Advances in Bioscience Education Summer Workshop Fluorescence and Electron Microscopy June 26 - 29,...
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Transcript of Advances in Bioscience Education Summer Workshop Fluorescence and Electron Microscopy June 26 - 29,...
Advances in Bioscience Education Summer Workshop
Fluorescence and Electron Microscopy
June 26 - 29, 2007
Biological Electron Microscope FacilityPacific Biosciences Research Center
University of Hawai’i at Manoa
What is a Microscope?
A tool that magnifies and improves resolution of the components of a structure
Has three components:
sources of illumination,
a magnifying system,
detectors.
Sources of Illumination
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
Images and pictures are your data!
Epifluorescence Microscopy
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
Better resolution
Serial optical sections can be collected from thick specimens
Live or fixed cell and tissue imaging
Laser Scanning Confocal Microscopy
Photos courtesy of Gregg Meada & Dr. Gert DeCouet, UHM
And Dr. Chris Yuen and Dr. David Christopher
Drosophila eye
Plant Protoplast
Epifluorescence vs. Confocal
Sample courtesy Gregg Meada & Dr. Gert DeCouet, UHM
Scanning Electron Microscopy (SEM)
View outer surface Coat specimen with
gold No sectioning High Mag (40x to
300,000x) High resolution (better
than 2 nm)
SEM Images
Transmission Electron Microscopy(TEM)
View inside cell via sectionsView inside cell via sectionsmagnification 120,000 Xmagnification 120,000 X
50,000X50,000X
Conventional TEM Micrographs
Skin
Bacteria in cell
Apoptosis
ChloroplastCollagen Virus in cell
Ultra-microtomy Ultrathin (60-90 nm)
sectioning of resin-embedded specimens
Several brands/models available
Cryotechniques
Ultrarapid cryofixation Metal mirror impact Liquid propane plunge
Freeze fracture with Balzers 400T
Cryosubstitution Cryoultramicrotomy –
Ultrathin frozen sections (primarily for antibody labeling)
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
Two-step Indirect Method for Immunolabeling
Fluorescent-conjugated secondary antibody attaches to primary antibody that is bound to antigen
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
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
Double-labeling Method Use primary antibodies
derived from different animals (e.g., one mouse antibody and one rabbit antibody)
Then use two different secondary antibodies conjugated with different sized gold particles
Preparation of Biological Specimens for Immunolabeling
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 OR Cryofixation
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
Embedding
Dehydrated tissue is embedded in a plastic resin to make it easier to cut thin sections
Steps in Labeling of Sections
Chemical fixation Dehydration, infiltration, embedding and
sectioning 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
Light Microscopes
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
Light beam excites the fluorochrome, raising it to a higher energy state,
As it falls back to it’s original state, it releases energy in the form of a light of lower E and longer wavelength than original beam of light
Primary Ab = PDIsecondary Ab = AlexafluorBlue light = exciting beamgreen and red light emitted
Know Your ArtifactsAutofluorescence And use them to your advantage!And use them to your advantage!
Green is label; orange-red is Green is label; orange-red is autofluorescenceautofluorescence
Acts as counterstainActs as counterstain
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
Choose Fluorochrome/Filter Combos
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
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
High pressure freezing:Plant tissue is flash frozen in a pressure bomb -197 C
Water in the tissue is replaced with acetone over 5 day period
Acetone saturated tissue is embedded in resin
Resin is cut in thin sections, 80 nm thick
Add antibodies - immunolabeling
Look under Electron microscope
Very Wrinkled
Chloroplast Carnage
Pretty badfixation
2nd time: stainings were done poorly, but there is hope…
Back to the drawing board to start over.But what to correct?What to do different?Will it improve?
Despite mistakes, keep moving forwardDespite mistakes, keep moving forwardand ignore doubt and negativism that comes with pressure.and ignore doubt and negativism that comes with pressure.
3rd time A charm
Excellent preservation AndImmunolabelingthe 3rd TIME
HIGHMAG
RE-search Not search
Research time is spent: 70% trouble-shooting 15% success 15% communicating success.
Must be repeated
ROOT
HOOK-o-PLASM
PDI inVacuole
200 nm
g
CNGC in Golgi Apparatus
c
200 nm
G
PDI in Golgi Apparatus
Dividing mitochondria
Channel located to the plasma membrane
Channel located to the plasma membrane -plasmolysis
We learn more from mistakes than successes…