MODULE 1: PART 1 – CONVENTIONAL TECHNIQUES IN MICROSCOPY

Slide 2: Several Types of Microscopy

 
 Light Microscopy:
o Involves light as source of energy
o Looking at things no smaller then 0.2 microns in diameter
o begin to see organelles, but not looking at vesicles or mitochondrial cristae
o parts of cells, tissues, groups of cells
 Fluorescence Microscopy:
o Casting monochromatic defined light onto specimen

Slide 3: Light Microscope

 Light Microscope:
o Incandescent bulb that creates light and casts image behind retina
o Start with light source and end up with the eye, light goes through diaphragm
o Eyes through optical lens
o Light comes through diaphragm (Sets diameter), allows for large or small amount
of light, then casts light on condenser (focuses light on specimen)
o Objective lens focuses image on back of eye
o Eyepiece: adds magnification
o Lens in eye used to focus on back of retina
o This microscope is used to look at live or fixed cells (expose tissues and cells to
certain chemicals to preserve, not going to die)
o Inverted microscopes: objective lenses are underneath and condenser is on top of
stage, this can manipulate petri dish of cells

Slide 4: Four types of LM

 Bright Field:
o When you open up condenser and blow right through it, you might find a bright
field image
o Using this commonly in the 50s
 Phase contrast:
o Convert phase differences into contrast; take differences in brightness and they
become differences in contrast - gives you detail

Slide 5: Four types of LM 

o If we have an unstained cell, light passing through will be modified, wave properties are
changed
 Before incident light hitting the cell compared to after, there is a phase shift in the wave
pattern
  Properties of light after they've interacted with cells is often exploited
Slide 6: Four types of LM 
 Differential:
o Almost 3-D
o See texture, lots of detail
 Dark Field:
o If we move the light source with reflectors and modify the light path, this creates
diff images by causing light to hit the specimen from diff angles
o Way of seeing details before phase contrast
o Utilizes scattered light

Slide 7: Fluorescence Microscopy

 Hitting a specimen and bouncing off then returning to the lens, not carried through the
specimen
 Uses light images to excite molecules that can emit light
 Specific wavelengths of light used
 Use filters to limit incident light to a specific wavelength
 Colour of light you use to stimulate specimen is not the same as the colour you will
observe through the microscope

Slide 8: Fluorescence Microscopy

 Commercially available dyes that we inject into the cell
 GFP (green fluorescence protein) when excited in microscope emits at the green
spectrum, have to use blue light to excite it so it can be absorbed

Slide 9: GFP in research

 
 create transgenic animals that express GFP in their genome
 Use GFP to track how cells divide, movement...

Slide 10: Tissue Prep

 Fix tissues and then cut very thin slices
 Embed in plastic material and produces block that helps retain tissues
 Microtome: cuts thin slices of the tissues and lays it on slide
 10 microns of tissues, almost a cell diameter thickness
 Staining using dyes to help get certain colours such as pink or purple

Slide 11: Immunofluorescence

 
 Immuno=antibodies
 Immunohistochemistry = use of antibodies in tissue
  Immunocytochemistry = use of antibodies in the cells
 Can exploit immune system of animals to exploit antibodies (mice, chicken, goats),
collect these antibodies to use in research - can help locate proteins
 Use of fixed tissues and permealize it with chemicals to get Hb across the membrane
 Treat them with primary Hb (directed against specific antigen, binds to antigen A which
binds to the secondary Hb (binds to primary Hb)
 Secondary Hb has fluorescence probes forming a cluster around every antigen A - can
identify proteins and subcellular structures
 Use Flu microscopy to identify structures and brightness (indirect histochemistry)

MODULE 1: PART 2 – HIGH RESOLUTION MICROSCOPY

Slide 2: Confocal Microscope

 Allows us to reduce background signal (noise), want to have very high signal to noise
ratio
 Want to find some way of imaging discrete aspect of your specimen (signal), everything
else is noise
 Useful for thick sections; cutting into sections and using for experiment (lots of work)
o Confocal saved time by using whole tissue instead of cutting sections
 Whole-mount: remove pieces of tissue through dissection w/o cutting small sections
 Very costly!!!

Slide 3: Confocal Microscope

 In image to the right: no incandescent bulb, laser light is bounced off mirror onto
specimen and returned to a detector; not transmitting light through specimen
 This microscopy, when using GFP or marker, taking laser light and using it to excite a
florescent molecule and during emission of photon, molecule relaxes during ground state
 Path of light coming from laser, bouncing off mirror and going all the way to the
specimen is the same from the specimen all the way to the detector
 Important: way in which we focus light onto the specimen is through the pinholes
(specific), bounces off specimen and focuses on detector
 Monochromatic polarized light
 Optical section: way of sectioning tissue by creating an optical slice (thinner than
micron), minimizes noise
 Have a Z formation (3-D), using motor can move specimen up and down and focus light
inside or outside
 Confocal microscope will take a raster scan and take images in organized pattern at diff Z
positions
 Ignores any noise in background and gets to point of focus
Slide 4: Confocal Microscope
 Green light reflected back from the specimen onto the detector (longer end of the
spectrum)
 Need blue light to produce stimulus
 Image shows on the left: when the center of tissue is focused on detector and the signal is
cast on detector, goes through mirror; blue light reflected off mirror and green light goes
through; any signal from center is focused on detector (no noise), anything above or
below doesn't get to the detector
 Let's us get clear image, let's us use whole tissue

Slide 5: Comparison of Techniques

 Confocal is much clearer, lots of detail

Slide 6: Two Photon Microscopy

 Non-linear devices (more than one photon in and limited amount getting out; linear is one
photon in and one out)
 Uses higher order light matter interactions; more than one photon being put out
 Used to help get very deep into tissue (millimetres); uses light in the near infrared part of
spectrum - penetrates deep into tissue
 Slide 5: Two Photon Microscopy

Slide 7: Two Photon Microscopy

 Need two photons to get into a fully excited state
 Emissions and excitation then occurs at longer wavelength
 Allows us to ensure we're getting discreet parts of tissue and reducing noise; signal to
noise ratio is higher
 Two or more photons arriving at where you're focusing the laser, lots of selectivity
because we're overwhelming the specimen with many photons (discrete point providing
signal)

Slide 8: Two Photon Microscopy

 Neocortex of mouse in diagram A
 Neurons injected with GFP and pipet used to record electrical stimuli
 In diagram B, actual neurons recorded
 Anesthetized animal where neurons are recorded

MODULE 1: PART 3 – ELECTRON MICROSCOPY

 

Slide 2:
  More complicated technique, longer to do
 If you want to look at subcellular parts or how diff cell types might interact, you can't see
synaptic density unless you use this microscopy
 TEM and SEM:
o Both use bombardment of electrons rather than light to produce image
o If you have two points separated by 0.1 nanometres, you can resolve them using
this technique
o Very time consuming but only approach used for some experiments

Slide 3:
 Light source is at top and and eye is at bottom for a transmission EM
 Specimen is at the bottom, electron gun is at the top (equivalent to the light source, but
emits electrons)
 Electrons bombard the specimen - acts as a vacuum so no other substances interact with it
 Electrons only interact with specimen and cast an image onto the film on the bottom
 Coils acts as a condenser lens (focuses light on specimen), focuses beam of electrons on
specimen and focuses image onto the film on the bottom
 Sample has to be fixed and stained; electrons pass easily through tissue, so we need to fix
them and stain them
 Have a film that is producing image based on shadow (which is produced by electrons)
 TEM is important for looking at sections of tissue

Slide 4:
 Use glutaraldehyde (specific to electron microscopy)
 Treat tissues and cells with glutaraldehyde and fixes them, prevents from decaying
 Glutaraldehyde at the subcellular level does better in fixing membranes and organelles
 Osmium tet is used to increase electron density
 Tissue then has to be dehydrated; water is removed from specimen and replaced with
liquid plastic resin
 Cut ultrathin sections that are handled using a a fiber to flip them on solvents
 Put on the copper grids (very small)

Slide 5:
 Usually black and white and colour is added afterwards (artificial)
 Dark grey indicates electron dense area and light grey indicated electron lucent area
 No staining = parts where electrons made it through to the detector and produced light on
film
 Black/stained area = electrons bounced off specimen and didn't make it through to the
film
 Can do serial section microscopy: take bunch of sections, put together and produce 3-D
images

Slide 6:
 Electron gun bombarding a sample with electrons, but electrons are just bouncing off
tissue
  Instead there's a specimen on bottom and all the electrons are being focused on it and
bouncing all over
 Detector that is receiving the electrons bouncing off and producing an image
 Produced a 3-D rendering
 SEM is about the surface of a specimen
 After fixation, coat tissues/cells with gold particles and put it on stage
 Scattering of electrons off the specimen that produces the image (doesn't pass through)

Slide 7:
 Not looking through the structure or running a section through the head
 Looking at surface features
 Use it to look at bacteriophage samples

MODULE 1: PART 4 – X-RAY CRYSTALLOGRAPHY

Slide 2:

 Best way to look at and solve the structure of proteins

 Need to understand the structure-function relationship; want to understand how proteins
interact with each other to produce mechanisms
 Proteins are macromolecules; x-ray crys. can help us get a finer resolution (0.1nm)
 Big challenge is crystallizing proteins; in order to do these experiments and solve
structures, you need enough pure protein
 First x-ray structure to be solved was for hemoglobin
 Hemoglobin: done in the absence of oxygen so that it would crystallize
 Once protein is crystallized, specimen is bombarded with x-rays
 Interpret structure based on interference patterns
 See diffraction patterns
 casting light through two adjacent holes in barrier will produce interference patterns on
the other side
 Banding light: where ripple patterns interfere produce more dense pattern on screen

Slide 3:
 X trays (short wavelength)
 Doesn't make it through everything
 Protein crystal is not one copy of protein but multiple copies in diff orientations and these
crystallize into solid structure; you get a common pattern that is the structure of the
protein and is isolated

Slide 5:
 Derived space filling model for DNA
  Film (x-ray) that reveals structure of DNA

Slide 6:
 Solved the first structure of an ion channel in a plasma membrane; provided
detailed structure at atomic level for pores in membrane that allowed for passage
of ions
 Green structures (Antibodies) included in the structure
 His innovation used antibodies to hold and position proteins in place as they
crystallized (hard to crystallize)