Microscopy and Microscopy and Specimen PreparationSpecimen Preparation

BIO3124Lecture #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 lightOptical behavior of light• Interaction of light with Interaction of light with

objectsobjects– 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 m

– Prokaryotes• Bacteria, Archaea• 0.2–10 m

– Viruses• 0.01-0.1 m

10 m

Lactobacillus lactis Amoeba proteus

Poliovirus

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

Incident angle

Normal

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

• working distance— distance between the front surface of lens and surface of coverslip or specimen when it is in sharp focus

Microscope ResolutionReducing the d value (higher resolution) means increasing the θ,

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 MicroscopesOptics 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

ParameciumIntracellular 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

Poliovirus infected HeLa T4 cellsReza Nokhbeh

Infected

GW bodies disintegrate as the result of Poliovirus infection virus and GW bodies are stained with fluorochrome conjugated specific antibodies

Electron Microscopy

• 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?

James Hillier (1915-2007)

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

Transmission Electron Microscopy (TEM)

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 phageTEM, 150,000x R. Nokhbeh , J. Trifkovic

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

DNA double helix

Atoms of MoS2, the bright spots are S atoms

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 groupsCationic (basic)Cationic (basic)  :: Positively charged.

– e.g. Methylene blue, Crystal violet, Safranine, Malachite green.

AnionicAnionic  (acidic):(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.Specimen staining.

Staining (Contd)

• Negative staining:Negative staining: – Background staining, not the specimen.

Methods

Simple StainingSimple Staining: • One type of stain.

• Cationic or Anionic stains.

• Able to determine the size, shape and the arrangment of bacteria.

Different Cell Morphologies

• Coccus:Coccus: – Sphere – 3 planes of division– Plane of division produces different arrangements of

cells. – Typical arrangements for different bacterial types.

• Bacillus:Bacillus:– Rods– One plane of division

Cocci

Diplococcus

Streptococcus(4-20)

Tetrad

Staphylococcus

Division axesDivision axes

Bacilli (Bacillus)

Diplobacilli

Streptobacilli

Other Cellular Forms

Curved rods (coccobacillus) Vibrio cholerae

SpiralsSpirochetes

Differential Staining Techniques: Gram Staining

• Bacteria divided into two groups:

• Gram Negatives: stain red Gram Negatives: stain red – Bacilli:Bacilli: Escherichia, Salmonella, Proteus, etc.– Cocci:Cocci: Neisseria and Pneumococcus.

• Gram Positives: stain blue/purple Gram Positives: stain blue/purple – Bacilli:Bacilli: Bacteria from the genera of Bacillus and

Clostridium– Coccus:Coccus: Streptococcus, Staphylococcus,

Micrococcus

Mechanism of Gram staining

1. Unstained

2. Crystal violet

3. Iodine

4. Destained (EtOH)

5. Safranin

Gram negativeGram negative Gram positiveGram positive

Typical examples of Gram staining reuslts