Advances in Bioscience Education

Summer Workshop
Immunolabeling for
Fluorescence and Electron Microscopy

June 27 - 29, 2006

Biological Electron Microscope Facility
Pacific Biosciences Research Center
University of Hawaii at Manoa
Biological Electron Microscope Facility
Pacific Biosciences Research Center, University of
Hawaii at Manoa
Instrumentation, service and training
State-of-the-art instruments for biological
microscopy
In operation since 1984
Personnel:
Dr. Richard D. Allen, Director
Dr. Marilyn F. Dunlap, Manager
Tina M. (Weatherby) Carvalho, M.S., Supervisor
Light and Electron Microscopy
Light microscopy
Glass lenses
Source of illumination is usually light of visible
wavelengths
Tungsten bulb
Mercury vapor or Xenon lamp
Laser

Electron microscopy
Electromagnetic lenses
Source of illumination is electrons
Hairpin tungsten filament (thermionic emission)
Pointed tungsten crystal (cold cathode field emission)
Lanthanum hexaboride
Epifluorescence Microscopy
Olympus BX51
upright microscope
Broad-band
epifluorescence
excitation and
detection
DIC optics
Optronics scientific
grade digital camera
Epifluorescence
Green photos courtesy Dr. Teena Michaels, KCC
Red photo courtesy Dr. Claude Jourdan-LeSaux
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
Olympus Fluoview
FV1000
Three colors + Trans-
mitted simultaneously
Excitation with 405,
458, 488, 515, 543, and
633 nm lasers
Various emission filters
Optical sectioning
3-D reconstruction
Stereo views
Animations

Laser Scanning Confocal Microscopy
Adjustable pinhole
aperture eliminates
out-of-focus glare
Better resolution
Serial optical
sections can be
collected from thick
specimens
Live or fixed cell
and tissue imaging
Photo courtesy of Gregg Meada & Dr.
Gert DeCouet, UHM
Drosophila eye
Epifluorescence vs. Confocal
Sample courtesy Gregg Meada &
Dr. Gert DeCouet, UHM
Field Emission Scanning Electron
Microscopy (FESEM)
Hitachi S-800 FESEM
High magnification (40x
to 300,000x)
High resolution (better
than 2 nm)
Easy to learn
Hi-res digital images
Prep equipment:
critical point dryer,
sputter coater

SEM Images
Transmission Electron Microscopy
(TEM)
Zeiss 10/A conventional
TEM
Excellent for training
Film only

LEO 912 Energy-Filtering TEM
In-column energy filter
(electromagnetic prism)
Ultrathin to 0.5 m sections
Contrast tuning
Elemental analysis with
electron energy loss
spectroscopy (EELS)
Elemental mapping with
electron spectrographic
imaging (ESI)
Eucentric goniometer stage
Digital images
Conventional TEM Micrographs
Skin
Bacteria in cell Apoptosis
Chloroplast Collagen Virus in cell
Negative Staining
Viruses, small
particles, proteins,
molecules

No sectioning

Same day results
EFTEM - Electron Spectrographic
Imaging (ESI) - elemental mapping
Calcium in
mitochrondria
from ischemic
brain
Iron in liver
EFTEM- Electron Energy Loss
Spectroscopy (EELS)
EELS spectrum
Ultramicrotomy
Ultrathin (60-90 nm)
sectioning of resin-
embedded specimens
Several brands/models
available
Cryoultramicrotomy

Cryotechniques
Ultrarapid cryofixation
Metal mirror impact
Liquid propane plunge
Freeze fracture with
Balzers 400T
Cryosubstitution
Cryoultramicrotomy
Ultrathin frozen
sections (primarily for
antibody labeling)
Cryo Examples
Freeze fracture,
deep-etch, rotary
shadow
Cryosection/im-
munogold label
Cryosubstitution
Image Manipulation and Analysis
Soft Imaging System
analySIS professional
software
EFTEM acquisition and
analysis
Light Microscopy
Images from other sources
Particle counting and
analysis
Feature extraction
Image and results database

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
Protein A Method: May be used as
secondary reagent instead of antibody
Direct Labeling Method
Labeled primary
antibody reacts
directly with the
antigen in the
histological or
cytological
preparation
Two-step Indirect Method
Fluorescent-
conjugated
secondary
antibody attaches
to primary antibody
that is bound to
antigen
Amplified Method
If the antibody
reporter signal is
weak, the signal
can be amplified by
several methods,
e.g., streptavidin-
biotin complex

Double-labeling Method
Use primary antibodies
derived from different
animals (e.g., one
mouse antibody and
one rabbit antibody)
Then use two
secondary antibodies
conjugated with
reporters that can be
distinguished from one
another
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
Can also do for LM
Preparation of Biological Specimens
for Immunolabeling
The goal is to 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 of whole mounts prior to
labeling for LM
Chemical fixation, dehydration, and embedment in
paraffin or resin for sectioning for LM or TEM
Chemical fixation for cryosections for LM
Cryofixation for LM or TEM

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

Preembedding or Postembedding
Labeling
May use preembedding labeling for surface
antigens or for permeabilized cells
The advantage is that antigenicity is more likely
preserved

Postembedding labeling is performed on
sectioned tissue, on grids, allowing access to
internal antigens
Antigenicity probably partially compromised by
embedding

Steps in Labeling of Sections
Chemical fixation
Dehydration, infiltration, embedding and
sectioning
Optional etching of embedment, permeabilization
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
Dilutions are Important
Typically should do an
extensive dilution series to
determine best
concentration of both
primary and secondary
antibodies

This shows an antibody at
concentrations of 1:100 and
1:2000
Know Your Artifacts
And use them to your advantage!
Green is label; orange-red is
autofluorescence
Acts as counterstain

Autofluorescence
Need to select label
that will be readily
distinguished from
autofluorescence
Several techniques
to quench
autofluorescence
What is a Microscope?
A tool that magnifies and improves resolution of
the components of a structure
Has three components: one or more sources of
illumination, a magnifying system, and one or
more detectors
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
Light Microscopes
Objective Lenses
Objective lens choice is important!
Not all objective lenses are created equal
The more correction a lens has, the less transmission
Resolution is dictated by Numerical Aperture (NA)
Talk to your microscope company representative
Light Microscopes - Resolution
Resolution depends
on the light gathering
of the objective, which
depends on the NA,
and on the light path,
which includes the
slide, sample,
mounting medium,
coverslip, and air or
immersion oil

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 Microscopes
Illumination light path is
the same as the sampling
light path
Need to maximize the light
throughput in both
directions no more than
22% of light will be
detected on a good day
Need to match refractive
indices (RI)
Use the best optics with the
fewest elements
Optical Choices for Fluorescence
Minimize the number of lens elements to increase light
throughput, but correct for spherical aberration

Optimize magnification and NA; best choice often a 60X
1.4NA plan objective

Only use magnification required to collect the information
needed

Use a mercury lamp for normal work and a xenon lamp for
quantitative studies
Kohler Illumination
Kohler illumination is essential for good
transmitted light contrast
Focus slide
Close field diaphragm
Focus diaphragm in field by adjusting condenser
height
Center diaphragm in field
Open diaphragm to fill field and recheck
centration
Adjust iris diaphragm (on condenser) to taste
(affects contrast and depth of focus)

Elements of Fluorescence Microscope
Light source
Mercury vapor
Xenon
Laser

Optical lenses

Optical filters

Detection system
Eye
Film camera
Digital camera
Photomultiplier tube (PMT)
Fluorescence
Photons of a
certain energy
excite the
fluorochrome,
raising it to a
higher energy
state, and as it falls
back to its original
state it releases
energy in the form
of a photon of
lower energy than
the excitation
energy.
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

Classification of Filters
Long pass passes longer wavelengths

Short pass passes shorter wavelengths

Band pass passes defined wavelengths

Dichromatic mirror transmits long
wavelengths, reflects shorter wavelengths
Choose Fluorochrome/Filter Combos
Spectral Characteristics of Probes
Omega Filters Curv-o-Matic
http://www.omegafilters.com/front/curvomatic/spectra.php
Other filter and microscope companies
Ideal Fluorochrome
Small size must get into cell
High absorption maximum sensitive to excitation
Narrow absorption spectrum excited by a narrow
wavelength
High quantum efficiency likely to fluoresce
Narrow emission spectrum so you can find it
specifically
Large Stokes shift emission curve far enough away
from excitation curve to minimize bleedthrough

Types of Fluorochromes
Simple dyes
Acridine orange, DAPI, Propridium iodide, Lucifer yellow

Physiological probes
Calcium green, Rhodamine 123, Fluorescein diacetate

Specific probes
Phalloidin, Lectins, GFP, Primary and secondary antibodies


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-of-
focus flare
Image is detected by
photomultiplier(s) and
digitized on computer
Compressed Z-stack Image
3-D reconstruction
Tilt and rotate
Stereo projection
Animation
Montage
Image
enhancement

Photo courtesy Dr. Alex Stokes, Queens Medical Center
Confocal Movies
Photo courtesy Dr. Alex Stokes, Queens Medical Center
Confocal Projects
Investigation of Wnt pathways in sea urchin
gastrulation (Dr. Christine Byrum/Dr. Athula Wikramanayake)
Localization of transmembrane proteins in airway
smooth muscle cells (Dr. Lynn Iwamoto, Kapiolani)
GFP in drosophila (Gregg Meada/Dr. Gert deCouet)
Neurohormones (Dr. Ian Cooke/Toni Hsu)
IL-10 receptors of lung fibroblasts (Dr. Claude Jourdan-
LeSaux)
Aggregation of acetylcholine receptors in muscle
cells (Drs. Jes Stollberg, UHM, and Michael Canute, HPU)

Differential Interference Contrast
(Nomarski)

Digital Imaging
Digital advantages include sensitivity,
speed, quantitation, feature extraction and
image analysis

CCD cameras - High resolution, slow
Video cameras Low resolution, fast
Photomultiplier tubes (PMTs) point
recorders, used for confocal

Digital Cameras
Need enough sensitivity for signal you want to
detect
Need enough speed for event you want to detect
Need enough grayscales 8 bits for
documentation, 12 bits for quantitation
Need enough resolution - the number of of pixels
must be sufficient to distinguish features of
interest, but too many pixels is a waste of data
space
Color is simply three black and white images
combined and useful primarily for image
processing

Optronics MacroFire Digital Camera
Extremely sensitive
2048 x 2048 pixels
Millisecond exposures
Firewire
Fits on both Olympus
compound and stereo
zoom microscopes
Suitable for BF, DF, and
Fluorescence
Also Optronics MagnaFire
SP 1280 x 1024 pixels and
Nikon Coolpix cameras
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
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
Colloidal Gold in TEM
Colloidal Gold in TEM

Double Immunogold Labeling of
Negatively Stained Specimens
Bacterial pili
serotypes dried
onto grid and
sequentially
labeled with
primary antibody,
then Protein-A-
5nm-gold and
Protein-A-15-nm-
gold before
negative staining
TEM Grids
TEM grids are 3
mm supports of
various meshes
You will handle
them by the
edges with fine
forceps
Colloidal Gold in SEM
Gold particles are often
difficult to see against
the membrane with
secondary electron
detection

Gold particles show up
brighter with
backscattered electron
detection
Preparation of Images for
Publication
Microscopy
Images are your
data!

Adjustment and
labeling of images
for figure plates
with Adobe
Photoshop

How to Contact the BEMF

Location: Snyder Hall 118 University of Hawaii at Manoa

Phone: 808 956-6251

FAX: 808 956-5043

URL: http://www.pbrc.hawaii.edu/bemf

E-mail: dunlap@pbrc.hawaii.edu
tina@pbrc.hawaii.edu
Acknowledgments

We thank all of the researchers who agreed
to let us use their images for this
presentation

Microscopy & Microanalysis 2005
July 31 - August 4, 2005
Hawaii Convention Center
Over 1100 talks and posters
Huge trade show featuring the latest in
microscopes and related instrumentation,
software, and support
Pre-meeting workshops
http://mm2005.microscopy.org