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 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, 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 Drosophila eye 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

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, 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 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