Advances in Bioscience Education Summer Workshop

1
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

2
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

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

• 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

Drosophila eye
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
Bacteria in cell
Apoptosis
Skin
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 1100
  and 12000

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 its 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
• http//www.omegafilters.com/front/curvomatic/spect
  ra.php
• 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 Stokes 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, Queens 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 gt1 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 Hawaii
  at Manoa
• Phone [phone]
• FAX [phone]
• URL http//www.pbrc.hawaii.edu/bemf
• E-mail dunlap_at_pbrc.hawaii.edu
• tina_at_pbrc.hawaii.edu

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
• http//mm2005.microscopy.org