Techniques - Histology

Front 1

Identify the 5 steps of tissue preparation, their purpose, and their general procedure.

Back 1

1) fixation: halts cell metabolism
after removal from source, specimen should be immersed in a fixative immediately. Fixatives can be formaldehyde, glutaraldehyde, osmium.

2) embedding: infiltrate water filled spaces with embedding medium
Before embedding, must dehydrate H2O (immerse in alcohol such as ethanol/acetone), clear the alcohol in preparation for the embedding process (by immersing in xylene). Finally, embedded with embedding medium (such as paraffin wax, plastic resin)

3) sectioning: slicing it- converting 3 dimensional into 2 dimensional
Can be whole mount (unsectioned), cross section, longitudinal section, or random. Thickness depends on imaging method.
**For sharper images, cut thinner sections.

4) staining: to enable interested parts to be visible, or else everything will look the same.
Can be specific- identified molecules or nonspecific- general.

5) imaging: allows small details and structure to be seen clearly.

Front 2

Name three types of microscope.

Back 2

1) compound light microscopy
2) confocal microscopy
3) electron microscopy

Front 3

How to calculate the magnification.

Back 3

Magnification= magnifying power of ocular piece (eye) X magnifying power of objective lens (piece right above the specimen)

Front 4

Define resolution (d) and its equation.

Back 4

resolution = the smallest distance (d) at which two objects can be successfully distinguished.
d = (0.61 x wavelength) / NA

Front 5

Better resolution means larger D or smaller D? What can you do to enhance resolution?

Back 5

d = (0.61 x wavelength) / NA

Better resolution means smaller d. You can increase numerical aperture or decrease wavelength.

Front 6

Define NA- Numerical aperture. Give equation. For better resolution, should the NA be larger or smaller?

Back 6

NA = (n)(Sin)(alpha)
n = refractive index (amt of light absorb)- varies with different medium: air, water, oil.
alpha= 1/2 of angular aperture (angle of light beam)
Numerical aperture is the measure of objective's ability to collect light from a specimen. Difference objective have different NA and working distance (how far they are from the cover slip).
For better resolution, the NA should be large. Large NA give small D - resolution.
* BIGGER NA IS ALWAYS BETTER

Front 7

Air, water, oil. Arrange in order of least to highest refractive index.

Back 7

Air < water < oil immersion
Oil immersion gives the highest because it captures more light. In other words, lights need to passed through more stuff. This will give a better NA --> better resolution.

Front 8

What is the relationship between resolution and wavelength?

Back 8

The higher the wavelength, the higher the resolution = the blurrier the image.
The lower the wavelength, the lower the resolution, the clearer the image.

Front 9

Tricky confused question:
Increased resolution or resolving power means- larger or smaller value of D (distance)?

Back 9

Increase resolution = enhancing it. This means SMALLER (D). On other hand, large resolution means LARGE (D).

Front 10

What is empty magnification?
How to calculate the maximum useful magnification?

Back 10

Empty magnification= an image is enlarged but no additional detail is resolved (shown).
Maximum useful magnification = 1000 x NA

Front 11

Two images given. Which one will have more details and a crispier image? Explain.
A: 20X objective, NA = 0.40, enlarged 10X, magnified 200
B: 4X objective, NA = 0.10, enlarged 50X, Magnified 200

Back 11

Case A will be in more detail and more crispier because- 1) LARGER NA is ALWAYS BETTER. 2) B is being magnified larger than its maximum useful magnification- which means it will lose significant crispiness.

Front 12

What is the function of a condenser in a microscope? How to manipulate it for more contrast in the image?

Back 12

Condenser controls the amount of light passing through. To increase contrast and sharpness, you should close the condenser and have less light pass through which make the parts appeared darker than surrounding and more easier to distinguished.

Front 13

What are two manipulations of bright field microscopy?

Back 13

Phase contrast and Differential Interference Contrast (DIC)

Front 14

What is Phase Contrast Microscopy useful for?

Back 14

Excellent for living tissue (unstained specimens with little contrast). This form of microscopy distinguished the boundaries of cells without manipulating it. Work by: things in cells moving manipulating lights. Organelles have different thickness, therefore different refractive index.

Front 15

Describe the principle of fluorescence microscopy. What are its advantages and disadvantages?

Back 15

Fluorescent molecules (fluorochrome) is introduced into the specimen. This allows us to tagged or mark structure in specimen of interests. Works by excitation -> loss of energy-> emission of the fluorochrome. Pros- good to visualize.

Front 16

What are the three fluorochromes mentioned in lecture?

Back 16

1)DAPI
2)FITC
3)RHODAMINE

Front 17

What are the two major problems with fluorescence microscopy?

Back 17

1) photobleach: if light is on it too long, fluorochrome will fade SOLUTION- Quantum Dots (can be toxic to cell)
2)MUST BE THINLY SECTION else--> Image degradation: blurring effect due to light scattering everywhere in many of the non-focal plane presents making it very hard to tell apart which is which. SOLUTION- Confocal Microscopy

Front 18

How does confocal microscopy works and what is its advantage?

Back 18

Works by collecting lights from one plane at a time so only light from that plane of focus will reached our eye.Image will then by much more crispier and in more details. VERY POWERFUL by collecting series of images from different focal planes, and assemble the image series to yield 3-D image.

Front 19

What are the two types of Electron Microscope?
HINT:
1- requires very thin section
2- sectioning not required because looking for surface structure.

Back 19

1) TEM- Transmission Electron Microscope
2) SEM- Scanning Electron Microscope

Front 20

Describe TEM (transmission electron microscopy).

Back 20

Required very thin section. Works similar to light microscopy, but instead of using light beam, electron beams are used. Specimen are stained with metals, those stain will appeared dark, unstained= light.

Front 21

Describe SEM (Scanning electron microscope)

Back 21

Purpose is to look at surface. Therefore, surface will be coated with metal, electron beams do not passed through specimen.

Front 22

What is the purpose of freeze fracture and what type of microscopy is often used for this?

Back 22

Freeze fracture is use to look at membranes and integral membrane proteins. Observed using SEM.
Procedure: freeze a fragment of tissue and fracture it to see membrane structure.

Front 23

Define:
Antigens
Epitopes
Polyclonal antibodies
Monoclonal antibodies

Back 23

Antigens = invasive foreign molecules.
Epitopes= regions of the antigens that are recognized as foreign. Site for antibodies to bind.
Polyclonal antibodies= using many different kinds of antibodies that can all recognized the same antigens - due to the different epitope sites on it.
Monoclonal antibodies= using just a single type of antibodies that recognized only one epitope on the antigen.

Front 24

What are the advantages and disadvantages of monoclonal & polyclonal antibodies?

Back 24

Monoclonal:
Advantage= more specific
Disadvantage= weak signal

Polyclonal:
Advantage: recognized more epitopes in tissue BUT
Disadvantage: less specificity.

Front 25

What is immunocytochemistry?

Back 25

A technique used to localize specific molecules in an organ, tissue, or cell. Primarily through exploit use as antibodies to target certain tissues.

Front 26

What is direct immunocytochemistry, its advantages and disadvantages?

Back 26

a visible marker is directly attached to antibody binding the antigen.
Advantage:
1) Specificity
2) Less background staining

Disadvantages:
1) Low sensitivity if antigen = low concentration in tissue
2) Need to directly conjugate markers to antibody- buy antibodies directly marked with a tag that is specific to them- very expensive option and a lot of work.

Front 27

What is indirect immunocytochemistry, its advantages and disadvantages?

Back 27

a visible marker that binds to secondary antibodies.
Advantages:
1) signals much more amplified
2) can use labeled secondary with different primary antibodies

Disadvantages:
1) Nonspecific background may increase
2) Takes longer to do
3) Needs more reagents.

Front 28

What are the tags we can used with immunocytochemistry? (3)

Back 28

1) Flurochrome
2) Enzyme (HRP)
3) Electron dense molecule (ferritin, gold)