Unit-II

Microscopes
• Bright field/Compound
• Dark Field
• Phase Contrast
• Fluorescece
• Electron Microscope (SEM and TEM)

Dr. Vikas Dutt

Assistant Professor
SSBSR, Sharda University
 Microscope
Device used for the magnification of the
sample.
Commonly used in laboratory to study the
specimen
 History of the Microscope
• 1585 –first compound microscope
• Not clear who invented
• Dutch spectacle maker Zacharias Janssen (b.1585) is
credited with making one of the earliest compound
microscopes
• Antoni van Leeuwenhoek (1632-1723): father of
microscopy. 1st observed the single-celled organisms in
pond water (protists and bacteria)
• 1655 – Robert Hooke used
a compound microscope
to observe pores in cork.

• He called them “cells”

 Microscope Vocabulary
•Magnification: The ability of a microscope
to produce an image of an object at a
scale larger (or even smaller) than its
actual size. Magnification is useful to see
more details of an object in the image
than when observing the object with the
naked eye.
•Resolution: Resolving power is the ability
to distinguish in an image adjacent points
of the object which are closely spaced
together. Usually these two terms are
used synonymously, however resolution
is the more practical one.
Compound Light Microscope
• It is a high power (high magnification) microscope
that uses a compound lens system.
• A compound microscope has multiple lenses:
• Objective lens (typically 4x, 10x, 40x or 100x) is
compounded (multiplied) by the eyepiece/Occular
lens (typically 10x) to obtain a high magnification of
40x, 100x, 400x and 1000x.
PRINCIPLE
• Specimens are visualized because of differences in
contrast (density) between specimen and
surroundings.
• Contrast differences arise because cells absorb or
scatter light to varying degrees
• Light pass through the thin transparent sample on slide,
and magnified image of the sample is obtained by the
objective lens. This image is known as the real image.
• The eyepiece or the ocular lens then magnifies the real
image and viewed as the virtual image.
• The compound microscope is also known as the
Bright-Field Microscope because the light passes
directly through the light source to the eye through the
two lenses.

Samples observed under compound microscope

 Mechanical components of Compound
Microscope

1. Ocular (eyepiece) lens

2. Objective turret or Revolver (to hold
multiple objective lenses)
3. Objective
4. Focus wheel to move the stage
5. Frame
6. Light source, a light or mirror
7. Diaphragm or condenser lens
8. Stage (to hold the sample)
9. Base
10. Phototube (for attaching a camera)
Advantages of Compound Microscope
• Due multiple lenses, one can obtain detailed information
about the sample.
• Own sources of light, user-friendly and easy to handle.
• Disadvantages of Compound Microscope
• Magnification of sample is possible only to a certain extent.
• Uses of Compound Microscope
• Pathology labs
• Forensic laboratories (human fingerprints).
• The presence of metals can be detected with the help of a
compound microscope.
• Basic observation of Bacteria and viruses.
• @ Schools and Universities for academic purposes.
 Dark Field Microscopy
• To view a specimen in dark field, an opaque disc is placed
underneath the condenser lens, so that only light that is
scattered by objects on the slide can reach the eye.
• Instead of coming up through the specimen, the light is
reflected by particles on the slide.
Principle of Dark Field
Microscopy

Frits Zernike a Dutch Physicist invented the Phase

Contrast Microscope and was awarded Nobel Prize
in 1953
 Phase Contrast Microscope (PCM)
• Frits Zernike a Dutch Physicist invented the Phase
Contrast Microscope and was awarded Nobel Prize in
1953
• A light microscopy technique used to enhance the contrast
of images of transparent and colorless specimens
• Phase differences are converted into amplitude variations
that can easily be detected.
• Does not require cells to be killed, fixed or stained, the
technique enables living cells, usually in culture, to be
visualized in their natural state
• Fluorescence staining can be used in combination with
phase contrast
• It is ideal for thinner samples, therefore an inverted
microscope system can be used for PCM.
 Principle of Phase Contrast Microscope
• Partially coherent illumination produced by the tungsten-
halogen lamp is directed through a collector lens and
focused on a specialized annulus (labeled condenser
annulus) positioned in the substage condenser front focal
plane.
• Wavefronts passing through the annulus illuminate the
specimen and either pass through undeviated or are
diffracted and retarded in phase by structures and phase
gradients present in the specimen.
• Undeviated and diffracted light collected by the objective
is segregated at the rear focal plane by a phase plate and
focused at the intermediate image plane to form the final
phase contrast image observed in the eyepiece
 Ciliate
(Oxytricha
saprobia)

Epithelial
cells
Bright field Phase Contrast
 Applications of phase contrast
microscope
To visualize transparent specimens, when high-
resolution is not required, including:
• Living cells (usually in culture)
• Microorganisms
• Thin tissue slices
• Fibers
• Subcellular particles, including organelles
 Fluorescence Microscope
• Fluorescence is one of the most commonly used physical
phenomena in biological and analytical microscopy for its
high sensitivity and high specificity.
• Fluorescence is a form of luminescence that through
microscopy allows users to determine the distribution of a
single molecule species, its amount and its localization
inside a cell.
• Colocalization and interaction studies
• Ion concentrations
• Intra- and Intercellular processes: Endocytosis and
Exocytosis
 Principle
• Use of fluorescent molecules
(fluorophores): for the labeling of
defined cellular structures.
• These molecules, such as green
fluorescent protein (GFP), absorb
light at a specific wavelength
(excitation) and emit it at a
specific higher wavelength
(emission).
• To visualize the molecule of
interest, fluorophore-coupled
specific antibodies or proteins, for
example, are transferred into the
cell
 Principle of Fluorescent Microscope
Higher intensity light illuminates the sample. This light excites fluorescent probe in the
sample, which then emit light of a longer wavelength. The image produced is based on
the second light source or the emission wavelength of the fluorescent probe - rather than
from the light originally used to illuminate, and excite, the sample.

19
 Advantages of Fluorescent Microscopy

• Live cell imaging

• Highly sensitive technique to detect as few as 50
molecules per cubic micrometer (mm3).
• Different molecules can now be stained with different
colors, allowing multiple types of molecule to be
tracked simultaneously.
• Have clear advantage over other optical imaging
techniques, for both in vitro and in vivo imaging.
 Electron Microscopy
• Electron microscopes use electrons instead of photons
(visible light) to image cells and structures.

• Electromagnets function as lenses in EM , whole system

operates in a vacuum.

• EM are fitted with cameras to take the photograph

• Two types of electron microscopes:

• Transmission electron microscopes (TEM)

• Scanning electron microscopes (SEM)

 Transmission Electron Microscopy (TEM)
▪ It is used to examine cells and cell structure at very high magnification and resolution,
even enabling one to view structures at the molecular level .

▪ This is because the high voltage electron beam is used to illuminate the specimen and
create an image. The wavelength of electrons is much shorter (about 100,000 times
smaller) than the wavelength of visible light and wavelength affects resolution.

▪ Electron beams were accelerated through a strong electromagnetic field, which

increases the microscope resolution by several orders of magnitude.

▪ Unlike visible light , electron beams can not penetrate very well. So, special
techniques of thin sectioning (thinner than 100 nm) are needed to prepare specimens
before observing them.

▪ To obtain sufficient contrast, the preparation are treated with stains such as osmic
acid, permanganate , uranium , lanthanum ; because these substances are composed of
atoms of high atomic weight , they scatter electrons well and thus improve contrast.
Electron
source

Evacuated
chamber

Sample
port

Viewing
screen
Advantages
• High-quality images can be obtained.
• TEMs have various applications at high spatial resolutions in different fields such as
scientific, educational and industrial fields.
• TEMs provide the highest magnification in microscope field.
• TEMs can provide information about surface features, shape, size and structure.
• Versatile imaging modes: dark/bright field and phase contrast (TEM).
Disadvantages
• The instruments are very large and expensive.
• TEMs require special housing and maintenance because they are sensitive to
mechanical vibration, fluctuation of electromagnetic fields, and variation of cooling
water.
• Sample preparations are very time-consuming.
• Potential artifacts can be generated by sample preparation.
• Special training is needed for tool operation and data analysis.
• TEM samples are limited to those that tolerate the vacuum chamber and are small
enough to fit in the chamber
• Possibility of electron beam damage, especially for light elements, biological samples,
and soft materials.
• Vacuum environment required–Limiting the ability to observe functional materials
under realistic “working” condition.
 Scanning Electron Microscopy
▪ This microscope is used to observed external features
(surface) of an organisms or cell.

▪ No need for thin sections.

▪ The specimen is coated with a thin film of a heavy metal

such as gold.

▪ An electron beam then scans back and forth across the

specimens. Electrons scattered from the metal coating are
collected and activate a viewing screen to produce an
image.
Cytoplasmic DNA
Septum Cell wall
membrane (nucleoid)
Advantages
• The SEM has a large depth of field, which allows more of a
specimen to be in focus at one time.
• The SEM also has much higher resolution, so closely spaced
specimens can be magnified at much higher levels.
• Provides information in microstructures, examine surface
contaminations, reveal spatial variations in chemical compositions,
and identify crystalline structures.
• Easy to operate with the proper training
Disadvantages
• Expensive, large and must be housed in an area free of any
possible electric, magnetic or vibration interference
• The preparation of samples can result in artifacts.
• risk of radiation exposure
• Limited to solid, inorganic samples small enough to fit inside the
vacuum chamber that can handle moderate vacuum pressure.