Light Microscopy

what is unique about light microscopy?

the can follow cells while they are still alive but you can’t view structures inside the cell because they are smaller then the level of detection of light microscopy

how did the microscope Robert Hooke used work?

mirror shine slight through sample

Which microscopy identifies more detail of structures?

electron microscopy can identify smaller structures and the detail of the cell

What are the types of light microscopy?

• transmitted light

• fluorescence

• Confocal

• super resolution

since cells are typically transparent, what can you do to make them visible?

• stain them with dyes like hematoxylin followed by eosin

• special optics like phase contrast, DIC, or dark field

What must you do if you are looking at cells in tissues?

fixed, embed in paraffin or plastic then section

• if tissue culture cells are live they are harder to work with

What is organ culture?

• taking out a whole organ from an animal

• difficult to do

• no blood flow → put into growth media

• keep the physiological conditions from a living organism

What is an explant culture?

• take organ and chop it into small enough pieces so that then you put it in the growth media it gets everywhere in the organ so blood and oxygen can more properly be supplied

• its easier to maintain in culture and still represents a 3D organization

What are continuous cell lines?

• you chop up the organ, then add trypsin which will digest the proteins sticking the cells together → broken apart them put on surface to then analyze

  • primary culture is taken directly from the tissue and has a limited lifespan (divide and undergo a crisis a some point causing most of them to die due to telomeres becoming shorter in regular cells → in germ cells telomeres keeps extending the telomeres)

    • only good for a certain number of divisions

  • continuous cell line divides forever → possible to freeze them

  • transform cell lines → undergone genetic and phenotypic changes that make them behave like cancer cells, allowing them to grow indefinitely and ignore normal growth controls

    • they lack contact inhibition

what is contact inhibition?

normal regulatory mechanism in which cells stop dividing when they come into contact with neighbouring cells

What are the kinds of tissue culture cells?

• primary cells - same cells obtained from the source

• non-immortalized cell lines - primary cells that reproduce and can be grown for many generations but stop at some point

• immortalized cell lines - mutations allowing them to grow indefinitely but have contact inhibition

• transformed cell lines - lose contact inhibition and have other abnormalities → cancer cells

How do you maintain a cell culture?

Artificial medium

• pH

• nutrients

• glucose

• serum (including growth factors)

• antibiotics (optional)

Temperature

• 37 Degrees Celsius, humidified environment

Sterile environment

What is fluorescence microscopy?

• fluorescent molecules can absorb light of higher energy (short wavelength) and emit light at lower energy (longer wavelength

• tissues and cells are irradiated with a blue-violet or even ultraviolet light so that the emission is in the visible part of the spectrum

• sensitivity is very high

• what is short or long depends on the dye used

Describe an Epi-fluorescence microscope (the three parts)

1. first barrier filter that lets through only blue light (wavelength between 450 and 490nm)

2. beam splitting mirror that reflects light below 510nm but transmits light above 510nm

3. second barrier filter which cuts out unwanted fluorescence signals passing the specific green fluorescein emission between 520 and 560nm

how to label proteins?

1. chemically label protein outside the cell and add it to the cell

2. label antibody against the proteins distain the cell

3. fuse protein of interest with GFP and express

What is direct immunofluorescence?

• used to detect a specific antigen in a sample using a single antibody that is directly labeled with a fluorescent dye

1. primary antibody that is already attached to a fluorescent tag is added to the sample

2. antibody binds to the target antigen (protein)

3. view under microscope → antigen appears glowing where antibody is bound

What is indirect immunofluorescence?

• used to detect and visualize a specific antigen/protein in a cell or tissue sample using fluorescent antibodies

1. primary antibody binds the antigen

2. secondary antibody binds to the primary antibody

3. detect fluorescence

example: if you want to detect tubulin in a cell the primary antibody used is anti-tubulin in a rabbit (not fluorescent) → secondary antibody used is fluorescent anti-rabbit IgG → microtubules light up under the microscope

What happens if the protein of intents is inside the cell?

the antibody will not be able to cross the plasma membrane

What happens when you add formaldehyde?

it cross links proteins at the lysine residue fixing the proteins in place → that kills the cell but the membrane is still intact so the protein can’t enter the cell

What are the steps to get the antibody in the cell?

1. use formaldehyde to kill the cell and stabilizes the cell

2. permeabilize the cell with detergent to put holes in the plasma membrane (the proteins won’t leak out but the antibodies can get in)

3. use direct or indirect immunofluorescence (you can label multiple proteins at once)

*secondary antibodies usually come from cows while the primary antibodies usually come from smaller animals like mice or rabbits

What are the advantages and disadvantages of direct immunotherapy fluorescence?

advantages:

• fast and simple

• lower background noise (less change of non-specific binding b/c no secondary antibody)

• more precise localization (signal comes directly from antigen-antibody interaction)

disadvantages:

• lower sensitivity

• more expensive

What are the advantages and disadvantages of indirect immunotherapy fluorescence?

advantages:

• higher sensitivity

• cost-effective

• flexible (easy to switch targets without relabeling antibodies)

disadvantages:

• more time consuming

• more probability for noise (secondary antibody may bind non-specifically)

What are the variations of immunofluorescence?

• fix/permeabilize with methanol

• skip permeabilization step (stain surface only) → if interested in only the surface

• use a small amount of gluteraldehyde as a fix → the only disadvantage is that it is fluorescent

• section the tissue prior to staining (unlike cells in tissue culture)

why can methanol be used as a fix and permeablize?

it can dissolve membranes and denature proteins

What is DAPI dye?

used to stain double stranded DNA

What is immunogold?

• electron microscopy technique used to detect and localize specific proteins using antibodies that are tagged with tiny gold particles instead of fluorescent dyes

• same idea as immunofluorescence

What are antibodies?

immunoglobulins produced by B-lymphocytese and plasma cells of vertebrates

• most antibodies used are of the IgG class

• they have two light and two heavy chains

what are some characteristics of antibodies?

• parts of the antigen that combine with the antigen binding sites are epitopes

• antibodies are often generated against a part of the peptide sequence of a protein (not the whole protein)

• possible to generate antibodies against small molecules like amino acids and monoamines if they are conjugated to a carrier protein

How are antibodies produced?

1. animals are injected with an antigen (the antigen can be conjugated into carrier protein or injected alone

2. the injection is given multiple times at specific intervals to build a strong immune response

3. animal immune system makes antibodies specific to that antigen

4. blood is taken from the animal, allowed to clot, them separated into serum and which contains the antibodies

5. the antibodies are collected and tested using immunochemistry

6. new antibody must be characterized to make sure it recognizes only the antigen it needs to

What is polyclonal vs monoclonal antibodies?

polyclonal:

• mixture of antibodies produced by many different B cells in an animal → they recognize multiple epitopes on the same antigen

monoclonal:

• identical antibodies produced from a single B cell clone → recognize one specific epitope on an antigen and are the product of a hybrid myeloma cell line (hybridoma)

• antibodies come only from rats and mice

how are hybridomas formed?

they are the result of the fusion of myeloma cell line with lymphocytes from an animal immunized with eh antigen

• fusing B cell with myeloma cell allows it to grow indefinitely

How are monoclonal antibodies prepared?

1. rats and mice are immunized with antigen multiple times

2. B cells are produced and make antibodies against the antigen

3. Animals are bled and sera tested by immunochemistry

4. B cells are fused with myeloma cells forming hybridoma cells

5. cells that don’t fuse are eliminated by allowing the fused cells to grow in HAT medium that kills the original myeloma cells (only the cells that have been fused survive) → unfused B cells will not survive on their own

6. hybrodiomas are tested (using ELISA) to find the ones that produced the desired antibody

What are the advantages of monoclonals?

• once generated, they can be obtained in high amounts at a low cost

• hybridomas are immortal cell lines

• monoclonals produce a highly specific staining

what is microinjection?

technique in which tiny, precise volumes of material are injected directly into a single cell

things that can be injected:

• DNA/plasmids

• RNA/mRNA

• proteins or enzymes

• drugs or dyes

• viruses

Where is it injected:

• cytoplasm

• nucleus

• embryo

Where was the green fluorescent protein (GFP) found?

in jellyfish

How do we get GFP into a cell? FIX THIS SLIDE!!!

1. get the plasma DNA into mammalian cell using liposomes (use transfection)

2. after 24hrs the GFP protein fusion will be expressed

3. cells can be visualized on the microscope

how are GFP tagged proteins constructed?

• involves creating a new artificial gene in which the DNA coding sequence for GFP is fused to the protein

• gene is usually created on a bacterial plasmid and must be introduced into the cell

• GFP easier to use in tissue culture than in animals

What are the advantages of GFP?

• can be used in living and fixed cells

• if DNA is introduced into cell, GFP tagged protein is produced by cell and is already fluorescent

• good for live imaging

• can be used with other colours of fluorescent proteins or in combination with immunofluorescence

what are the disadvantages of GFP?

• sometimes GFP fusion protein does not fold properly

• protein may be present in cell in higher than physiological amounts

• endogenous protein in cell is not visualized

• expression of GFP proteins in whole animals is more difficult in tissue than culture cells

What is digital wide field microscopy?

a type of light microscopy where a camera (digital detector typically CCD or CMOS) captures the entire field of view and once → image is then processed digitally to improve clarity, contrast or extract measurements

• the camera is typically attached to fluorescence microscope

• fluorescence in images can be accurately quantified

• digital images are made up of pixels

• camera can detect out-of-focus and in-focus light

Describe greyscale and colour images

• greyscale images or images with one colour have one number associated with each pixel

• microscope camera used for fluorescence image are greyscale only, however multiple images can be taken of the same field using different emission filters → images can then be viewed side-by-die or easily combined to produce colour images

Why are out of focus structures a problem for thick samples?

in thick samples the light from planes above and below the focal plane reach the camera → out-of focus light overlaps with the in focus image making it harder to distinguish real structures

What is confocal microscopy?

technique that produces sharp, high-resolution images of thick samples by eliminating out-of-focus light. It allows you to see thin optical slices of a specimen and even build 3D images

• traditional microscopes can only produce high quality pictures at high magnification only when the specimen is this → this technique uses lasers, fluorescence optics and computers to avoid this limitation

How does confocal microscopy work?

1. you have a focused laser that scans the specimen point by point and only a small spot of the sample is illuminated at a time

2. light emitted from the focal plane passes through a pinhole to the detector

• light coming from out-of-focus plane is blocked by the pinhole which removes blur and improves clarity

3. computer assembles all scanned points into a sharp digital image → you can construct a 3D image of the sample

What are the biological uses of confocal microscopes?

• 3-D or single slices from fixed samples including thick samples

• 3-D or single slices from living cells

• time lapse in living cells

• photo bleach techniques

• quantitative information on any of the above

What is photobleaching?

• the permanent loss of fluorescence when a fluorescent molecule (fluorophore) is exposed to light for too long or too high intensity

1. fluorophore absorbed light and gets excited (when excited it can react with molecular oxygen irreversibly destroying the fluorophore)

2. instead of safely releasing the light, some molecules undergo a chemical change

3. chemical change permanently destroys their ability to be fluorescent

• this matters for microscopy because the image fades over time and data can be lost

• stays excited for 1-10 nanoseconds before re-emitting

What is FRAP?

• fluorescence recovery after photobleaching

• used to study the movement and mobility of molecules inside living cels

• cell labeled with fluorescence → strong laser is used to photo bleach a small region of the cell (fluorescence disappears) → overtime unbleached fluorescent molecules move into the bleached area → return of fluorescence is recorded and graphed

• first used to detect diffusion of molecules in the plasma membrane

• you can determine rate of diffusion

• can determine whether a protein is mobile or stays in place

• can also be used to see the movement of proteins between organelles

What is the biggest thing that can be measured by FRAP?

diffusion of molecules

• diffusion does not require energy however proteins moving in membrane-bound vesicles requires energy

• diffusion rate can be quantified as a diffusion coefficient

What is the minimum resolution of a light microscope?

0.25 microns

What is the smaller things we can see in a light microscope?

single atoms as long as it produces enough fluorescent signal to see

What is the mini of resolution?

the smallest distance between two points that can still be distinguished

What is near field microscopy?

• a microscopy technique that allows imaging at a resolution much smaller than the normal light limit

• regular light microscopes cannot resolve objects smaller than 200nm because of the refraction limit of light

• near field microscopy overcomes this by using light in the near field → light that exists only a few nanometers from the surface and has not yet diffracted

How does near field microscopy work?

• a very tiny probe scans across the surface

• probe kept close to the sample

• light interacts with the sample in this region

• detector collects the signal to form high resolution image

since the probe is so close, the resolution depends on the probe size rather than the wavelength of light (tip in their sample was 30nm)

• image acquisition is slow

What is PALM/STORM?

• also allows you to see objects smaller than 200nm

• They work by precisely locating single fluorescent molecules and building a high-resolution image over time

How does PALM/STORM work? GENERAL

1. The sample is labeled with special fluorescent molecules that can be turned on and off

PALM: have photoactive GFP which is not fluorescent until you expose it to a small amount of ultraviolet light

STORM: same sample placed in a special buffer that drives most of the fluorescent dye molecules into a dark state

2. Only a small random subset of fluorophores is activated at one time (so their light spots don’t overlap)

3. Each molecule appears as a tiny blurry dot, but its center position can be calculated very precisely (nanometer accuracy)

4. Those molecules are turned off (or bleached), and a new subset is activated

5. Thousands of images are collected and combined to reconstruct a super-resolution image

• improves location determination accuracy of the molecules

• in PALM you only see each area once

how are PALM and STORM different?

PALM woks with special photoactivatable GFP’s

a small portion of paGFP can be turned on by ultraviolet light, images and bleached

What are the limitations of PALM and STORM?

• not compatible with confocal microscopy because confocal isn’t good with dim images

• best with thin samples (TIRF can help but limits observation to the bottom of samples)

• often used on sections prepared for electron microscopy (san sub for immunogold if you are willing to image same section on two microscopes

• very difficult with living cells