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Advances in Bioscience Education
Summer Workshop
Fluorescence and Electron Microscopy
June 26 - 29, 2007
Biological Electron Microscope Facility
Pacific Biosciences Research Center
University of Hawai’i at Manoa
What is a Microscope?
 A tool that magnifies and improves resolution of the
components of a structure
 Has three components:

sources of illumination,

a magnifying system,

detectors.
Sources of Illumination
 Light microscopes use a beam of light for illumination
and include fluorescence and confocal microscopes
 Electron microscopes use electrons as a source of
illumination and include transmission and scanning
electron microscopes.
Light and Electron Microscopes
 Lenses are
used to control
a beam of
illumination,
magnify, and
direct an image
to a detector
Images and pictures are your data!
Epifluorescence Microscopy
Common Fluorescence Applications
Localize/identify specific organelles
Detect live cells vs. dead cells, necrotic vs.
apoptotic cells
Determine cell membrane permeability
Localize antigen-specific molecules
Multiple labeling
Laser Scanning Confocal Microscope
 Better resolution
 Serial optical sections
can be collected from
thick specimens
 Live or fixed cell and
tissue imaging
Laser Scanning Confocal Microscopy
Drosophila eye
Plant Protoplast
Photos courtesy of Gregg Meada & Dr. Gert DeCouet,
UHM
And Dr. Chris Yuen and Dr. David Christopher
Epifluorescence vs. Confocal
Sample courtesy Gregg Meada &
Dr. Gert DeCouet, UHM
Scanning Electron Microscopy (SEM)
 View outer surface
 Coat specimen with
gold
 No sectioning
 High Mag (40x to
300,000x)
 High resolution (better
than 2 nm)
SEM Images
Transmission Electron Microscopy
(TEM)
View inside cell via sections
magnification 120,000 X
50,000X
Conventional TEM Micrographs
Bacteria in cell
Apoptosis
Skin
Collagen
Chloroplast
Virus in cell
Ultra-microtomy
 Ultrathin (60-90 nm)
sectioning of resinembedded specimens
 Several brands/models
available
Cryotechniques
 Ultrarapid cryofixation
 Metal mirror impact
 Liquid propane plunge
 Freeze fracture with
Balzers 400T
 Cryosubstitution
 Cryoultramicrotomy –
Ultrathin frozen
sections (primarily for
antibody labeling)
Immunolocalization




LM
Fluor/confocal
TEM
SEM with
backscatter
detector
Approaches to Immunolabeling
Direct Method: Primary antibody contains
label
Indirect Method: Primary antibody
followed by labeled secondary antibody
Amplified Method: Methods to add more
reporter to labeled site
Two-step Indirect Method for
Immunolabeling
Fluorescentconjugated
secondary
antibody attaches
to primary antibody
that is bound to
antigen
Immunolabeling for Transmission
Electron Microscopy
 Normally do Two-Step
Method
 Primary antibody
applied followed by
colloidal gold-labeled
secondary antibody
 May also be enhanced
with silver
Colloidal Gold Immunolabeling for
TEM
Colloidal gold of defined sizes, e.g., 5 nm,
10 nm, 20 nm, easily conjugated to
antibodies
Results in small, round, electron-dense
label easily detected with EM
Can be enhanced after labeling to enlarge
size for LM or EM
Double-labeling Method
 Use primary antibodies
derived from different
animals (e.g., one
mouse antibody and
one rabbit antibody)
 Then use two different
secondary antibodies
conjugated with
different sized gold
particles
Preparation of Biological Specimens for
Immunolabeling
 Preserve tissue as closely as possible to its
natural state while at the same time maintaining
the ability of the antigen to react with the antibody
 Chemical fixation OR
 Cryofixation
Chemical Fixation
 Antigenic sites are easily denatured or masked during
chemical fixation
 Glutaraldehyde gives good fixation but may mask
antigens, plus it is fluorescent
 Paraformaldehyde often better choice, but results in
poor morphology , especially for electron microscopy
 May use e.g., 4% paraformaldehyde with 0.5%
glutaraldehyde as a good compromise
Embedding
 Dehydrated tissue is embedded in a plastic resin
to make it easier to cut thin sections
Steps in Labeling of Sections
 Chemical fixation
 Dehydration, infiltration, embedding and
sectioning
 Blocking
 Incubation with primary antibody
 Washing
 Incubation with secondary antibody congugated
with reporter (fluorescent probe, colloidal gold)
 Washing, optional counterstaining
 Mount and view
Controls! Controls! Controls!
Omit primary antibody
Irrelevant primary antibody
Pre-immune serum
Perform positive control
Check for autofluorescence
Check for non-specific labeling
Dilution series
Light Microscopes
Light Path in Fluorescence
 Light delivered
through excitation
filter and then
objective lens to
specimen where it
is absorbed;
 emitted light goes
back through
objective lens
through barrier
filter and emission
filter and then to
detector.
Fluorescence
 Light beam excites
the fluorochrome,
raising it to a higher
energy state,
 As it falls back to
it’s original state, it
releases energy in
the form of a light of
lower E and longer
wavelength than
original beam of
light
Primary Ab = PDI
secondary Ab = Alexafluor
Blue light = exciting beam
green and red light emitted
 And use them to your advantage!
 Green is label; orange-red is
autofluorescence
 Acts as counterstain
Know Your Artifacts
Autofluorescence
Fluorescence
 Fluorochromes are
excited by specific
wavelengths of light
and emit specific
wavelengths of a
lower energy
(longer wavelength)
Filter Cubes for Fluorescence
 Filter cubes
generally have an
excitation filter, a
dichroic element,
and an emission
filter
 The elements of a
cube are selected
for the excitation
and fluorescence
detection desired
Choose Fluorochrome/Filter Combos
Laser Scanning Confocal Microscopy
 Fluorescence technique
 Uses laser light for excitation
 Improves image resolution over conventional
fluorescence techniques
 Optically removes out-of-focus light and detects
only signal from focal plane
 Can construct an in-focus image of considerable
depth from a stack of images taken from different
focal planes of a thick specimen
 Can then make a 3-D image that can be tilted,
rotated, and sliced
Principal Light Pathway in Confocal
Microscopy
 Laser light is scanned pixel
by pixel across the sample
through the objective lens
 Fluorescent light is reflected
back through the objective
and filters (dichroic mirrors)
 Adjustable pinhole apertures
for PMTs eliminate out-offocus flare
 Image is detected by
photomultiplier(s) and
digitized on computer
TEM
 Transmission Electron
Microscope
 Illumination source is
beam of electrons from
tungsten wire
 Electromagnetic lenses
perform same function
as glass lenses in LM
 Higher resolution and
higher magnification of
thin specimens
Specimen Preparation for TEM
 Chemical fixation with buffered glutaraldehyde
 Or 4% paraformaldehyde with >1% glutaraldehyde
 Postfixation with osmium tetroxide
 Or not, or with subsequent removal from sections
 Dehydration and infiltration with liquid epoxy or
acrylic resin
 Polymerization of hard blocks by heat or UV
 Ultramicrotomy – 60-80nm sections
 Labeling and/or staining
 View with TEM
High pressure freezing:
Plant tissue is flash frozen in a pressure bomb
-197 C
Water in the tissue is replaced with acetone
over 5 day period
Acetone saturated tissue is embedded in resin
Resin is cut in thin sections, 80 nm thick
Add antibodies - immunolabeling
Look under Electron microscope
Very
Wrinkled
Chloroplast
Carnage
Pretty bad
fixation
2nd time: stainings were done poorly, but there is hope…
Back to the
drawing board
to start over.
But what to
correct?
What to do
different?
Will it
improve?
Despite mistakes, keep moving forward
and ignore doubt and negativism that comes with pressure.
3rd time
A charm
Excellent preservation
And
Immunolabeling
the 3rd TIME
HIGH
MAG
RE-search Not search
Must be repeated
Research time is spent:
70% trouble-shooting
15% success
15% communicating success.
ROOT
HOOK-o-PLASM
PDI in
Vacuole
CNGC in Golgi Apparatus
g
200 nm
PDI in Golgi Apparatus
c
G
200 nm
Dividing mitochondria
Channel located to the plasma membrane
Channel located to the plasma membrane -plasmolysis
We learn more from mistakes than successes…