Advances in Bioscience Education Summer Workshop

Immunolabeling for Fluorescence and Electron Microscopy

June 27 - 29, 2006

Biological Electron Microscope FacilityPacific Biosciences Research Center

University of Hawai’i at Manoa

Biological Electron Microscope Facility

Pacific Biosciences Research Center, University of Hawai’i 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, KCCRed 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 FV1000Three colors + Trans-mitted simultaneouslyExcitation with 405, 458, 488, 515, 543, and 633 nm lasersVarious emission filtersOptical sectioning3-D reconstructionStereo viewsAnimations

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 µm 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 it’s 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 Stoke’s 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, Queen’s 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, slowVideo cameras – Low resolution, fastPhotomultiplier 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 Hawai’i 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