Week 2

Lectures 1 + 2

EM > LM > naked eye

Light Microscopy
 types
o conventional
o fluorescence - high
powered lamps with
limited wavelengths
o confocal
o two photon

Structure
 diaphragm - regulate how much light to come through
 condenser - focus light on specimen
 objective - focus specimen on your eye
o lens you look through. change focal distance to make it clearer
to your eye
 focal knob moves either the objective or the plate where the specimen
sits on
 inverted microscope - for isolated cells
o objective and condenser switched positions
 upstanding microscope - for tissue and fixed cells

Types
1. Bright Field
a. transmitted light
b. light goes through specimen

2. Phase Contrast
a. add a phase plate in the light path causing interference
b. converts phase differences into changes in brightness
c. light wave function changes as it passes through a medium
d. causes a shift in troughs and peaks - phase shift in unstained cells
as light passes through

3. Differential Interference Contrast (DIC)

a. almost 3D imaging
b. creates shadows
c. phase shift for different contrasts
d. use prisms to separate light into different rays then bring them back together
 Week 2
Lectures 1 + 2

4. Dark Field
a. turning angle of light shows only scattered light
b. make light hit specimen from different side, not bottom
c. see changes in contrast
d. can see organelles

Fluorescence Microscopy
 photon changes wavelengths
o e.g. GFP is blue then emits green light
o stimulate a specimen with one color so that it emits another color
o shoot it with strong blue, it absorbs photon, emits weak green
o absorb at short, emit at high wavelength
 shoot light that bounces back to lens
 high intensity light source needed as high magnification needs more light
 filters cut the unwanted light wavelengths
 mirrors reflect certain wavelengths and let some pass through
 Week 2
Lectures 1 + 2

Tissue Preparation
 microtone slices specimen into thin slices
 fixation - expose cells to chemicals to preserve and stabilize
o alcohols, aldehydes, acids
 embedding - after fixation. embed soft tissue in some polymer so that you can cut it
o substitute to water in it for alcohol or some other solvent
o plastic or polyethylene glycol
 stain tissues – expose to dyes
o hematoxylin, eosin, antibodies

Immunofluorescence
 use antibodies to target/label parts
o inject antigen into host
o host makes antibodies against it
o extract and purify
1. fixed tissue is treated with primary antibody
2. antibody binds to antigen on/in cell
3. secondary antibody with fluorescent marker binds to primary
 indirect immunohistochemistry as you identify a secondary antibody that is attached to
the one that you care about
o helps amplify the signal as many of the secondary attach to 1 primary

Hi-Res Microscopy (LM) - non conventional

Confocal Microscopy
 advantage
o can use whole tissues without dissection
o results have little noise, high signal
 disadvantage
o about 10x conventional microscope price. starts at 500k
 Week 2
Lectures 1 + 2

 uses pinholes to remove stray beams of light helping increase resolution

o only light focused on the pinhole enters the detector
 same focal distance between laser and mirror with mirror and detector
 very precise beam of light
o can control level where light hits so you can scan different depth sections creating
a 3D render of specimen
o raster scan
 shoot short wavelength light that bounces off mirror to specimen
o specimen emits long wavelength light after excitation that goes through mirror
 a lot better than conventional - images are sharper
 Week 2
Lectures 1 + 2

Two Photon Microscopy (nonlinear)

 linear v nonlinear microscopy
o linear - photons in = photons out
o nonlinear/two photon - photons in exceeds photons out
 moving from ground to excited state needs two+ photons
 weaker energies of light used so more needed to raise molecule to excited
state
 2 photons hit simultaneously - 1 longer wavelength photon comes out
o depends on density of photons arriving onto the tissue simultaneously
 goes deeper into tissue - up to 1mm
o use light the near infrared region - very high frequency
 multiphoton microscopy has a very fine point causing only a specific tissue to receive
photons
o cuts out a lot of background noise
o only a fine point where the molecule gets excited
 lasts about 100fs
 no pinholes needed

Electron Microscopy
 using electrons instead of photons as source
 light photons can start getting disoriented and limitation with equipment
 can get 2000x light microscopy
 very time consuming

Transmission EM
 good for tissue sections - intracellular organelles, etc
 electron gun shoots electrons
 condenser lens focuses beam
 done in a vacuum\
o high voltage 105V
 2 magnetic coils act as
o condenser lens - focus electrons on specimen
o objective lens - focus electrons from specimen
onto film
 Week 2
Lectures 1 + 2

 sample has to be fixed and stained so electrons interact properly

o glutaraldehyde to fix it
o osmium tetroxide used to make electron dense areas
o dehydrate it with alcohol/solvent and fix with a liquid plastic resin gives extra
support
o cut ultrathin (under 50nm) sections
o place on copper grid
 Micrograph - shadows form where electrons don't make it through are the image
o electron dense - gray output
 parts would absorb electron casting a shadow onto the film
o black - electrons didnt make it through
o electron loose - light gray
 not many parts to absorb so more electrons move through
o since you slice something you can rebuild it by stacking the images onto each
other like a sliced sausage

Immunogold EM
 use gold to bounce electrons back
 creates very dark spots

Scanning EM
 good for surface structures
o 3D like images
 no electron passing through
o electrons bounce off in all directions
o detects all around to pick up info
 coat specimen with gold then scan
 Week 2
Lectures 1 + 2

 SEM
o superficial structural image
 DIC
o blurrier
 TEM
o only 1 slice
o does show the areas where its connected to the ground
o not making contact

Ion Imaging
 color changes based on Ca concentration
 focus on changing calcium levels
 ion-selective indicators emit light depending on local ion concentrations.
 changing in electron voltage calcium enters the cell
 dye emits light at very specific wavelengths displaying as waves
o as more calcium builds in cell it gives off more light at wavelengths
 dye fluoresces when in contact with calcium
 sperm changes membrane potential by flooding with calcium, blocking other sperm from
entry

X-Ray Crystallography
 best way of getting protein structure
o structure - function important
 good at going through tissue but not bone
o x ray doesn't interact with a lot of matter
 disadvantage
o needs a lot of sample and crystallized
1. crystal bombarded with x-rays
2. get structure of sample from interference
 synchrotron - produce x rays as beams
o bombard crystallize protein
o crystal lattice structure causes small gaps for x ray to pass through
o where waves interact - interference happens displaying patterns
 Week 2
Lectures 1 + 2

 many copies of sample in crystal

o find common pattern to find the structure of protein
 increasing magnifications
o protein folding
o groups of atoms
o individual atoms
o electron density
 x-ray bombardment to get images of protein structures through shadows
 one of the main reasons we know the conformation of DNA
 MacKinnon used antibodies to stabilize ion channels to get the first ever structure of K
channels