Unit 1 Topic 1: Microscopy techniques

Magnification vs resolution

• Can increase resolution by decreasing wavelength or increasing Numerical aperture (Nsina (a))

• Smallest wavelength of visible light is 450 nm

• Best media for visualization is immersion oil (N = 1.5)

• Maximum half-angle (of human eye*) is sin(70˚) = 0.94

• D= (0.61)(450)/(1.5)(0.94)=194 nm - 0.2 microns

• lower D= greater resolution

Light microscopy advantages

• Live samples

• Cheaper

• Usually safer to use

• • More ways to optimize

• Less training involved

Electron Microscopy advantages and disadvantages

-greater resolution

-more expensive

-has to be done on dead/ preserved samples

-more dangerous

-heavy metal solutions used ; toxic

Bright Field Microscopy : phase contrast

Emphasizes changes in refraction of light within sample- light has to slow down as travel through matter.

Lighter regions are where there is less material seen

Accumulation of proteins at boundaries

Can see more in phase contrast; more detailed

Tells more of what’s happening internally

Bright field microscopy : Differential Interference contrast

Emphasizes changes in refraction of light at sample-(oil, water ,air emmersion) media boundary

Gives good information about surfaces,

Bright field microscopy : Polychromatic polarization microscopy ( MPP)

Makes use of the ability of some materials (ex collagen) to double refract light (property called birefringence)

Naturally refract light ( show rainbow like images on top of sample)

Optical ( microscope lens)

Sample ( physical and chemical ) optimizations : Fixation

cross-link macromolecules and permeabilize cells for staining; kills samples -Fixation freezes sample ; gives snapshot of whats going on

-Keeps sample stable and sturdy

-Kills sample

-Facilitates addition of stain; make sample more porous so stain can penetrate easily ▪ Formaldehyde/formalin for light microscopy (protein) -technically a gas, when mixed with water makes formalin

▪ Glutaraldehyde for electron microscopy because stronger fixative (protein) -stronger version of formaldehyde; used for electron microscopy -can be damaging to sample

-Osmium tetroxide (membrane lipids) -heavy metal ; preserves membrane lipids

▪ Ethanol/alcohol for dehydration (nucleic acids)

-safer -can cross proteins in live or dead samples

-cross links nucleic acids

-can dry out sample; does not preserve 3D structure

sample ( physical and chemical) optimizations : embedding and sectioning

Embedding and Sectioning: Necessary for thick samples; kills samples

Embedding :Suspending sample in solid matrix

Sectioning: slicing sample at various sizes

sample ( physical and chemical) optimizations : staining

• may be done on live or perserved samples

• Hematoxylin and eosin ( H&E) : acidic molecules (ex DNA) appear blue-violet, and basic molecules (ex membrane proteins) appear pink -one stains positively charged molecules ( blue/purple) DNA -other stains negatively charged molecules like proteins ( pink)

• methylene blue: acidic molecules (ex bacteria) appear blue or as bioindicator of damage; less penetrance than H&E -stains acidic ( negatively charged ) molecules : molecules with DNA -because it is non-toxic, can be applied to living organisms -bio-indicator of damage. Live cells metabolize and detoxify methylene blue

• ▪benzidine: heme-containing proteins

• congo red: amyloid deposits

Light microscopy techniques: fluorescent microscopy

Fluorescent Molecule (fluorophore or fluorochrome) absorbs light at one wavelength (excitation wv) and emits light at a specific longer wavelength (emission wv)

- Fluorochrome is the specific molecule/protein responsible for fluorescence Advantages

advantages and disadvantages of fluorescent microscopy

Advantages:

-specific; can target RNA sequences, carbohydrate complexes. Targeting based on certain sequence -quantifiable: show amount of protein present: more fluorescence= more protein

-multiple “colors” available; some are naturally occurring like GFP in jellyfish

Disadvantages:

-autofluorescence : other things in sample can fluoresce

-blurring: usually computers can refine blurring, but can take time to resolve photobleaching: overexposing sample to light causing fluorochrome to burn out

fluorescent microscopy techniques: indirect immunofluorescence

– Uses antibodies to target a protein and fluorochromes to target the antibody

- Using a secondary antibody

- More expensive

- Create antibody to target protein

- In direct – uses fluorochrome

Indirect: second antibody to first antibody that has fluorochrome amplifies signal, more antibodies= fluorochromes you can put into sample= more fluorescence

Fluorescent tags

– Genetically modify (recombinant) cells to express fluorochrome sequence at end ( C or N) of protein in living cell

-attach fluorochrome to protein that is “turned on” and expressed

-attach to Carbon or Nitrogen end of DNA sequence

-grow and culture cells to produce fluorescent tags

Fluorescence microscopy techniques: confocal laser scanning microscopy (CLSM)

– focuses on a chosen plane of a thick specimen while rejecting the light that comes from out of focus areas

-angles light to virtually cut slice into sample

-if thick specimens are used for CLM the image is blurred above and below the plane of focus

-CLSM makes it possible to focus on a chosen plane of a thick specimen while rejecting other parts

Fluorescent microscopy techniques: Fluorescence resonance energy transfer ( FRET)

Fluorescence of one protein causes another, nearby protein to fluoresce

• Requires fluorophores to be close together

• When the two proteins are close together, the energy transfer goes from one to the other = fluoresce at different wv due to exciting other fluorochrome

Fluorescent microscopy techniques: Fluorescent recovery after photobleaching

Deliberately overexpose sample to light (photobleaching) then observe restoration of fluorescence

• Can quantify rate and amount of recovery

• Wait to see equilibrium re-established; allow to see how fluorescent tagged proteins move in/out cell

• Ex: want to measure rate of transport or conversion

Fluorescent microscopy techniques: Ion sensitive Fluorescent dyes

-bind to ions(positive or negative) compared to proteins, when binds = fluoresce

Fura-2 fluoresces when it binds to calcium

-low Ca2+=blue

-med Ca2+=green

-High Ca2+=yellow/orange/red

o SPQ 6 stops fluorescing when it binds chloride

o Zinquin fluoresces when it binds to zinc

o Several that are sensitive to pH changes

Fluorescent microscopy techniques : optogenetics

Using light to control biochemical reactions ( ion channels)

• Permits temporal control

• Shining light can cause certain ion channels to open; allows for control over time of certain chemical processes

• Using light as trigger induces chemical reactions

What is the main difference between EM (electron microscopy) and LM (light microscopy) ?

·       The main difference between EM and LM is the Electromagnet used for microscopy

·       In light microscopy it is visible light

·       EM uses smaller wv of light

·       Has to do with where detector/receiver of electron is

-for TEM, e pass through sample to get to detector. For things that are large and electron

-gives good internal information

dense repel electrons- get dark regions ( not a lot of electrons)

      - SEM detector is to the side of detector, catch electron bouncing off and hit plate

- Don’t give as much resolution, but better resolution of surface

- Get good idea of what surface looks like

-SEM gives 3D image of surfaces, while TEM gives 2D

immunoelectron microscopy

·       Electron-dense markers (ex. gold) are attached to an antibody.

·       Using antibodies to mark where protein target is

o   Marks where proteins are in TEM

o   Electrons ( dark circles where electrons aren’t penetrating sample ) are where the antibodies are

o   Localizing antibodies to particular protein

cryoelectron tomography

·       Freeze sample (-196 degrees C) instead of fixing and staining

·       Stabilizes structure so not losing or distorting shape of organelles

·       Has even better resolution than standard TEM