1. Advances in Bioscience Education Summer Workshop
Immunolabeling for
Fluorescence and Electron Microscopy
June , 2006
Biological Electron Microscope Facility
Pacific Biosciences Research Center
University of Hawai’i at Manoa

2. 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

3. 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

4. Epifluorescence Microscopy
Olympus BX51 upright microscope
Broad-band epifluorescence excitation and detection
DIC optics
Optronics scientific grade digital camera

5. Epifluorescence Green photos courtesy Dr. Teena Michaels, KCC
Red photo courtesy Dr. Claude Jourdan-LeSaux

6. 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

7. 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

8. 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

9. Epifluorescence vs. Confocal
Sample courtesy Gregg Meada & Dr. Gert DeCouet, UHM

10. 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

11. SEM Images

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

13. 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

14. Conventional TEM Micrographs
Skin
Bacteria in cell
Apoptosis
Chloroplast
Collagen
Virus in cell

15. Negative Staining Viruses, small particles, proteins, molecules
No sectioning
Same day results

16. EFTEM - Electron Spectrographic Imaging (ESI) - elemental mapping
Calcium in mitochrondria from ischemic brain
Iron in liver

17. EFTEM- Electron Energy Loss Spectroscopy (EELS)
EELS spectrum

18. Ultramicrotomy
Ultrathin (60-90 nm) sectioning of resin-embedded specimens
Several brands/models available
Cryoultramicrotomy

19. Cryotechniques Ultrarapid cryofixation
Metal mirror impact
Liquid propane plunge
Freeze fracture with Balzers 400T
Cryosubstitution
Cryoultramicrotomy – Ultrathin frozen sections (primarily for antibody labeling)

20. Cryo Examples Freeze fracture, deep-etch, rotary shadow
Cryosection/im-munogold label
Cryosubstitution

21. 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

22. Immunolocalization LM
Fluor/confocal
TEM
SEM with backscatter detector

23. 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

24. Direct Labeling Method
Labeled primary antibody reacts directly with the antigen in the histological or cytological preparation

25. Two-step Indirect Method
Fluorescent-conjugated secondary antibody attaches to primary antibody that is bound to antigen

26. Amplified Method
If the antibody reporter signal is weak, the signal can be amplified by several methods, e.g., streptavidin-biotin complex

27. 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

28. 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

29. 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

30. 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

31. 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

32. 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

33. 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

34. 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

35. Know Your Artifacts And use them to your advantage!
Green is label; orange-red is autofluorescence
Acts as counterstain

36. Autofluorescence
Need to select label that will be readily distinguished from autofluorescence
Several techniques to quench autofluorescence

37. 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

38. Light and Electron Microscopes
Lenses are used to control a beam of illumination, magnify, and direct an image to a detector

39. Light Microscopes

40. 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

41. 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

42. 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.

43. 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

44. 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

45. 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)

46. Elements of Fluorescence Microscope
Light source
Mercury vapor
Xenon
Laser
Optical lenses
Optical filters
Detection system
Eye
Film camera
Digital camera
Photomultiplier tube (PMT)

47. 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.

48. Fluorescence
Fluorochromes are excited by specific wavelengths of light and emit specific wavelengths of a lower energy (longer wavelength)

49. 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

50. 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

51. Choose Fluorochrome/Filter Combos

52. Spectral Characteristics of Probes
Omega Filters Curv-o-Matic
Other filter and microscope companies

53. 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

54. 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

55. 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

56. 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

57. Compressed Z-stack Image
3-D reconstruction
Tilt and rotate
Stereo projection
Animation
Montage
Image enhancement
Photo courtesy Dr. Alex Stokes, Queens Medical Center

58. Confocal Movies
Photo courtesy Dr. Alex Stokes, Queen’s Medical Center

59. 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)

60. Differential Interference Contrast (Nomarski)

61. 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

62. 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

63. 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

64. 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

65. 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

66. 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

67. Colloidal Gold in TEM

68. Colloidal Gold in TEM

69. 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

70. TEM Grids TEM grids are 3 mm supports of various meshes
You will handle them by the edges with fine forceps

71. 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

72. Preparation of Images for Publication
Microscopy – Images are your data!
Adjustment and labeling of images for figure plates with Adobe Photoshop

73. How to Contact the BEMF
Location: Snyder Hall 118 – University of Hawai’i at Manoa
Phone:
FAX:
URL:

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

75. 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