Microscopy I

Light and Electron

Microscopy
Replica of van
Leeuwenhoek’s
(1632-1723)
microscope
constructed c.
1670. Moody
Medical Library,
Univ. Texas
Medical Branch,
Galveston, TX
 Replica of Culpepper tripod
microscope built c. 1725 by
Edmund Culpepper (1670-1738).
Collection of Moody Medical
Use the Library, Univ. Tex., Galveston,
TX. (Replica by Replica Rara Ltd.
information in Antique Microscopes)

this tutorial to
supplement the
visuals in lab
and the
information in
Chapters 1, 8
and 9 in your
lab manual .
 Contents
I. Types of Microscopy
A. Light Microscopy
1. Brightfield
2. Darkfield
3. Phase Contrast
4. Polarizing
5. DIC
6. Epi-fluorescence
7.Confocal laser scanning
B. Electron Microscopy
1. Scanning
2. Transmission
II. Specimen Preparation Replica of Marshall
Microscope, c. 1700, by John
A. Light Microscopy Marshall (1663-1725).
B. Scanning Electron Microscopy Collection of Moody Medical
C. Transmission Electron Microscopy Library, Univ. Texas,
Galveston, TX
Microscopes have long been essential
tools of cell biologists. This tutorial
provides a brief overview of types of
microscopes commonly used in biological
studies and general techniques for
preparing specimens for the various types
of microscopy. The two broad categories
of microscopy we are concerned with are:
Old monocular
brightfield
Light Microscopy (LM) microscope with
fixed stage and
and mirror.

Electron Microscopy (EM)

Light Microscopy
Bright field Microscopes--the
most common general use
microscopes. Bright field
microscopes are named
because the microscopic “field”
is bright, while the object being
viewed is dark.
- Simple design
- Light directed at specimen is absorbed to
form image
- Unstained specimens have poor contrast
Binocular, bright field microscope with
- Stained specimens show excellent
movable stage, dioptic adjustment,
contrast
condenser and iris diaphragm, and
- Ideal for stained bacteria, cells, tissues
built-in light source. These are used
- High N.A., good resolution
as clinical, research and student
- Bright background, dark specimen
microscopes.
- tungsten or halogen light source
 Section of gut tissue
Bright field images-- containing ciliated parasites

Stained blood cells in

peripheral blood smear

Flagellate --Trichomonas
The basic design of
bright field
microscopes has been Light
modified for special
uses. Inverted
microscopes (right)
allow viewing of cells in
Objectives
flasks, welled-plates, or
other deep containers
that do not fit between
the objectives and Inverted microscope.
stage of standard BF Position of light source and
objectives is “inverted”--
microscopes. light source is above
specimen and objective
lenses are located beneath
the stage.
Dark field Microscopes (DF)
The dark field microscope creates a dark
background to allow viewing of small unstained
objects, such as motile bacteria, that would be
difficult to view in a bright field. The central
portion of the light is blocked so that only
oblique light strikes the specimen, scattering
light rays that then enter the objective to form
the image.

- A method from the 19th century

- Bright specimen, dark background
- Light not scattered by the specimen bypasses the
objective, therefore making the “field” dark.
- Can see very small objects but resolution is
variable
-High contrast, good for unstained, live, and motile
specimens
Darkfield Images--

Leptospira, a spirochete,
viewed with darkfield
microscopy

The algae Hydrodictyon

reticulatum viewed with
darkfield microscopy. Photo
by Dr. D. Folkerts, AU.
Phase Contrast--Converts differences in
refractive index in a specimen to differences in
image brightness.
The central portion of the light source is blocked,
creating a ring of light from the condenser that
illuminates the specimen. The light waves refracted
by the specimen are slowed by a phase retardation
plate (in phase objectives) increasing the difference
in wavelength between refracted and unrefracted
rays (which do not pass through the phase plate).
When the refracted and and unrefracted waves are “Phase”
focused, they produce an interference due to the objectives
difference in wavelengths--this is seen as
differences in brightness in the specimen.
- technology from 1940’s
- provides high contrast, good resolution
Annular
- good for bacteria, flagella, cilia, organelles such as ring
mitochondria
- good for unstained or live mounts
- phase halos (artifacts) occur
Phase Contrast Image (left)--compared with DIC
image (right)

Unstained squamous epithelial cells observed with

phase contrast microscopy (above) and DIC microscopy
(right). Differences in refractive index in various
regions of the cells account for contrast in the images.
(From Becker et al., The World of the Cell, Benjamin/Cummings
Publ. Co., 2000.)
Polarization Microscopy
Detects specimens that are birefringent
(have the characteristic of double
refraction, i.e. the velocity of light Polarizer
refracted by a substance is not the same
in all directions). The specimen is placed
between two polarizers crossed at 90o to
each other (one in condenser and one in
Specimen
objective).
- bright image, dark background
- used for substances with highly organized Polarizer
molecular structure, such as crystals,
minerals
- can be quantitative
Polarizing Microscopy images--many crystals and
minerals display characteristic patterns in polarizing light

Left, portion of Martian meteorite that has been ground to 0.3

mm depth and viewed under polarizing microscopy (From Calvin J.
Hamilton). Top right, BF view of renal epithelial cells containing
fat globules; bottom right, high magnification view of same
cells viewed with polarizing microscope and showing typical
“maltese cross” formation displayed by fat in polarized light.
(From R. Hyduke, U. Iowa Hospitals & Clinics.)
Epi-fluorescence Microscopy
Allows the detection of molecules and ions
within cells. Fluorescent dyes absorb short
wavelengths of light and emit longer UV light
source
wavelengths. Barrier filters and a dichroic
prism select the excitation wavelength that
strikes the specimen and exclude the Barrier
excitation wavelength from the detector, filters and
allowing only emitted light to reach the dichroic
detector (oculars). prism
- uses uv light source = mercury or xenon arc
lamp.
- high contrast, high resolution image
- special fluorescent dyes used to locate
“molecules” in a specimen
- black background, bright-stained specimen
- no condenser required, light comes from above
(“epi”) specimen
- multiple fluorescent probes available
- detects small quantities, molecules; can use
antibody staining techniques
 Epi-fluorescent images--The locations of specific molecules can
be identified using fluorescent probes. In immunofluorescence
techniques, antibody probes that bind to the molecules and
secondary antibody labeled with a fluorescent dye are used.

Above--Immunofluorescence of Tetrahymena, a
ciliated protozoan, using the fluorescent dye,
fluorescein isothiocyanate, which emits “green light.”
Above left, cell is stained with anti-basal body
antibody; above right, cell is stained with antibody
against cell membrane surface antigen. Compare the
information obtained using the fluorescent technique
with the BF view of Tetrahymena (far right) stained
with hematoxylin.
Epi-fluorescent image showing metaphase in newt lung cell. Three
fluorescent probes--specific for DNA, keratin, and tubulin--were
used. Blue, metaphase chromosomes (DNA); red, keratin filaments;
yellow, spindle apparatus made of microtubules. From ASCB, Bethesda, MD;
image by Rieder and Hughes, NY State Dept. Health, Albany, NY.
Differential Interference Contrast
(DIC) Microscopy--also called
Normarski optics
Resembles phase-contrast but more sensitive--
gives higher resolution. Uses polarizing lenses Recombining
prism
like the polarizing microscope, and therefore can Mercury
be quantitative. The halogen light beam is lamp
polarized, split by a beam splitter (Wollaston
prism), and passed through the specimen. The Beam splitter
split beams are recombined by a second prism in (Wollaston
the objective. Any change in a light wave due to prism)
Polarizing
passage through a specimen causes interference filter
as the beams recombine, producing differences
in image brightness corresponding to the
refractive index of specimen structures.
Microscope having both
- produces 3-D images epi-fluorescence and
- excellent resolution, high NA, high contrast DIC capabilities.
- good for unstained specimens, live mounts; can
see membranes within cells
- detects changes in refractive index of specimen
Hoffman modulation contrast microscopy (HMCM)
is the poor man’s DIC. Contrast is not as good as in DIC.
DIC image--
comparison of DIC and
fluorescent images O
Top, DIC image of N
cricket ovariole
showing oocytes N
(O), oocyte nuclei B
(N), and Balbiani
bodies (B), also
called mitochondrial
clouds. Note three-
dimensional
appearance.
Bottom, epi-
fluorescent image
of Balbiani bodies B
stained with
rhodamine 123, a
fluorescent stain
specific for
mitochondria.
Confocal laser scanning microscopy
A laser is focused at a plane in
the specimen and scans the
specimen in a horizontal (XY)
plane. Only light from the plane Laser/scanner
of focus reaches the detector.
The scanned image (an optical
section) is digitally recorded.
Images from consecutive focal
planes can be recorded, and
composite or 3-D images can
be digitally created.
- krypton/argon laser
- high resolution, sharp image
- high sensitivity
- Can be used in reflectance or
fluorescence mode Bio-Rad confocal microscope
- eliminates background showing fluorescent-labeled
cells on monitor
interference
Confocal image--

Above, BF
image of
Trichomonas.
Posterior
flagellum
(green arrow)
can be seen
but axostyle
(red arrow) is
unstained.

Left, image composed from 13 optical sections of Trichomonas

immunostained with anti-tubulin and fluorescein isothiocyanate (FITC).
The green structures consist of microtubules. Pf, posterior flagellum;
Af, anterior flagella; C, costa; and axostyle (red arrow). Compare with
BF image (right). (Photo by B. Estridge)
 Electron Microscopy
Scanning Electron Microscopy (SEM)
Transmission Electron Microscopy (TEM)
Scanning Electron Microscopy (SEM)
Fixed, dehydrated specimens are mounted on
stubs and surface-coated with gold, palladium or
rhodium. The specimen is placed in a vacuum
and an electron beam scans back and forth over
it. Electrons that bounce off the metal-coated
specimen surface are collected, converted to a
digital image and displayed on a TV-like monitor.
- Electron beam is focused using a magnetic field
- SEM provides a 3-D image
-Gives information about external topography of
specimen
-Much higher resolution and magnification than
possible in LM
 SEM images--comparison of SEM images (top)
with images obtained using DIC microscopy (bottom).

Both DIC microscopy and SEM reveal specimen surface topography. Much
greater detail is obtained using SEM because of greater resolution and
magnification capability. Top, SEM of Trichomonas (left) and Tetrahymena
(right); bottom, DIC images of Trichomonas (left) and Tetrahymena (right).
Transmission Electron Microscopy
(TEM)
Fixed, dehydrated specimens are embedded in a
resin, hardened, sectioned, stained with heavy metals
such as uranium and lead, and inserted into the
electron column in the microscope. The electron beam
is absorbed or deflected by the heavy metal stains
and shadows are cast onto film or a phosphorescent
plate (image is a shadow) at the bottom of the column.
- 2-D image
- reveals internal cell structure
- high resolution, high magnification
- electron beam is focused by magnetic field
Comparison of TEM (right) and SEM (left) images

Trichomonas as viewed with SEM (left) and TEM (right).

Note that with SEM details of external structure are
visible while in TEM internal structures are revealed. N,
nucleus; G, Golgi; H, hydrogenosome (equivalent of a
mitochondrion); F, flagellum; Rf, recumbent flagellum; B,
cross-sections of basal bodies of three anterior flagella.
TEM images--

RER

Nu

CB
N

TEM photos of rat liver cell (left) and pancreatic acinar cells (right)
revealing internal cell structure. N, nucleus; Nu, nucleolus, RER,
rough endoplasmic reticulum; CB, cell boundary; Z, zymogen granules;
M, mitochondrion.
Specimen Preparation

Light Microscopy
Scanning Electron Microscopy
Transmission Electron Microscopy
 General Schematic for Preparing Specimens for
Light Microscopy
Live mounts Fixed (preserved) Specimens
(Histology)
View Vital stain 1) Fix
View 2) Dehydrate
3) Infiltrate and Embed
4) Section
5) Mount
6) Stain
7) View
 Live Mounts
To view organisms, tissues, or cells
in as close to the natural state as
possible, unstained live mounts are used.
Viewing time of live mounts is limited.
Unstained specimens have low contrast.
Supravital stains may be applied to
provide more contrast or identify certain
components--these are stains that are
not harmful to living cells.
 Fixed (Preserved) Specimens for
Histology--the Study of Tissue
1) Specimens may be preserved using chemicals
such as formalin, acetic acid, ethanol, and
methanol. Fixation immobilizes molecules such as
proteins and lipids.
2) Fixed specimens are dehydrated by serial
transfer through an ascending alcohol series, to
100% alcohol.
3) Specimens are infiltrated with melted
paraffin, paraffin substitute, or plastic and
placed in a mold to harden.
 4) Specimens are cut into 5-10 um thick
sections using a steel knife or razor on an
instrument called a microtome.

Specimen
Holder
Knife
holder

Vibratome--uses a
vibrating knife to cut
sections
 5) Sections are then mounted on slides,
6) Stained to achieve contrast or identify
cell structures or components, and
7) Viewed microscopically
Many variations in technique are used to prepare
specimens for light microscopy. Some omit the
dehydration, infiltration, embedding and sectioning steps
and use aqueous staining systems for viewing whole mounts
(unsectioned tissues or cells).
Freezing may be used instead of chemicals to fix
tissues that need to be examined quickly or that have
components damaged by the chemicals. Examples are
frozen biopsies and tissues in which heat-labile structures
are to be stained. Frozen specimens are sectioned using a
cryotome, a microtome encased in a freezing chamber.
Permanent slides may be made from paraffin sections but
not from frozen sections.
Schematic for Preparing Specimens for Scanning
Electron Microscopy (SEM)
1) Fix

2) Dehydrate

3) Mount on stubs

4) Sputter coat

5) Observe
1) Fixation--fixatives used are glutaraldehyde,
paraformaldehyde, osmium tetroxide.
2) Dehydration is accomplished by carrying the
specimens through an ascending alcohol series, to 100%
alcohol (i.e., no water), then to an organic solvent such as
acetone or propylene oxide. Specimens for SEM may also
be processed in a critical point drying apparatus.
3) Specimens are mounted on aluminum stubs using
sticky tape.

Applying specimens to stubs

Example arranged in a holder.
of one
type of
stub

Stubs

4) A sputter coater coats the specimen with gold,

palladium or rhodium in a special chamber to cover the
specimen with a 10-20 nm thick metal layer
5) The stub is inserted into the SEM, scanned and
observed on a video display

Electron
column

Display of
scanned
image

Specimen
Insertion
Schematic for Preparing Specimens for Transmission
Electron Microscopy (TEM)
1) Fix

2) Dehydrate

3) Infiltrate -- Embed

4) Section

5) Apply sections to grids

6) Stain

7) Observe
1) Fixation--specimens are fixed in glutaraldehyde, or
paraformaldehyde-glutaraldehyde mixtures, followed by
osmium tetroxide.
2) Dehydration is accomplished by carrying the
specimens through an ascending alcohol series, to 100%
alcohol (i.e., no water), then to an organic solvent such as
acetone or propylene oxide.
3) Specimens are then infiltrated with an epoxy or plastic
resin and placed in plastic molds to harden.
Molds

Specimen
Hardened
Specimen specimen
ID blocks
removed
from molds
4) An instrument called an ultramicrotome is used
to section the specimen.

Specimen
Holder
Knife
holder
 Glass or diamond knives are used to cut the ultrathin
sections.
Closeup view of
ultramicrotome
specimen holder

3 mm Resin
Diamond Specimen
in epoxy block
knife containing
specimen
Knife
edge
Ultrathin
sections,
approx. 1
mm wide
5) Sections are transferred to tiny metal grids for support
(the equivalent of the function of the glass slide in LM).

Closeup view of
one type of grid

Grid and
forceps

6) Heavy metal stains such as uranyl acetate and lead

citrate are applied to make certain structures electron
dense.
7) Grids are then inserted into the transmission electron
microscope and observed.

Electron
Additional Column
image display

Specimen
Insertion

Phosphorescent
plate for viewing
image