Microscopy and
Specimen Preparation
BIO3124
Lecture #2
Objectives and reading
• Reading: Ch.2
1. Principles of optical resolution of microorganisms
– Optical behaviour of light
– Detection, magnification and resolution
– How to increase resolution
• Optical parameters
• Increasing contrast
2. Microscopic systems and their applications
– Bright field
– Dark field
– Phase contrast
– Fluorescence and Confocal
– EM
– Scanning Probe (AFM, STM)
Optical behavior of light
• Interaction of light with
objects
– Absorption: object will
appear dark
– Reflection
– Refraction: basis for
magnification
– Scattering: when size of
object is close to the
wavelength of light
• Principle: to be resolved
the wavelength should
be smaller than the size
of object
Detection, Magnification and Resolution
Definitions
• Optical systems: detect, magnify, resolve
– Eye, simple lens, microscopes
– Use electromagnetic radiation, eg. visible light, laser
or electron beam
• Detection: ability to determine the existence of an
object
• Resolution: ability of an optical
system to distinguish two small close objects
– Human eyes limit of resolution is 150 µm
• Magnification: ability to increase the apparent size of
the image of an object
Observing Microbes
• Microscope needed to see smaller objects
– Eukaryotic microbes
• Protozoa, algae, fungi
• 10–100 mm
– Prokaryotes
10 mm
• Bacteria, Archaea
• 0.2–10 mm
– Viruses
Lactobacillus lactis
• 0.01-0.1 mm
Poliovirus
Amoeba proteus
Magnification
• Microscopes: Magnify image to match the limit of
resolution of eye retina ie. 150 um
• Magnification’s contribution to resolution is limited
• Distortion due to light wave interference
• Empty magnification: magnification that does not improve resolution
• Magnification is due to refraction
• Refraction: light is refracted (bent) at the varying density
interfaces
• Light travels slower, waves compressed (higher
frequency)
• Depends on refractive index of object
Lenses and the bending of light
• Refractive index (RI)
– how greatly a substance slows the velocity of light
• Direction/magnitude of bending depends on the RI
• A lens behaves like a prism
Normal
Incident angle
Refracted angle
Refraction and Magnification
 Image forms at crossing refracted light originated from the object
 Magnification ratio depends on the position of object with respect
to the lens
Watch tutorial
Summary: optic principles
Light Microscopes
 Compound microscopes
– image formed by action of 2 lenses
 Bright-field microscope
 Dark-field microscope
 Phase-contrast microscope
 Fluorescence microscope
The Bright-Field Microscope
• Dark image against a brighter bkg
• Several objective lenses
Parfocal: stays focused
when objectives
changed
• total magnification
(max 1000-fold)
– product of the
magnifications
of the ocularrlenses
and the objective lenses
Microscope Resolution
• Optical parameters affecting resolution
• Shortest distance resolved by an optic system (d) is
expressed by:
• Abbe equation: d=0.5λ/n.sinθ
• λ= wavelength, n= refractive index, θ= angle of apreture
– shorter wavelength  greater resolution
– Numerical aperture: NA= n.sinθ
– Smaller d value = more powerful optic system
Microscope Resolution
Reducing the d value (higher resolution) means increasing the θ,
• working distance
— distance between the front surface of lens and
surface of coverslip or specimen when it is in
sharp focus
Microscope Resolution
• Effect of refractive index:
NA= n.sinθ
The Dark-Field Microscope
• Image is formed by light reflected or refracted by
specimen
• Interference by the bkg light eliminated
• produces a bright image against a dark bkg
• to observe living, unstained preparations
– For eucaryotes has been used to observe internal
structures
– For procaryotes has been used to identify bacteria
such as Treponema pallidum, the causative agent of
syphilis
Dark field microscopy: Light path
Spider Light stop:
produce annular ring of
light, no light from the
centre enters objective
• only light passing
through object enters the
objective lens
• bkg stays dark,
specimen shines
Dark field microscopy
Example of an insect larva
examined in a dark field
microscope
The Phase-Contrast Microscope
 first described in 1934 by Dutch
physicist Frits Zernike
 enhances the contrast btw intracellular
structures that have slight differences
in their refractive indices
 excellent tool to observe living cells
– bacterial components such as
endospores and inclusion bodies
– Eukaryotic organelles
Frits Zernike (1888-1966)
Optics of Phase Contrast Microscopes
Phase contrast image of HeLa cells
HeLa cells
Reza Nokhbeh
Phase contrast microscopy
P.aeruginosa
Sporulating bacterium
Contrast between spores and
Vegetative forms
Paramecium
Intracellular organelles
contrasted
The Fluorescence Microscopy
• specimens usually stained with
antibodies tagged with a fluorophore
• Excitation light: ultraviolet, violet, or
blue light activates fluorophore tagged
cells
• Emission light: longer wavelength,
enters objective
• bright image of the object resulting
from the fluorescent light emitted by
the specimen
• Applications: medical microbiology
and molecular biology
The Fluorescence Microscope
Excitation and Emission lights
Poliovirus interferes with the integrity of SiRNA centres
 GW bodies disintegrate as the result of Poliovirus infection
 virus and GW bodies are stained with fluorochrome conjugated specific antibodies
Infected
Poliovirus infected HeLa T4 cells
Reza Nokhbeh
Electron Microscopy
James Hillier
(1915-2007)
• Ernst Ruska and Max Hall in Germany
finished the first prototype in 1931
• Eli Franklin Burton (1847-1948) and his
students, James Hillier, Cecil Hall and Albert
Prebus, built the first functional EM in 1938
at Toronto university
• Louis de Broglie’s principle that electron
particles also have electromagnetic (wave)
property
• accelerated electronic beam in microscopy
would enhance resolution, why?
Transmission Electron Microscopy (TEM)
 wavelength of electron beam is
much shorter (0.005 nm or 5 A˚)
than light, i.e. much higher
resolution
 Magnification is 100,000 to
200,000
 Resolution approaches 0.5 nm,
ie about 1000-fold higher than
light microscopes
Principles of light microscopy applies to TEM
Thermionic Electron Gun
~300 Kev monochromatic beam
The Scanning Electron Microscopy (SEM)
•
uses electrons scattered
from the surface of a
•
specimen to create
image
produces a
3-dimensional
image of specimen’s
surface features
Examples of TEM and SEM micrographs
P. acens lytic phage
R. Nokhbeh , J. Trifkovic
TEM, 150,000x
New Techniques in Microscopy
 Confocal laser scanning
microscopy (CLSM) and
scanning probe microscopy
 have extremely high
resolution
 Expanded the resolution to
molecular and atomic levels
i.e. 1-100 A
Confocal Microscopy
Confocal Laser Scanning Microscope (CLSM)
 laser beam used to illuminate a variety of planes
in the specimen, exciting fluorophore
 computer compiles images to generate 3D image
 used extensively to observe biofilms
 Also used in studying the sub-cellular structures
 Light is only gathered from the plane of focus
Confocal scanning laser microscope
• blurring does not happen since signal is gathered by scanning a thin
layer of specimen, plane of focus, at each round
Scanning Probe Microscopy
• Atomic Force Microscope (AFM)
– Vertical movement of probe is
followed by a laser beam
– probes surfaces that are not charged
Atomic Force Microscope
Membrane integral aquaporin protein captured by AFM
α-synuclein protein fibers.
Misfolded fibers are incolved
in Parkinson disease
Human mitotic chromosome spread
Scanning Probe Microscopy
• Scanning Tunneling Microscope (STM)
• Measures the surface features of specimen by moving a sharp
probe over the surface
– steady current (tunneling current) maintained between microscope
probe and specimen
– up and down movement of probe as it maintains current is
detected and used to create image of surface of specimen
– Magnification: 100 million times, capable of detecting the surface
atoms
Scanning Tunneling Microscope
Atoms of MoS2, the
bright spots are S atoms
DNA double helix
Silicon surface atoms enlarged 20 million times
individual surface atoms and the bonds that hold them in place
Preparation and Staining of Specimens
 Staining techniques are applied to increase the contrast
 increases visibility using bright field microscopes
 accentuates specific morphological features
 preserves specimen (due to fixation)
Fixation
 preserves internal and external structures and stabilizes
them in position
 organisms usually killed and firmly attached to microscope
slide
• heat fixation – routinely used in procaryotes,
preserves overall morphology but not internal
structures
• chemical fixation – used for larger, more delicate
organisms
 protects fine cellular substructure and morphology
Dyes
 Dyes
• Ionizable dyes have charged groups
Cationic (basic) : Positively charged.
– e.g. Methylene blue, Crystal violet, Safranine,
Malachite green.
Anionic (acidic): Negatively charged
– e.g. Nigrosin black, Indigo ink.
Simple and Differential staining
 Simple staining
– a single stain is used
– use can determine size, shape and arrangement of
bacteria
 Differential staining
divides microorganisms into groups based on their
staining properties
– e.g., Gram staining
– e.g., acid-fast staining
Staining
• Positive staining: Specimen staining.
Staining (Contd)
• Negative staining:
– Background staining, not the specimen.
Methods
Simple Staining:
• One type of stain.
• Cationic or Anionic stains.
• Able to determine the size, shape and
the arrangment of bacteria.
Different Cell Morphologies
• Coccus:
– Sphere
– 3 planes of division
– Plane of division produces different arrangements of
cells.
– Typical arrangements for different bacterial types.
• Bacillus:
– Rods
– One plane of division
Cocci
Division axes
Diplococcus
Streptococcus
(4-20)
Tetrad
Staphylococcus
Bacilli (Bacillus)
Diplobacilli
Streptobacilli
Other Cellular Forms
Curved rods (coccobacillus)
Vibrio cholerae
Spirals
Spirochetes
Differential Staining Techniques: Gram Staining
• Bacteria divided into two groups:
• Gram Negatives: stain red
– Bacilli: Escherichia, Salmonella, Proteus, etc.
– Cocci: Neisseria and Pneumococcus.
• Gram Positives: stain blue/purple
– Bacilli: Bacteria from the genera of Bacillus and
Clostridium
– Coccus: Streptococcus, Staphylococcus,
Micrococcus
Mechanism of Gram staining
1. Unstained
2. Crystal violet
3. Iodine
4. Destained
(EtOH)
5. Safranin
Typical examples of Gram staining reuslts
Gram negative
Gram positive