
by a secondary antibody, which binds to a
primary antibody, which, in turn, binds to
the antigen. (credit a: modification of work
by Centers for Disease Control and
Prevention; credit b: modification of work by
Centers for Disease Control and
Prevention)
 Confocal Microscopes (1 of 3)

• A confocal microscope uses a laser to

scan multiple z-planes successively.

• This produces numerous two-dimensional,

high-resolution images at various depths,
which can be constructed into a three-
dimensional image by a computer.
 Confocal Microscopes (2 of 3)

• Confocal microscopes are very useful for

examining thick specimens such as
biofilms, which can be examined alive and
unfixed.
 Confocal Microscopes (3 of 3)

Figure 2.20 Confocal microscopy can be used to visualize

structures such as this roof-dwelling cyanobacterium biofilm. (credit:
modification of work by American Society for Microbiology)
 Electron Microscopy (1 of 12)

• An electron microscope (EM) uses short-

wavelength electron beams rather than
light to increase magnification and
resolution.

• An EM can produce a sharp image that is

magnified up to 100,000x.
 Electron Microscopy (2 of 12)

• EMs can resolve subcellular structures as

well as some molecular structures (e.g.,
single strands of DNA).

• Electron microscopy cannot be used on

living material because of the methods
needed to prepare the specimens.
 Electron Microscopy (3 of 12)

• There are two basic types of electron

microscopes:
– Transmission electron microscope (TEM)
– Scanning electron microscope (SEM)
 Electron Microscopy (4 of 12)

• The TEM uses an electron beam from

above the specimen that is focused using
a magnetic lens and projected through the
specimen onto a detector.

• Electrons pass through the specimen, and

the detector captures the image.
 Electron Microscopy (5 of 12)

• For electrons to pass through the

specimen in a TEM, the specimen must be
extremely thin (20–100 nm thick).
 Electron Microscopy (6 of 12)

Figure 2.21 A transmission electron microscope (TEM).

 Electron Microscopy (7 of 12)

• Scanning electron microscopes form

images of surfaces of specimens, usually
from electrons that are knocked off of
specimens by a beam of electrons.

• This can create highly detailed images

with a three-dimensional appearance that
are displayed on a monitor.
 Electron Microscopy (8 of 12)

• Specimens are typically dried and

prepared with fixatives that reduce
artifacts, such as shriveling, that can be
produced by drying, before being sputter-
coated with a thin layer of metal such as
gold.
 Electron Microscopy (9 of 12)

• SEMs can be used to view the surfaces of

larger objects as well as the surfaces of
very small samples.

• Some EMs can magnify an image up to

2,000,000x.
 Electron Microscopy (10 of 12)

Figure 2.22 Electron microscopes use magnets to focus

electron beams similarly to the way that light microscopes use
lenses to focus light.
 Electron Microscopy (11 of 12)

Figure 2.23 These schematic illustrations compare the

components of transmission electron microscopes and
scanning electron microscopes.
 Electron Microscopy (12 of 12)

Figure 2.24 (a) This TEM image of cells in a biofilm shows well-defined
internal structures of the cells because of varying levels of opacity in
the specimen. (b) This color-enhanced SEM image of the bacterium
Staphylococcus aureus illustrates the ability of scanning electron
microscopy to render three-dimensional images of the surface structure
of cells. (credit a: modification of work by American Society for
Microbiology; credit b: modification of work by Centers for Disease
 **Microscopic Techniques**
Various techniques enhance the quality and specificity of images produced by microscopes. These
techniques may involve sample preparation, staining, or the use of different types of light or
electrons.

**1. Staining Techniques:**

- Staining is used to enhance contrast in biological specimens. Common stains include:
- **Gram Staining:** Differentiates bacteria into Gram-positive (purple) and Gram-negative (pink)
based on their cell wall composition.
- **Hematoxylin and Eosin (H&E):** Widely used for histological samples, with hematoxylin
staining cell nuclei blue and eosin staining the cytoplasm pink.
- **Fluorescent Stains:** Such as DAPI (stains DNA) and Rhodamine (stains actin filaments).
**2. Polarized Light Microscopy:**
- Polarized light microscopy uses polarized light to study materials with birefringent
properties, such as crystals, minerals, and collagen fibers. The interaction of polarized
light with anisotropic structures reveals information about the orientation and physical
properties of the sample.

**3. Live-Cell Imaging:**

- Live-cell imaging techniques allow scientists to observe biological processes in real-
time. These methods often involve using phase-contrast or differential interference
contrast (DIC) microscopy, along with fluorescence microscopy to tag specific
What is cryo EM?
Cryo EM is an advanced imaging technique
used to elucidate the three-dimensional
(3D) structure of biological molecules and
complexes at near-atomic resolution. In this
technique, the sample is rapidly frozen to
temperatures below -150 °C, trapping it in
vitreous ice. The sample is then imaged
from different angles using an electron
microscope, where a beam of electrons
passes through the specimen, resulting in a
set of two-dimensional (2D) projections.
Advanced computational algorithms are
then used to reconstruct a 3D model of the
sample from these projections