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TNS > TNS Exam 2 > Flashcards

TNS Exam 2 Flashcards

1
Q

What is Magnification?

A

How much larger a sample appears compared to it’s original size

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2
Q

What is resolution?

A

The minimum distance that two points can be separated, but are still considered two points

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3
Q

What is the diffraction limit of resolution?

A

It leads to a limit of optical resolution. Airy Patterns and Disk can lead to convolution of points in the image

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4
Q

What are the factors that resolution depend on?

A

The wavelength of light and Numerical Aperature

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5
Q

Why is wavelength a limiting factor for resolution?

A

DIfferent colored wavelengths have different limits of resolution

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6
Q

What is Numerical Aperture?

A

It is the measure of a microscope’s ability to gather light, or the range of angles it can accept/emit light. It depends on the angle of the light entering and the refractive index of the medium

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7
Q

What does refractive index show?

A

It shows the changes in speed of light that traveling through a medium

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8
Q

How does angle of light affect numerical aperture?

A

A broader light cone means more rays of light. This causes a higher numerical aperture.

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9
Q

Two important wavelengths for fluropores?

A

Excitation and Emission Frequency. Excitation frequency is the light that excites the fluoropore while Emission frequency is the light that the fluropore emits in response

  • Excitation: high energy and intensity
  • Emission: low energy and intensity
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10
Q

How does fluoresence microscopy work?

A

Light excites the fluoropore, the fluoropore absorbs the energy and emits a new photon in another wavelength in response

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11
Q

Components of the microscope?

A
  • Camera: captures images
  • Eyepieces: 2nd stage of magnification
  • Excitation and Emission filters: only certain wavelengths can reach sample and detector
  • Shutter: controls if sample is exposed to light
  • Stage: where to put the sample
  • Mercury lamp source: bright white light that’s filtered for fluorescence
  • Objective lenses: multiples lenses with different levels of magnification that’s rotated
  • Focus Knob: moves the stage or objective lens in Z-axis to focus the image
  • Transmitted light source: used in standard light microscopy
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12
Q

How does a microscope work?

A

Condenser focuses light on the sample. The objective and ocular lenses are used to focus and magnify the image

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13
Q

What is Stereo microscope?

A

It’s useful for dissection and 3-dimensional viewing.

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14
Q

What is brightfield microscopy?

A

In brightfield microscopy, parallel rays are passed through the sample or reflected off the sample. In this technique, there is no contrast enhancement (stains and coloring agents used)

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15
Q

What are microscopy techniques that enchance contrast?

A

Phase Contrast, Darkfield, and Differetial Interference Contrast Microscopy

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16
Q

Phase Contrast Microscopy

A

This technique uses the different refactive index that different matterials have. Since parts of the cell have different refractive indices, these differences are amplified to make some parts of the cell brighter.
- doesn’t need treatment of sample

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17
Q

Darkfield Microscopy

A

This technique uses illuminating rays to scatter light because different regions of the cell scatter light differently. Parts of the cell that scatter less light, like the Cytoplasm, appear dark. Organelle/structures that scatter more light appear bright.

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18
Q

Differential Interference Contract Microscopy

A

This technique uses polarized light and light scattering. Scattering light throws off the angle of polarization, which makes structures stand out against the dark background and seem more 3-dimensional

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19
Q

Fluoresence microscopy

A

It uses fluorophores. It absorbs light at a specific frequency and emits light in another longer frequency.

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20
Q

Dichroic Mirror

A

Long wavelengths of light have lower energy and are transmitted by the dichroic mirror. Short wavelengths of light have higher energy adn are reflected by the dichroic mirror

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21
Q

Two Photon Microscopy

A

This is used for the imaging of living tissue or very thick samples.

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22
Q

How does the energy of a light photon relate to frequency and wavelength?

A

A photon with high frequency has high energy and a short wavelength. A photon with low frequency has low energy and a long wavelength.

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23
Q

Electron Microscopy

A

It has a smaller diffraction limit of resolution which means superior resolution of sample. This technique requires fixation and thick sections. For sectioning, the samples need to be dehydrated, stained and embedded in epoxy medium.

24
Q

WHat is the preparation of TEM (transmission EM) sample?

A

It uses ultamicratome and needs to have extremely thin sections

25
Q

Scanning electron microscope (SEM)

A

This microscope uses an electron beam to scan the surface of a sample and study its topography. It uses 3D images but can’t view living specimens

26
Q

Transmissin electron microscope (TEM)

A

In this microscope, electrons pass through the sample and show an image of the internal details. It uses thin slices but has less focus.

27
Q

Steps to prepare tissue?

A

Fixation and Perfusion, Embedding and Freezing, Sectioning and Mounting

28
Q

What is Fixation?

A

It is when chemical methods are used to preserve, stabilize and strengthen the specimen.

29
Q

Two categories of fixation?

A

Crosslinking and Dehydration

30
Q

Perfusion and Immersion

A

Perfussion is when you pump saline into the ventricles, then pump solution into the left ventricle. The right atrium is cut for relief of pressure and exsanguination. Immersion is when you summerge the specimen in the solution.

31
Q

How does fixation work? Crosslinking and Dehydration?

A

Crosslinking Fixation uses fomaldehyde or Glutaraldehyde, and these cause binding between the proteins. Dehydration fixative disrupts the lipids and reduces solubillity of the proteins, which cause the proteins to go out and fix into place.

32
Q

What happens after Perfussion?

A

Embedding and Freezing

33
Q

What is Embedding?

A

It is when the brain is surrounded in a substance that creates a hard shell, which stabilizes the structure of the brain

34
Q

What is Freezing?

A

It is when the brain needs to be post fixed in sucrose solution or saline sp tha tissue isn’t destroyed by water crystallization.
- many techniques use frozen brains

35
Q

Sectioning of brain

A

Brain is sliced and mounted on slides. It can be sectioned along any dimension

36
Q

Cryostat

A

Roatry microtome used for obtaining single samples

37
Q

Microtome

A

Equipment used to cut thing into very thin slices

38
Q

Vibratome

A

Similar to microtome but uses a vibrating blade to cut thin slices of tissue

39
Q

Cell body stains

A

It uses basophiilic stains to bind and stain acidic molecules

40
Q

Nissl stains

A

Nissl stains label RNA in the cell, and neqjrons stand out due to enriched ribosomes and rough ER

41
Q

Fiber stains

A

Fiber stains label white matter tract by staining myeline

42
Q

Golgi stains

A

Silver staining technique used to see nervous tissue under light microscope

43
Q

In Situ Hydridization

A

This allows one to see the mRNA for a specific protein. For this to happen, the sequence of the protein is needed and it uses cDNA. It also shows where specific mRNAs were produced

44
Q

How does In Situ Hydridizatiom use cDNA?

A

cDNA find and sticks to mRNA sequences. The cDNA is tagged with a probe in order to stand out.

45
Q

Anterograde and Retograde

A

Anterograde: dendrite to cell body to axon
Retrograde: axon to cell body to dendrite

46
Q

Antibodies

A

They identify and neutralize foreign objects in the body, and are mediators of the humoral immune system
- also called immunoglobulins

47
Q

Structure of antibodies

A

2 indentical heavy chains and 2 identical light chains, constant region and variable region

48
Q

Paratope and Epitope

A

Paratope is the part of the antibody that finds and sticks to antigens. Epitope is the part of the antigen that antibodies stick to.

49
Q

Why is paratope and epitope important?

A

Paratope functions like a lock, and epitope is a key. This system can tag something using the epitope to be destroyed or to block its function. It can also be used to tag any proteins/chemicals that have the specific epitope

50
Q

Primary and Secondary antibodies

A

Primary antibodies bind to the antigen while secondary antibodies bind to primary.

51
Q

Monoclonal and Polyclonal anitibodies

A

Monoclonal: identical to each other and produced by single cell
Polyclonal: different from each other and produced by many cells, and isolated from blood

52
Q

Electrophysiology presentation

A
  • Animal: rhesus monkey
  • Structure: MT/V5 (Medial Temporal Visual Area)
  • Explores how microstimulation of neurons affect visual fields of monkey
  • Uses LED dots as targets
  • Conclusion: monkeys are more likelu to choose preferred direction of stimulus caused by microstimulation (preferred LED), and visual perceptiom can be controlled/changed theough manipulation of neurons
53
Q

Behavior presentation

A
54
Q

Microscopy presentation

A
  • Uses electron microscopy tomography: reconstructes 3D structures from 2D projections collected at different tilt angles
  • Findings: AZM is a multifunctional organelle, and EM allows for detailed analysis of organelles that are too small to see otherwise
  • AZM: helps with anchoring Ca2+ channels, docking/fusion of vesicles with membrane, maintenence of spacing between channels and vesicles
55
Q

Stereotaxic presentation

A
  • Two photon imaging
  • Animal: Mice
  • Mice had YFP (yellow fluorescent protein) in layer 5 of thr cortex

TNS (3 decks)

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