in Advances 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 + Transmitted 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
Drosophila eye
Photo courtesy of Gregg Meada & Dr. Gert DeCouet, UHM
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
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 resinembedded 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/immunogold 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
Fluorescentconjugated 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., streptavidinbiotin 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-offocus 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 JourdanLeSaux)
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 Or not, or with subsequent removal from sections
Postfixation with osmium tetroxide
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-A5nm-gold and Protein-A-15-nmgold 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: [phone] FAX: [phone] URL: http://www.pbrc.hawaii.edu/bemf E-mail: [e-mail] [e-mail]
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