7.1 Visualizing Cells

Question 1

What are the smallest things we can really see with a light or electron microscope?

Answer 1

Large macromolecular complexes and only their general shape.

Question 2

Material we can’t see through light or electron microscopes need to be seen how?

Answer 2

Things smaller than large macromolecular complexes has to be inferred from experimental data like the scattering of x-rays that pass through crystals of the protein

Question 3

What is resolution and how is it determined?

Answer 3

Resolution is determined by the wavelength of light and the numerical aperture of the objective lens. Resolution IMPROVES if D gets SMALLER
Resolution is directly proportional to the lens numerical aperture
Resolution is inversely proportional to the wavelength used for imaging

Question 4

What is the equation used to determine the resolution? What does each unit stand for?

Answer 4

D = .61 λ / n sin θ 
 λ = wavelength of light
  n = refractive index  
θ = half angle of lens
D= diameter of smallest  resolvable object (object you can see with an imaging system)

Question 5

The diameter (D) is going to get smaller or bigger depending on what factors?

Answer 5

Smaller if wavelength gets smaller. Bigger if wavelength gets bigger

Question 6

What’s the difference between blue/violet light versus red light? How does this affect resolution?

Answer 6

Light is measured in wavelengths
blue/violet are short about 180nm or 0/38 micrometers (waves are closer)
Red lights are long, longer than 700nm, which is twice the size of blue (waves are farther apart)
Light/blue will have better resolution than red since wavelength is on top of the equation for determining diameter of smallest resolvable object
One way to think about this is shown in the lower left hand side of this slide. If you have a small object being imaged with long wavelength light, a photon of light going by has very few opportunities to interact with the object before it’s gone, and light that has a shorter wavelength has more chances to interact with the object and this interaction is what allows us to see something

Question 7

What does it mean if D (diameter) is big or small in terms of resolution?

Answer 7

BIG: It means that the smallest object the system can resolve is also big and therefore the resolution is actually poor or bad.
SMALL: that means the resolution of the system is high or good.

Question 8

Large refractive index (n) will produce what values of D? If you have a lens with air n=1, water n 1.3, which will have a better resolution?

Answer 8

Smaller values of D since n is at the bottom of the equation.
water immersion lens has a greater resolution bb/c it can resolve smaller D’s than the same lens if there was air between the lens and the specimen

Question 9

Sign of theta in the resolution equation equals what?

Answer 9

Theta is determined as one half of the angle of a cone of light that the lens is using to collect light from the specimen.

Question 10

What is the numerical aperture of a lens (NA)?

Answer 10

The value of n times sin of theta is called the numerical aperture (NA) of a lens and the NA
Low resolutions lenses have NA values of less than 1
High resolution lenses can have NA values as high as 1.5

Question 11

Currently, what are the highest available NA lenses? What can you see with this?

Answer 11

About 0.2 microns
Good enough to see mitochondria but not internal details or smaller things such as ribosomes
You need electron microscope to see smaller

Question 12

What are the advantages of using long wavelength light for imaging?

Answer 12

Cy5 is a dye of long wavelengths. Long wavelength light is less damaging and less easily scattered than short wavelength light and can illuminate deep structures (can penetrate more deeply into a tissue than short b/c shorter waves are filtered out or absorbed early on during their traversal of tissue.
Less harmful to living specimens than short wavelength light. E.g., ultraviolet waves have short wavelengths of light cause cancer and are harmful but infrared lamps are not.

Question 13

What is magnification and why is it important?

Answer 13

Magnification becomes important when we consider how to record an image of something that we have resolved

Question 14

What is the difference between resolution and detection?

Answer 14

resolution is how clear something is seen. Objects smaller than the resolution limit can be detected but are not resolved (aka we can’t see them clearly)

Question 15

A minimal adequate magnification is one that allows what?

Answer 15

The smallest object you want to resolve to fall on 3 discrete elements of the imaging device
The smallest spot in an image that can be recorded accurately needs to fall on an area of the detector that is 3 detector elements wide and 3 detector elements tall.

Question 16

What are several ways light or electrons may interact with a specimen (5 ways)?

Answer 16

1) No interaction - the wavelength goes through the object/specime without being affected by it in any way
2) Absorbed - light could be absorbed by a specimen. If all the light is absorbed by a spot in the cell and you look at the cell from the other side, that spot will be black and if only some colors of light are absorbed and the remaining colors allowed to pass through then you would see a spot that’s colored.
3) Reflected - light can be reflected by a specimen. Living cells generally don’t reflect a lot of light
Reflection technique: specimens can be coated in a layer of metal atoms and these will then reflect electrons and that’s a reflection technique.

4) Refracted - light is being bent and changes direction as it passes through the specimen.Light is also slowed down while it is in the specimen (waves are compressed together). This means that when the light leaves the specimen its wavelength may be shifted relative to those of a photon of light that did not interact with the specimen.
5) Emitted - used for fluorescence imaging methods

Question 17

What happens when light is refracted?

Answer 17

Light is bent and changes directions as it passes through the specimen.
Slows down in specimen
Wavelength shifted relative to those photons that did not interact w/specimen

Question 18

What are stains?

Answer 18

Compounds that absorb light or electrons. Stains are used for absorption methods. Different components of a cell can be selectively stained..

Question 19

Black stains absorb what kind of light?

Answer 19

All colors of light

Question 20

Colored stains absorb..

Answer 20

Some color of light, others to pass

Question 21

What is the purpose of staining tissue?

Answer 21

Good for showing structure of cell but not informative w/regards to specific things in cells

Question 22

What are antibodies used for?

Answer 22

To detect specific cell components- they selectively bind to a very unique target, sometimes as specific as just a few amino acids in a sequence on a particular protein.

Question 23

Explain the method of using antibodies on animals (indirect or two antibody approach):

Answer 23

1) obtain a highly purified protein and then inject into an animal (rabbit, rat or mouse)
2) after enough time has passed for the animal to generate an immune response, the antibodies are isolated - (through blood plasma or spleen cells.)
2) a second antibody is produced, which detects antibodies made from the host animal. E.g., if you raised your initial antibody in a rabbit you might have a second antibody that was raised in a goat (used is the antibody generating protein for rabbits to amplify effect by using two layers of antibodies) (amplification method)
3) second antibody is generated with labels such as fluorescent chemicals or enzymes or secondary antibodies as opposed to having to do it specifically for every primary antibody that’s out there

Question 24

What are the advantages of using an indirect or two-antibody approach for detecting specific components of a cell?

Answer 24

1) The amplification method
2) Many primary antibodies are raised from one kind of animal, (rabbit or mouse) therefore you can generate second antibodies of large amounts for general use against say rabbit antibodies and these are less expensive to make than primary

Question 25

how can antibodies be used to detect specific things in protein?

Answer 25

Using enzyme linked antibodies. These produce colored products. If you
Label a secondary antibody with some kind of an enzyme you can use that enzyme to generate insoluble components which are then going to be revealing the location of the primary antibody

Question 26

What are the limitations with using stains?

Answer 26

1) most stains are toxic and the techniques used to apply them to cells kills them
2) you can’t make images of living cells. Living cells are almost colorless and nearly transparent

Question 27

What is Brightfield Imaging? When does it work well? When doesn’t it work well?

Answer 27

Technique is used for looking at stained specimens. Light slows down as it passes through a cell and it can be bent or refracted and this refracted light carries info about the contents of the cell.

1) You have a collecting light, which collects light from an illumination source and focuses a cone of light onto the specimen and the the objective lens (lens that you’re seeing with) captures the light passing through the specimen, whether or not it interacted w/the specimen in any way. This is a characteristic of Brightfield imaging

When looking at the images, cells are barely visible against the very large amount of non-interacting light that has passed through the specimen but not been absorbed by it. The cell is basically the same brightness overall as the background which is how this process gets its name.

Question 28

What is one technique used to deal with issues from Brightfield Imaging?

Answer 28

Looking at live specimens through Darkfield Imaging

Question 29

What is Darkfield Imaging?

Answer 29

1) shine light on the specimen with a collecting lens but in such a way that only refracted light enters the objective lens, only sees the refracted light.

There’s a ring-shaped opening in a metal plate that is placed between the collecting lens and the light source and what it results in is that you shine a hollow cone of light on the specimen, instead of a solid cone of light. The objective lens on the other side of the specimen is then fitted inside of the area of the hollow cone on the opposite side of the specimen and so light from the source that doesn’t interact with the specimen basically also doesn’t
interact or is not captured by the objective lens
Light from the source that doesn’t interact w/specimen is also not captured by obj. lens. objective lens is put in a situation where it can detect some of the light that’s refracted by the specimen.

b/c the background is dark, it’s the light that’s going past the objective lens, that’s why it’s called Darkfield microscopy and because the objective lens is only seeing the light that did interact with the specimen, it’s possible to use relatively sensitive cameras to detect the small number of photons that did interact w/the specimen and were refracted towards the objective lens.

Question 30

How are living cells seen clearly?

Answer 30

Using phase contrast microscopy to image the difference between refracted and non-refracted light

Question 31

What is phase contrast microscopy?

Answer 31

Most important technique used for looking at LIVING CELLS. This method takes advantage of the fact that light slows down when it is refracted as it passes through a specimen. When light emerges from the specimen is phase shifted relative to the light that didn’t interact with the specimen.

METHOD:
1) separates the light that was refracted by the specimen from the much larger amount of light that comes from the light source that passed through the specimen and was not refracted.

2) light that passed through the specimen w/o interacting w/it is reduced in overall brightness by passing it it through a neutral density filter (like a pair of non-polarizing sunglasses)
3) There’s another lens, the eyepiece lens, which brings the two light paths back together so they can interact again.

b/c light passing through the specimen may have been slowed down by refraction, it’s possible that the light waves from the two different pathways will either constructively or destructively interfere w/each other. This can create bright or dark regions.

The illumination elements needed to perform phase contrast microscopy are the illuminating side, phase annulus, and the phase ring.

The illuminating side is below the specimen

Question 32

What is the difference between phase contrast microscopy and darkfield imaging?

Answer 32

The ring-shaped opening, which is called a phase annulus, is smaller than the one used for Darkfield Imaging and it’s small enough that light passing through it is collected by the edges of the objective lens, instead of missing the objective lens entirely as it was in Darkfield Imaging. The light that’s refracted by the specimen is also collected by the lens, but it’s collected in the center region.

Within the phase contrast microscope is a second optimal element called a phase ring that’s coated w/ a filter that acts to reduce the light that didn’t interact w/the specimen. The light that did interact with the specimen goes straight up the middle and it is not reduced in any way. The refracted and non-refracted light are finally recombined by the last lens on either the eye or detector. You can see the diff in phase as the two light beams interact.

Question 33

What is a stokes shift and how is this related to fluorescences?

Answer 33

Fluorescence is a characteristic of some molecules that can absorb the energy of a light photon and then re-release it in the form of a second photon of light. Emitted photon has less energy than the photon that was absorbed b/c energy of the first photon is always lost during the process. Loss of energy results in a change in wavelength or color of light towards the red end of the visible light spectrum. Fluorescence molecules absorb and emit lights as a probability.

Fluorescence molecules absorb high energy light and then emit less energetic, longer wavelength light. The shift in wavelength between absorbed and emitted light is called the Stokes shift

Question 34

What three optical components are unique to fluorescence microscopes? What do these optical components do?

Answer 34

1) Excitation filter: select a wavelength light close to excitation maxima of the fluorescent molecule you’re trying to look at
2) Emission filter aka barrier filter: allows only light emitted by the specimen to be seen by detector
3) Dichroic beam splitter: reflects short wavelengths of light but allows long wavelengths to go through it.

These optical components take advantage of the Stokes shift.

Question 35

Describe how fluorescence microscopes work

Answer 35

Use excitation, emission, and dichroic filters to take advantage of the strokes shift.
1. Excitation filter: First barrier filter; lets through only blue light with a wavelength between 450 and 490 nm.

2. Dichroic Beam splitter: Beam-splitting mirror: reflects light below 510 nm but transmits light above 510 nm.
   fancy mirror which reflects short wavelengths of light, but allows long wavelengths of light to go through it.
3. Emission Filter or barrier filter: Second barrier filter: cuts out unwanted fluorescent signals, passing the specific green fluorescein emission between 520 and 560 nm. Emission filter
   Filter set depends on the fluorescent molecule being used.

Question 36

How can fluorescent dyes be developed to recognize specific and unique cellular components like nuclei acids or DNA?

Answer 36

b/c chemical components of some fluorescent stains bind preferentially to specific cellular components. These would be visualized using a different set of fluorescent components (excitation, emission, and dichroic beam splitters would be unique for each color). Toxins are actually often used to visualize structures in cells b/c many of them bind to unique molecular targets.

Question 37

Why are fluorescent molecules linked to antibodies for?

Answer 37

Fluorescent molecules can be linked to antibodies and then used to determine the LOCATION of proteins and cell components.
Can help identify proteins and DNA

Question 38

How can we visualize the distribution of individual RNA molecules?

Answer 38

Using fluorescence in situ hybridization (FISH), which uses synthetic fluorescent RNA probes to detect compatible mRNA in cells and tissue

Question 39

How does FISH work?

Answer 39

1) synthesize an RNA molecule that is fluorescently labeled and has a sequence that is exactly complementary to the target RNA molecule or to a piece of it.
2) specimen is incubated w/the fluorescent RNA molecules that you synthesized at a high temperature and then partially cooled to allow the fluorescently labeled RNA to hybridize w/its complementary sequence on RNA molecules in the cell.
The overall idea is for any unbound labeled RNA to be washed away, leaving behind fluorescent RNA molecules that are stuck or hybridized to exactly the sequence they were designed to interact with.
This method can identify cells in an organism or tissue that are expressing unique RNA molecules

Question 40

How can fluorescent molecules be used to reveal the presence or concentration of small molecules and ions?

Answer 40

Using light emitting dyes that reveal changes in ion concentrations. Example using fluorescent protein called aequorin, which releases a photon of light when calcium binds to it.

Question 41

What is green fluorescent protein or GFP? What is one thing you can do when inserting it into animals.

Answer 41

Gene coding sequence for a protein expressed in jellyfish
GFP is a beta barrel protein structure with three amino acids in its center that join their side chains to create a small fluorescent molecule. The Gene coding sequence can be introduced into many different settings including animals.

1) introduce cells derived from animals with GFP into other animals and then follow their fate in a living cell.
2) Express GFP in specific cell or tissue using gene promoters
3) GFP fusion proteins can be used to label proteins in living cells.
4) GFP-fusion proteins can be used for FRAP
5) Photo-activation using a unique GFP protein

Question 42

What is a promoter?

Answer 42

Regulatory DNA segment that promotes expression of a gene coding sequence.

Animals that have GFP express GFP under the control of a general promoter which was active in all of the cells of the animal
you can also create DNA sequences that place the expression of GFP under the control of other promoters, which may not be expressed throughout the organism

Question 43

What can green fluorescent fusion proteins be used for?

Answer 43

To label all proteins in living cells
Recombinant DNA
Relocating of GFP-tagged proteins in muscle cells

Question 44

What is photo activation? What is the benefit of using photo activated subunits over frap?

Answer 44

Photo activation is used when some GFP proteins have been modified by mutation and produce some GFPs that are non fluorescent until they are briefly illuminated by a short pulse of a short wavelength of light and after this they remain fluorescent. This technique is more difficult than FRAP b/c it uses a unique GFP protein and you have to have special equipment to activate the protein in a specific location in cells but it has advantages over FRAP.

in FRAP, the protein of interest that you are actually looking at is the bleached subunit, which becomes increasingly difficult to detect over time against the bright background of non-bleached subunits. In the case of photo-activated subunits, the subunits that you’re interested in looking at are the only ones that are bright and it’s easier to detect them over long periods of time because the background proteins, which were never activated, are simply not visible.

Question 45

Describe electron microscopy

Answer 45

EM uses magnets instead of lenses to focus the beams of electrons the design of the system directs electrons through a specimen to a detector of some sort on the other side of the specimen. (transmission EM and Scanning EM)

Uses electrons to resolve fine structures of the cell

Question 46

What’s the difference between transmission electron microscopy and scanning electron microscopy?

Answer 46

Electrons can Transmit through the specimen or bounce off the specimen
Transmit: transmission electron microscopy
Bounce: Scanning electron microscopy

Question 47

What is a disadvantage to electron microscopy?

Answer 47

1) Biological specimens do not absorb or refract electrons very well and therefore in order to visualize components of cells using electron microscope the specimens have to be stained w/heavy metals that absorb light.
Metals used for stains can be highly toxic. Cells and tissues have to be killed and thoroughly preserved in order to process them to see things.

2) Electrons don’t travel far in anything other than a vacuum so most specimens must be placed in a vacuum and this would kill most living specimens
3) YOU CAN’T STUDY LIVE CELLS. Only used to study dead cells

Question 48

What type of metals are used to stain specimens in electron microscopy?

Answer 48

Heavy metals like lead, osmium, uranium, gold, silver and tugsten

Question 49

What is electron or rotary shadowing? How does it work?

Answer 49

WHAT: This technique allows development of three dimensional type images

HOW: specimens are prepared for staining by fixing the proteins to make a mass that is resistant to harsh environments (such as found inside an electron microscope)

2) the specimens are then placed in a vacuum chamber that has a metal filament on one side of it and then the chambers’ evacuated and the filament is heated up.

3) b/c this is in a vacuum, metal atoms start to fly off of the filament and they coat things that they hit in a directional matter and this is the apparatus is called a sputter coater.
the atoms that are emitted from the filament strike specimens at a particular angle and contours on the specimen that face the filament will absorb a high number of the atoms, while contours that are facing away from the filament don’t receive as many atoms, because they’re simply facing away, and the result is a specimen that is darker on one side of a feature that was facing the filament and lighter on the other side.

Question 50

What is metal shadowing used for?

Answer 50

Applied to surface structures or interior structures using freeze fracture and freeze etching methods. it involves freezing the specimen with liquid nitrogen and then breaking the specimen. You then use a sputter coater to get three dimensional view of the inside of the structures. In freeze etched you partially evaporate the ice that was formed by freezing the specimen w/o melting and then do shadow of the specimen that’s been evaporated.

Question 51

How can you look at things that are inside the specimens?

Answer 51

Freeze-etching technique and freeze fractured technique
freezing the specimen very quickly by plunging it into something like liquid nitrogen and then breaking the specimen, typically with a very sharp razor blade
The broken surface then can travel, the break can travel through cells and into their interior and of course you can use the surface as the material that you use in your sputter coater, and so you get a three dimensional view of the inside of structures.

Question 52

What is negative staining?

Answer 52

An alternative method to the shadowing technique that will give you three dimensional information about macromolecular complexes.
Common method to look at viruses
Negative staining involves soaking a specimen in a solution that has one of these dissolved metal stains and then the excess solution is removed and this leaves behind a small amount of stain that dries onto the specimen and the surrounding surface, and crevices or edges of three dimensional objects will retain stain and appear darker while parts that stick up away from the surface will not have been stained and that’ll appear lighter. Commonly used to look at viruses.

Question 53

How has negative staining been rejuvenated?

Answer 53

The use of computers to analyze image data, which produces very detailed, 3D information about specimens.
Combines two dimensional images and looks at structure from all different directions

Question 54

How can antibodies be used for electron microscopes?

Answer 54

antibodies can be attached to metal (gold or tungsten) particles to detect specific cellular components. It is possible to localize specific proteins within the structure being examined. Pulse of labeled material would move through different cell compartments and allow you to identify location of labeled material at different times, and order of flow through cell compartments, as well as how fast things were moving through one compartment.

Question 55

What is pulse-chase radion labeling?

Answer 55

Can be used with electron microscope to get an idea about the series of events in a dynamic process

Question 56

What is scanning EM?

Answer 56

used to generate three dimensional images of specimens. It’s a reflection technique in microscopy.

The specimens are coated with electron reflecting material, usually gold. in something like the sputter coater, except that all sides of the specimen are coated (rotate the specimen while it’s in this chamber. )

Then you place a detector for electrons and instead of placing it at the bottom of the electron microscope where it will collect all of the electrons, you now place this detector to one side. The electron beam is then scanned across the specimen in an orderly manner and the detector measures how many electrons are being reflected toward it and then the electronics of the system correlates the position of the beam with the intensity of the signal to produce an image.

When the electron beam strikes the specimen in a location such that it bounces electrons toward the detector, you get a bright spot in the final image and when it strikes a place in the specimen that’s pointed away from the detector, you get a dark spot.

it’s a favorite technique for looking at relatively large structures like whole insects and plants at high resolution