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…