ID. Microscopy and imaging: http://www.microscopyu.

com/
Types of microscopes
1) Light.
a. A standard bight-field light microscope allows us to
examine light transmitted through a specimen.
Preparation of specimen
-fixative like formaldehyde
cross-links amino groups on adjacent
molecules, so the molecules are not
washed out.
-embedding to support
tissue.
-sectioning on a microtome,
which cuts thin sections with a knife.
-sometimes staining to
enhance contrast.
Viewing:
-light focused on specimen
with condenser lenses
-objective and eyepiece
lenses focus image In the eye.

Optical methods to enhance contrast of specimens allow structures in

living cells to be visualized without the use of stains that typically
require cells to be dead.
-Phase contrast converts phase shifts in light passing through
a transparent specimen to brightness changes in the image.
-Differential interference contrast (DIC) uses polarized light to
produce contrast by showing the refractive index gradients of different
areas of a specimen.
Bright field

Phase Contrast

DIC

b. In fluorescence microscopy, fluorescent molecules which are attached to

particular parts of the cell are excited with light of one wavelength, and the
position of the fluorescent molecule is visualized from the light emitted at
a longer wavelength.
DNA and the mitotic spindle detected with
different dyes

c. A confocal microscope is a more sophisticated fluorescence

microscope, which scans a tightly focused laser beam over the
specimen and filters out light that comes from outside the focal
plane. Very sharp images are assembled from these scans by
computer.

-In deconvolution microscopy, a series of images are taken at

different focal planes with a conventional fluorescence microscope. A
computer algorithm removes the blur from each focal plane
computationally.

-Two photon excitation microscopy excites fluorophores with two photons

instead of one. Because this happens mostly in the focal plane of the lens, a
pinhole aperture is not needed for optical sectioning, and and absorbance of
photons does not occur out of the focal plane, leading to a capacity to
penetrate to greater depths and avoidance of photobleaching.

2) Electron microscopes have higher resolving power (can see smaller

objects) because the wavelength of the electron is smaller than the
wavelength of light.

-A
transmission
electron
microscope
(TEM) focuses a
beam of
electrons on a
specimen
stained with
heavy metals to
scatter
electrons, and
detects the
electrons which
pass through
(metal stain
appears dark).

-A scanning
electron
microscope
(SEM) scans a
beam of
electrons over
the surface of
the specimen
coated with
heavy metals,
and detects
excited
electrons which
are emitted, to
compile a
surface view of
the structure.

3) In an in situ hybridization, the labeled nucleic

acid probe, which is typically produced from a cloned
gene, hybridizes with mRNA in fixed and permeablized
cells or tissue.

The signal tells you how much complementary mRNA is

present and where it is expressed.

e.g, pax6 mRNA

in early chicken
embryo

4) Antibody detection methods can also be used in

situ (in tissues) to determine the amount and location
of the protein.
a. E.g., a cell surface protein which causes cells to stick
together is detected at the cell surface by a fluorescently
labelled antibody which binds to the protein, or with
secondary fluorescent antibody that binds to the constant
region of the primary antibody.
Green excitation,
red emission

Other applications of fluorescence

b. Detection of GFP fusion proteins, to detect tissue-specificity, time of
expression, and subcellular localization of proteins in real time (fusion protein
confers a localization signal, while promoters confer tissue-specific and
temporal control).
c. fura-2, whose fluorescence increases at some wavelengths when it is bound
to Ca++ but does not change at other wavelengths, therefore can be used to
quantify and localize increases in cytosolic Ca++ (measure ratio of emissions at
two wavelengths).
A wave of Ca++ moves
across a fertilized egg,
starting that the point of
sperm fusion

d. Fluorescence (or Forster)

Resonance Energy Transfer can be
used to detect protein/protein
interactions. If two proteins come
close enough together (<5nm) to
allow one fluorescent tag (e.g., CFP,
a genetically engineered form of
GFP that emits with a maximum at
480nm) to excite the tag on the
other protein (e.g., YFP, which is
genetically engineered to absorb at
wavelengths overlapping the CFP
emission spectrum, and to emit at
535nm), FRET is detected.
-Efficiency of FRET proportional
1/r6, where r= distance between donor
and acceptor.
-FRET can be used to detect
intracellular 2nd messengers, in
conjunction with proteins that change
conformation when bound with
messenger and alter proximity of
CFP/GFP.

e. Separation of a specific cell type, e.g. with a fluorescence-activated

cell sorter (FACS).

Light scatter detects size

of cell.
Fluorescence
might come from a
fluorescently
tagged antibody
molecule that
binds to a specific
protein on one
type of cell,
or from a dye that
binds to DNA.