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Optical Microscopy
Resolution, magnification, lenses, focal point, foal length, NA,
minimal limit/ distance of resolution, wavelength, refractive index
of the medium and object.
Combination of objective and eyepiece focuses image to the eye

Obj

Compound microscope
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Microscopy

Image

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How can we use the properties of light to

create contrast?
Which properties can
be used?
Absorption
Scattering
Refraction
Phase
Polarization

Contrasting techniques

Brightfield

DIC

Phase contrast

Darkfield

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The Light Microscope

many types
bright-field microscope
dark-field microscope
phase-contrast microscope
fluorescence microscopes

are compound microscopes

image formed by action of 2 lenses

The Bright-Field Microscope

Principle: Light is transmitted through the
sample and absorbed by it.
produces a dark image against a brighter
background
has several objective lenses
total magnification - product of the magnifications
of the ocular lens and the objective lens
Application: Only useful for specimens that can be
contrasted via dyes. Very little contrast in unstained
specimens.
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Brightfield

Piece of artificially grown skin

Cross section of sunflower root

(www.igb.fhg.de/.../dt/PI_BioTechnica2001.dt.html )

(http://www.zum.de/Faecher/Materialien/beck/12/bs12-5.htm)

all our microscopes can be used for brightfield

working distance
distance between the front surface of lens and surface of cover
glass or specimen

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The Dark-Field Microscope

Principle: The illuminating rays of light are directed
through the sample from the side by putting a dark
disk into the condenser that hinders the main light
beam to enter the objective. Only light that is
scattered by structures in the sample enters the
objective.
produces a bright image of the object against a
dark background
Application: used to observe living, unstained
preparations

Symbiotic Diatom colony

(www1.tip.nl/~t936927/making.html)
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The Phase-Contrast Microscope

Introduced by Dutch physicist Frits Zernike, 1930 (a
Nobel laureate in 1953)
enhances the contrast between intracellular structures
having slight differences in refractive index
excellent way to observe living cells
Phase contrast shows differences in as difference in
contrast
Phase plates produce dark and light effects for the light
falling in objectives
A halo contrast around the specimen, due to
the difference in RI of cell and surrounding
medium, obscures interphase details
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Phase contrast microscopy

Phase contrast
compound
Microscope

Simple
compound
Microscope

Microscopy

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Phase contrast microscopy enhances image quality

wrong phase stop

brightfield

Bright field

right phase stop

Phase contrast

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D(ifferential) I(nterference) C(ontrast)

Principle: Also known as Nomarski microscopy. Uses polarized light for
illumination. Synchronizing of the different phases of incident and
transmitted light is done by a set of prisms and filters introduced into the
light path.
creates image by detecting differences in refractive indices and thickness of
different parts of specimen

excellent way to observe living cells

The Fluorescence Microscope

exposes specimen to ultraviolet, violet, or blue
light
specimens usually stained with fluorochromes
eg. FITC, DAPI, Hoechst, Mitotracker Red/
Orange, wavelength specific dyes (alexa fluor
488/562/594/647), etc.
shows a bright image of the object resulting
from the fluorescent light emitted by the
specimen
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Fluorescence microscopy
A fluorophore-based microscopy

FITC (Fluorescein
isothiocyanate)

GFP (Green
fluorescence protein)

Texas Red

Luciferin
Rhodamine

Cyanin dyes
Microscopy

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Vishal Saxena: unpublished data

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How fluorophores are important in fluorescence microscopy

Fluorophore-conjugated antibody stained samples are used in
fluorescence microscopy

Primary antibody
Antibody-fluorophore
conjugate

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Microscopy

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Fluorescence microscopy

Light microscopy

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Fluorescence microscopy

Microscopy

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Newer Techniques in Microscopy

confocal microscopy and scanning probe
microscopy
have extremely high resolution
can be used to observe individual atoms

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Confocal Microscopy
confocal scanning laser microscope
laser beam used to illuminate spots on
specimen
computer compiles images created from each
point to generate a 3-dimensional image

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Confocal microscopy (Principled by Marvin Minsky, 1957)

Conventional microscope "sees into the specimen as far light can penetrate

20 m

Optical microscopy

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Plane of focus
X and Y

Microscopy

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Confocal microscopy (Principled by Marvin Minsky, 1957)

Conventional microscope "sees into the specimen as far light can penetrate
Confocal acquires in-focus images with depth selectivity (called optical
sectioning)

Optical microscopy

LASER Confocal microscopy

20 m

Plane of focus
X and Y

Plane of
focus
X, Y and Z

Merging of optical sections generate 3D topography

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Microscopy

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Confocal microscopy of Plasmodium-infected mosquito gut cells

Upper side

Fluorescent
microscopy

Middle

Bottom side

LASER Confocal microscopy

Side view
Microscopy

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Scanning Probe Microscopy

scanning tunneling
microscope
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
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Scanning Probe Microscopy

atomic force microscope
sharp probe moves over surface of specimen at
constant distance
up and down movement of probe as it
maintains constant distance is detected and
used to create image

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Polarization Contrast
Principle: Polarized light is used for illumination. Only when the vibration direction of the polarized
light is altered by a sample placed into the light path, light can pass through the analyzer. The
sample appears light against a black background. A lambda plate can be used to convert this
contrast into colours.
Application: Polarization contrast is used
to look at materials with birefringent
properties, in which the refractive index
depends on the vibration direction of the
incident light, e.g. crystals or polymers.

Analyzer
Lambda
plate

Brightfield

Polarization Polarization
contrast
contrast with
Lambda plate

Polarizer

we do not have microscopes set up for polarization contrast

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Electron Microscopy
beams of electrons are used
to produce images
wavelength of electron beam
is much shorter than light,
resulting in much higher
resolution
They are vacuum-enclosed
devices to avoid any loss of
electron energy
Types
Transmission electron
microscope (TEM)
Scanning electron
microscope
(SEM)
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Electron microscope
- the specimen is coated with electron-dense metal particles

- Optical lenses are replaced by magnetic field

Magnetic field
----

Light

Lens

Electron
beams

Specimen

Light microscope

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----

Electron microscope

Microscopy

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The Transmission Electron Microscope

electrons scatter when they pass through
thin sections of a specimen
transmitted electrons (those that do not
scatter) are used to produce image
denser regions in specimen, scatter more
electrons and appear darker

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TEM
Electron gun

e- beam passes through thin section of specimen to generate internal

details
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Microscopy

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The Scanning Electron Microscope

uses electrons reflected from the surface of
a specimen to create image
produces a 3-dimensional image of
specimens surface features

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SEM

e-

beam falling on specimen surface produces secondary electrons

(SE), which generate surface architecture

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Microscopy

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Observe the difference between the image from TEM and SEM
SEM

WBC

TEM

Useful to explore the internal

structure of a cell

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Generates detailed surface

architecture of a cell

Microscopy

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Specimen Preparation
analogous to procedures used for light
microscopy
for transmission electron microscopy,
specimens must be cut very thin
specimens are chemically fixed and stained
with electron dense material

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Preparation and Staining of Specimens

increases visibility of specimen
accentuates specific morphological features
preserves specimens

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Sample preparation for microscopy

- Fixation
- Embedding
- Sectioning
- Mounting

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Microscopy

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Fixation
process by which internal and external
structures are preserved and fixed in
position
process by which organism is killed and
firmly attached to microscope slide
heat fixing
preserves overall morphology but not internal
structures

chemical fixing
protects fine cellular substructure and morphology
of larger, more delicate organisms
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Fixation
Cross-linking of biological macromolecules to freeze specimen structure
Glutaraldehyde : Cross-links proteins

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Osmium tetraoxide: Cross-links lipids

Microscopy

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Embedding
Placing fixed sample into a matrix

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Microscopy

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Sectioning
cutting thin strips
Thickness of sections
For light microscopy
Thick sections (~1 micron)

For TEM
Thin sections (100-500 Ao)

Microtome for sectioning

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Microscopy

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Mounting : placing sample for visualization

Light microscopy
Specimen is colored with dyes
mounted on glass slides

EM
Specimen is colorless

Post stained with electron-dense material (Uranyl

acetate, Uranyl nitrate, Lead citrate)

Mounting on copper grids

Samples visualization
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Microscopy

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Dyes and Simple Staining

dyes
make internal and external structures of cell
more visible by increasing contrast with
background
have two common features
chromophore groups
chemical groups with conjugated double bonds
give dye its color

ability to bind cells

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Dyes and Simple Staining

simple staining
a single staining agent is used
basic dyes are frequently used
dyes with positive charges
e.g., crystal violet

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Differential Staining
divides microorganisms into groups based on
their staining properties
e.g., Gram stain
e.g., acid-fast stain

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Gram staining
most widely used differential staining
procedure
divides Bacteria into two groups based on
differences in cell wall structure

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primary
stain

mordant

decolorization
counterstain
positive
negative

Figure 2.14

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Figure 2.15c

Escherichia coli a gram-negative rod

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Acid-fast staining
particularly useful for staining members of the
genus Mycobacterium
e.g., Mycobacterium tuberculosis causes tuberculosis
e.g., Mycobacterium leprae causes leprosy

high lipid content in cell walls is responsible for

their staining characteristics

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Specialized applications of TEM

- Shadow casting
- Negative staining
- Freeze-fracturing

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Microscopy

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Staining Specific Structures

Negative staining
often used to visualize capsules surrounding
bacteria
capsules are colorless against a stained
background

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Negative staining

Electron-dense material (Ex. Phosphotungustic acid) sprayed around

the sample

Electron micrograph reveals white object in dark surroundings

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Microscopy

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Ebola

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Staining Specific Structures

Spore staining
double staining technique
bacterial endospore is one color and vegetative
cell is a different color

Flagella staining
mordant applied to increase thickness of
flagella
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Other preparation methods

shadowing
coating specimen with a thin film of a heavy metal

freeze-etching
freeze specimen then fracture along lines of
greatest weakness (e.g., membranes)

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Shadow casting

For small particles (virus or

macromolecules)

Metal (Cr or Pt) vapors deposited on

sample at precise angle

Electron micrograph is printed in reverse

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Microscopy

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Fly head

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Freeze-fracturing
Visualizes 3D contours of a specimen

Knife

Specimen fractured along naturally weak points (Eg. membranes

around organelles) under frozen condition

Freeze
fracturing

Sectioning

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Microscopy

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How freeze-fracturing is achieved

Sample is fast cooled to -130 oC in liquid Freon

Frozen tissue is fractured at -100 oC

Vacuum sublimation of H2O from fractured surface

Shadow casting by (C + Pt) on fractured surface

C-Pt replica (a ghost of original template) remains

after tissue is dissolved, to generate 3D contour

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@ Produces

Microscopy

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an amorphous solid VITREOUS ICE instead of ice crystals

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Freeze-fracturing : Some examples

Nuclear pores

E. coli

Plasma membrane

RBC membrane
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Microscopy

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Advancements in electron microscopy

High Voltage electron microscopy (HVEM)

High energy e- beam is employed to resolve the structure of thick sample
(E.g. whole cell)

Scanning tunneling microscopy (STM)

Used for imaging surfaces at the individual atomic level with 0.1 nm lateral
x 0.01 nm depth resolution

Reflection electron microscope (REM)

A type of TEM where reflected beam of scattered electrons is detected to
study microstructure of magnetic domains

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Microscopy

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Advancements in electron microscopy

Atomic or scanning force microscopy (A/SFM)

Used for imaging and manipulating atoms and structures on a variety of
surfaces

Cryo-electron microscopy or electron cryo-microscopy (Cryo-EM)

A TEM where sample is studied at LN temperatures to visualize unstained
or unfixed specimens in their native environment

Cryo-electron tomography (CET) reconstructs a 3D view of sample

from 2D images
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Microscopy

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Q??

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Microscopy

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Magnification
ocular power = 10x
low power objective = 20x
high power objective = 50x
a) What is the highest magnification you
could get using this microscope ?
500x
Ocular x high power = 10 x 50 = 500. (We
can only use 2 lenses at a time, not all
three.)
b) If the diameter of the low power field is 2
mm, what is the diameter of the high
power field of view in mm ?

.8 mm
The ratio of low to high power is 20/50. So at high
power you will see 2/5 of the low power field of
view (2 mm). 2/5 x 2 = 4/5 = .8 mm
c) in micrometers ? 800 micrometers
To convert mm to micrometers, move the decimal 3
places to the right (multiply by 1000). .8 mm x 1000
= 800 micrometers
d) If 10 cells can fit end to end in the low power field
of view, how many of those cells would you see
under high power ? 4 cells.
We can answer this question the same way we go
about "b" above. At high power we would see 2/5
of the low field. 2/5 x 10 cells = 4 cells would be
seen under high power.

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