MICROSCOPY
1. Introduction
Microscopy is the science of using microscopes to view objects that are invisible to the naked
eye.
A microscope magnifies, resolves, and renders details of a specimen to the human eye or a
camera.
A good microscope must:
1. Produce a magnified image of the specimen.
2. Separate fine details in the image (resolution).
3. Render these details visible to the eye or camera.
2. History of the Microscope
Year Scientist(s) Contribution
13th — Spectacles in Italy offered basic
century magnification.
1590s Janssen brothers Built the first compound microscope
(~10×).
1609 Galileo Galilei Improved lenses and created the
“occhiolino”.
1665 Robert Hooke Published Micrographia; coined the term
“cell”.
1670s A. van Leeuwenhoek Observed bacteria, protozoa, sperm;
Father of Microbiology.
1820– Joseph Lister Developed achromatic lenses, reduced
1830s aberrations.
1930s Frits Zernike, Ernst Ruska, Invented phase contrast, TEM, and SEM
Manfred von Ardenne respectively.
3. Parts of a Compound Microscope
Part Function
Ocular Lens (Eyepiece) Magnifies image from objective; usually 10×.
Body Tube Holds eyepiece and objectives at proper distance.
Nosepiece Revolving part holding objective lenses.
Objectives (Scanner, Low, High Provide primary magnification (4×, 10×, 40×,
Power) 100×).
Stage & Clips Platform to place and hold slides.
Condenser & Diaphragm Controls light amount and focus on specimen.
Coarse & Fine Adjustment Knobs For rough and precise focusing.
Light Source / Mirror Illuminates specimen.
Arm & Base Support and carry structure of the microscope.
4. Optical Principles
Magnification-
Total Magnification = (Objective Lens) × (Eyepiece)
Example:
Eyepiece (10×) × Objective (40×) = 400×
Field of View
Circular visible area under the microscope. Field of view decreases as magnification increases.
5. Types of Microscopes
Type Description / Use
Simple Microscope Single lens (like magnifying glass).
Compound Two lens systems (ocular + objective), 1000–1500×
Microscope magnification.
Electron Microscope Uses electron beam, up to 1,000,000× magnification.
6. Types of Microscopy techniques
1. Phase contrast microscopy
Phase contrast microscopy is a powerful optical technique that converts small, invisible phase
shifts in light passing through a transparent specimen into visible differences in brightness
(amplitude). This contrast-enhancing method, invented by Frits Zernike in 1934, is essential
for visualizing unstained, living cells and other transparent specimens that lack sufficient
contrast in a standard bright-field microscope.
Principle of Operation
Unstained biological specimens (often called phase objects) do not absorb much light, so they
appear transparent. However, the different structures within the specimen (like the nucleus or
organelles) have varying refractive indices and thicknesses. When light passes through these
structures, its speed is slightly altered, creating a small, unnoticeable shift in the light's phase.
The phase contrast microscope uses two special components to make this phase shift visible:
1. Condenser Annulus (Annular Ring): Located in the condenser, this is an opaque disk
with a transparent ring. It produces a hollow cone of light that illuminates the specimen.
2. Phase Plate: Located at the rear focal plane of the objective lens, this is a transparent
disk with a groove or ring of material that matches the size and position of the image
of the condenser annulus.
The Mechanism
The light is split into two components:
• Undeviated (Direct) Light: Light that passes straight through the specimen's clear
background. This light passes through the phase ring on the phase plate.
• The phase plate is designed to either advance or retard this direct light's phase. It also
slightly dims this light to match the intensity of the diffracted light.
• Diffracted Light: Light that is scattered or diffracted by the specimen's structures. This
light passes outside the phase ring.
• By manipulating the phase of the direct light, a total phase difference is created between
the undeviated and diffracted light rays. When these two light waves recombine
(interfere) at the image plane creates:
• Destructive Interference: Results in a darker image of the specimen's structure against
a brighter background (known as positive phase contrast).
• Constructive Interference: Results in a brighter image of the specimen's structure
against a darker background (known as negative phase contrast).
This interference transforms the invisible phase shifts into visible differences in light amplitude
(brightness), creating the high-contrast image.
Key Uses and Applications
The major advantage of phase contrast microscopy is its ability to visualize specimens without
the need for toxic stains or fixation, allowing for the study of natural, dynamic processes.
Application Area Specific Uses
Cell Biology Observing living cells in their natural state (in culture) and
monitoring cellular events such as cell division (mitosis), cell
motility (movement), and overall cell morphology.
Microbiology Visualizing microorganisms (like bacteria and protozoa), as well as
internal structures like endospores and flagella, without staining.
Subcellular Examining intracellular components such as nuclei, mitochondria,
Structures and vacuoles in living cells at relatively high resolution.
Histology/Pathology Viewing thin, unstained tissue slices and identifying cellular details
in clinical or diagnostic settings (e.g., live blood cell analysis).
Advantages and Limitations
Advantages (Pros) Limitations (Cons)
Non-Invasive: Cells do not need to be Halo Artifact: Images often display a halo
killed, fixed, or stained. (bright ring) around the boundaries of structures
with high phase shifts, which can obscure detail.
Live Cell Imaging: Allows for the Thickness Limitation: Not ideal for thick
observation of dynamic biological specimens; image clarity is reduced by phase
processes over time. shifts from out-of-focus planes.
High Contrast: Produces high-contrast Resolution Limit: The annular ring slightly
images of transparent objects that would reduces the numerical aperture (NA) of the
be invisible in bright-field microscopy. objective, which can limit the ultimate
resolution.
2. Dark Field Microscopy
Principle of Operation
Dark Field Microscopy is an optical technique that exploits light scattering to render unstained,
transparent specimens visible as bright objects against a black field.
The Mechanism
The technique requires a modified illumination system:
1. Condenser Stop: A special condenser uses an opaque stop (a dark disk) to block the
central rays of light.
2. Oblique Illumination: This setup creates a hollow cone of light that illuminates the
specimen at a high, oblique angle.
3. Exclusion of Direct Light: All light that passes directly through the clear specimen
background misses the objective lens, leaving the field of view dark.
4. Scattering and Image Formation: When the light cone hits the specimen's structures
(e.g., cell boundaries, granules), the light is scattered (diffracted, refracted, or reflected).
This scattered light enters the objective lens and forms the image, making the specimen
appear bright (self-luminous) against the dark background.
Key Uses and Applications
Dark Field Microscopy is primarily used for observing living, highly transparent, and
often motile specimens.
Application Area Specific Uses
Microbiology Visualization of spirochetes (e.g., Treponema pallidum), which are too
thin to be seen clearly by bright-field microscopy.
Live Cell Viewing unstained, living bacteria and protists, and observing their
Observation movement (motility) and external features.
Material Science Examining fibers, mineral crystals, aerosols, and colloids, where edges
and surface texture are the primary features of interest.
Thin Structures Ideal for detecting the presence of sub-resolution particles and fibers
due to the high contrast generated by scattering.
Advantages and Limitations
Advantages (Pros) Limitations (Cons)
High Contrast: Provides a vivid image Dust Artifacts: Dust, dirt, and air bubbles scatter
of the specimen as bright on a truly light and appear as bright, confusing artifacts.
black background.
Non-Invasive: Requires no staining or Low Light Intensity: The final image is formed
fixation, making it excellent for only by scattered light, requiring a very bright
observing living, delicate organisms. light source, which can sometimes damage live
specimens.
Simple Conversion: A standard bright- Internal Detail: Less effective for revealing
field microscope can be converted with internal structures compared to phase contrast or
a simple stop or a dark-field condenser. DIC.
Resolution: Can detect objects just Thick Specimens: Cannot be used effectively
below the theoretical limit of resolution with thick specimens, as excess scattered light
for light microscopy. will "whiten" the dark background.
3. Fluorescence Microscopy
Fluorescence microscopy uses the phenomenon of fluorescence to create high-contrast images
of specific structures in a specimen, often appearing as bright, coloured features against a dark
background.
Principle of Operation
The principle relies on special chemical compounds called fluorophores (or fluorochromes)
that are used to label the target structures (like proteins, DNA, or organelles).
The Mechanism
1. Excitation: A powerful light source (usually a mercury/xenon lamp or a laser) emits
high-energy, short-wavelength light (e.g., UV or blue light). This light is directed onto
the specimen.
2. Absorption & Excitation: The fluorophores in the specimen absorb the high-energy
light, which promotes their electrons to a higher energy state (excited state).
3. Emission: The excited electrons rapidly drop back down to their stable ground state. In
the process, they release the excess energy as a new photon of light, which is always
lower-energy and longer-wavelength (e.g., green or red light). This shift in wavelength
is called the Stokes Shift.
4. Signal Separation: The microscope uses specialized filters to separate the intense,
short-wavelength excitation light from the much weaker, long-wavelength emitted
(fluorescent) light. Only the emitted fluorescent light is allowed to reach the detector,
creating a highly specific image of the labeled structures.
Key Components
Most fluorescence microscopes are epi-fluorescence types, where the excitation and emission
happen through the same objective lens. A critical component is the filter cube, which typically
contains three parts:
Component Function
Excitation Filter Only transmits the specific wavelength of light needed to excite the
fluorophore, blocking all other wavelengths from the light source.
Dichroic Mirror Sits at a angle; reflects the short-wavelength excitation light down
toward the sample, but transmits the long-wavelength emission light
up toward the detector.
Emission Filter Blocks any residual excitation light and transmits only the longer-
(Barrier Filter) wavelength fluorescent light emitted by the specimen, ensuring a dark
background.
Key Uses and Applications
Fluorescence microscopy is crucial in life sciences because of its high specificity and
sensitivity, allowing researchers to track individual molecules within a cell.
Application Area Specific Uses
Molecular Identifying and localizing specific proteins (e.g., using
Localization Immunofluorescence), nucleic acids (DNA/RNA), or organelles.
Live Cell Imaging Observing dynamic processes in living cells, such as cell motility,
protein movement, ion transport, and cell division (mitosis), often
using Genetically Encoded Fluorescent Proteins (like GFP).
Diagnostic/Clinical Rapid detection of microorganisms (e.g., Treponema pallidum in
syphilis testing) and in techniques like FISH (Fluorescence In Situ
Hybridization) for genetic analysis.
Advanced Imaging Forming the basis for high-resolution techniques like Confocal
Microscopy (for 3D imaging/optical sectioning) and TIRF (for
imaging cell surfaces).
4. Differential Interference Contrast (DIC) Microscopy
Differential Interference Contrast (DIC) microscopy, also known as Nomarski interference
contrast (NIC), is an optical microscopy technique that enhances the contrast in unstained,
transparent specimens by converting invisible differences in light phase into visible differences
in intensity (brightness) and pseudo-three-dimensional relief.
Principle of Operation
The core principle of DIC microscopy is interferometry and the conversion of Optical Path
Length (OPL) gradients into amplitude (intensity) variations.
1. Polarization and Beam Splitting: Plane-polarized light is split into two orthogonally
polarized, closely spaced rays (the ordinary and extraordinary rays) using a Nomarski
prism (condenser prism). This small spatial separation is called shear.
2. Phase Shift Induction: The two rays travel through adjacent, closely spaced points in
the specimen. Since the specimen has variations in refractive index or thickness, the
two rays travel different Optical Path Lengths. This difference in OPL causes one ray
to be delayed relative to the other, creating a phase shift.
3. Beam Recombination and Interference: A second Nomarski prism (objective prism)
recombines the two sheared rays. Because of the phase shift experienced in the
specimen, the recombined rays interfere with each other.
4. Conversion to Visible Contrast: The recombined, elliptically polarized light then
passes through an analyser, which is oriented perpendicular to the first polarizer. This
analyser converts the phase differences into detectable amplitude variations.
5. Image Formation: Areas where the OPL gradient is steep (e.g., cell boundaries) result
in maximum interference and appear bright or dark, creating a characteristic shadow-
cast or pseudo-3D relief image.
Key Components
The DIC microscope requires specialized components to convert a standard brightfield
microscope into a DIC system:
Component Location Function
Polarizer Before the condenser Generates the initial plane-polarized light.
Condenser Prism In the condenser Splits the polarized light into two
(Nomarski) orthogonally polarized, slightly sheared
rays.
Objective Prism Behind the objective lens Recombines the two sheared rays after
(Nomarski) they pass through the specimen.
Analyzer Between the objective Filters the recombined light to convert the
prism and the interference effects (phase shifts) into
eyepiece/camera visible intensity variations.
Applications
DIC microscopy is highly effective for imaging transparent and unstained samples. Its
advantages include high resolution, good contrast, and the absence of halo artifacts common in
phase contrast.
• Live Cell Imaging: Observing unstained, living biological samples like cells, protozoa,
and organelles in real-time without the need for toxic stains.
• Optical Sectioning: Using high numerical aperture optics, DIC provides excellent
axial resolution, allowing a clear image of a thin focal plane within a thick specimen
(optical sectioning).
• Material Science: Characterizing surfaces in opaque materials (using reflected light
DIC), such as semiconductors, precision mechanical components, and ultra-thin films
to visualize surface roughness, scratches, and step heights.
• Tissue Analysis: Examining unstained tissue sections and embryos.
5. Confocal Laser Scanning Microscopy (CLSM)
Confocal Laser Scanning Microscopy (CLSM) is an advanced optical imaging technique that
provides high-resolution, high-contrast images of specimens, particularly thick and
fluorescently labelled samples. Its key advantage over traditional wide-field microscopy is its
ability to perform optical sectioning, which allows for the non-invasive creation of thin, in-
focus slices of a specimen, enabling detailed 3D reconstruction.
Principle
The core principle of CLSM is the use of point illumination and a spatial pinhole for point
detection, which work together to reject out-of-focus light The name "confocal" stems from
this configuration, as the pinhole for the light source and the pinhole for the detector are in
optically conjugate planes (i.e., they are focused on the exact same spot, or focal point, in the
specimen)
1. Point Excitation: A laser is focused by an objective lens onto a tiny, diffraction-limited
spot within the sample.
2. Point Detection with Pinhole: The light emitted or reflected from the illuminated spot
travels back through the objective lens. Before reaching the detector, the light must pass
through a small aperture called the pinhole.
3. Out-of-Focus Rejection: Only the light originating from the focal plane (the in-focus
spot) is sharply focused and passes through the pinhole to reach the detector. Light from
regions above or below the focal plane (out-of-focus light) is blocked by the pinhole,
thus eliminating the blur or haze common in conventional microscopy.
4. Image Construction (Scanning): Since only a single point is illuminated and detected
at any given time, the laser beam is systematically scanned across the sample (raster
scanning, point-by-point, line-by-line in the X-Y plane) using high-speed mirrors. A
computer then compiles the light intensity data from each point to construct a complete,
clear, two-dimensional image (an optical section).
5. 3D Imaging: To create a 3D image, the focal plane is shifted in the Z-direction (depth)
and the scanning process is repeated to acquire a stack of optical sections. These slices
are then computationally re-assembled into a three-dimensional reconstruction.
Main Components of a CLSM
A typical Confocal Laser Scanning Microscope consists of several specialized components:
Component Function
Laser Provides a highly focused, intense, monochromatic light source
for specimen illumination (excitation). Different lasers are used
for different fluorophores.
Scanner Consists of galvanometer mirrors that precisely deflect the laser
beam to scan the focal spot across the specimen in the X and Y
directions.
Objective Lens Focuses the laser light onto the specimen and collects the
emitted light.
Dichroic Mirror (or Separates the excitation laser light (which is directed to the
Beamsplitter) sample) from the longer-wavelength emission light (which is
directed to the detector).
Pinhole The critical component placed in front of the detector. It acts as
a spatial filter, blocking out-of-focus light and allowing only
light from the focal plane to pass.
Detector Highly sensitive detector that captures the light passing through
(Photomultiplier Tube the pinhole, converting the light signal into an electronic signal
or PMT) (a pixel of the final image).
Computer System Controls the scanning process, collects the signal data from the
detector, and reconstructs the point-by-point data into a
complete image.
Advantages and Applications
CLSM is a fundamental tool in life sciences and materials science due to its unique capabilities.
Advantages
• Optical Sectioning: The ability to acquire thin, in-focus slices from thick specimens
without physical slicing.
• 3D Imaging: Allows for the reconstruction of detailed three-dimensional
representations of structures.
• Improved Resolution and Contrast: Eliminates out-of-focus background
fluorescence, leading to sharper images, especially in the axial (Z) direction.
• Live Cell Imaging (with certain types like Spinning Disk CLSM): Allows for the
observation of dynamic processes in living cells over time.
• Multi-Colour Imaging: Can simultaneously image multiple fluorescent labels within
a single specimen.
Applications
• Cell and Molecular Biology: Studying cell morphology, organelle distribution, protein
localization, and dynamic cellular processes (e.g., cell signalling, protein transport).
• Neuroscience: Imaging complex neural networks and observing calcium signalling in
neurons.
• Developmental Biology: Imaging thick specimens like embryos in 3D.
• Clinical/Medical Imaging (e.g., Dermatology): Non-invasive "optical biopsy" of skin
and real-time visualization of skin layers for cancer detection and monitoring.
• Materials Science: Analysing the topography, surface structure, and internal structure
of materials (e.g., polymers, ceramics).
6. Electron Microscopy
Electron microscopy (EM) is a technique that uses a beam of accelerated electrons as a source
of illumination to create highly magnified images of specimens, enabling visualization of ultra-
fine details down to the nanoscale.
This technique achieves much higher resolution and magnification than traditional light
microscopes because the wavelength of an electron is much shorter than that of visible light.
Principle
In a typical electron microscope, an electron beam is produced by an electron gun (usually a
heated tungsten filament) and then accelerated by a high voltage. Instead of glass lenses used
in light microscopes, electromagnetic lenses are used to focus and manipulate the electron
beam because electrons are charged particles.
1. Electron Beam Generation: An electron gun generates a stream of electrons.
2. Focusing: Electromagnetic lenses condense and focus the beam.
3. Interaction: The focused electron beam interacts with the specimen inside a high-
vacuum chamber.
4. Signal Detection: Detectors capture the resulting electrons or X-rays that are scattered
or transmitted from the specimen.
5. Image Formation: The signals are processed to form a highly magnified image on a
screen or computer monitor.
Main Types of Electron Microscopes
The two most common types of electron microscopes are the Transmission Electron
Microscope (TEM) and the Scanning Electron Microscope (SEM).
Transmission Electron Microscope (TEM)
Feature Description
Principle The electron beam passes (transmits) through a very thin specimen.
Information Provides information on the internal structure (ultrastructure), crystal
structure, and composition.
Image Type 2D projected image.
Sample Prep Requires ultra-thin sections (typically less than 150 nm thick) to allow
electrons to pass through.
Magnification Very high (e.g., up to 50 million times).
Scanning Electron Microscope (SEM)
Feature Description
Principle A focused electron beam scans the surface of the specimen, and detectors
collect secondary or backscattered electrons emitted from the surface.
Information Provides highly detailed images of the surface topography and
composition.
Image Type 3D view of the surface.
Sample Prep Can image larger, thicker samples; often requires a thin conductive coating
(e.g., gold) on non-conductive materials.
Magnification High (e.g., up to 1–2 million times).
Applications
Electron microscopy is a vital tool across numerous fields:
• Biology/Medicine: Visualizing the detailed internal structure of cells (organelles),
viruses, protein molecules, and diagnosing certain diseases (e.g., tumours, pathogens).
• Materials Science: Analysing the microstructure of metals, ceramics, and polymers;
studying crystal defects, grain boundaries, and nanomaterials.
• Geology/Forensics: Studying the structure of rocks and minerals and analysing trace
evidence like gunshot residue or dust.