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Fundamentals of neuroscience > Imaging in the NS (cells of NS) > Flashcards

Imaging in the NS (cells of NS) Flashcards

1
Q

Why do we want to see cells?

A
  • Cell morphology
  • Cell projections & connections
  • Protein expression
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2
Q

How can we highlight specific proteins using fluorescent microscopy?

A

Different stains are used for different wavelengths

Means we can use different stins to highlight certain proteins & can change the waveength of the microscope for the specific marker we want

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3
Q

How does fluorescent microscopy work?

A
  • EXCITATION light is shone onto sample
  • Excitation filter determines wavelength of excitation light
  • Excitation light is absorbed by the fluorescent tag
  • Electrons are EXCITED
  • Emission light is released at the electrons relax
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4
Q

What are the parts of a wide-feild fluorescent microscope?

A
  • Arc lamp
  • Excitation diaphragm
  • Excitation filter
  • Camera
  • Objective
  • Emission filter
  • Sample

Refer to image

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5
Q

How does confocal microscopy?

A

-Excitation light from a laser is shone onto the sample

-Excitation pinhole creates a very narrow beam of the excitation light onto the sample

-Excitation light is absorbed by the fluorescent tag in particular plane of field

-Electrons are excited

-Emission light is release as the electrons relax

-Emission filter is selective for the light emitted the specific plane

-Allows for 3D images

-Takes pictures of layers (planes)

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6
Q

What sort of image does SEM make?

A

3D image of the surface

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7
Q

How do SEM microscopes produce an image?

A

Electrons bounce off the surface

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8
Q

What sort of image does TEM microscopes produce?

A

Internal strucutre

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9
Q

How does a TEM microscope proudce an image?

A

Electrons pass through sample

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10
Q

Summarise a light microscope

A
  • AKA bright field
  • Visible light
  • Stains and dyes
  • Whole cell morphology
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11
Q

Summarise fluorscent light microscopes

A
  • AKA wild field
  • Visible lgiht (excitation & emission)
  • Fluorescent markers
    -Whole cell morphology, protein expression
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12
Q

Summarise confocal microscopes

A
  • Visible light (excitation and emission)
  • Fluorescent markers
  • Whole cell morphology, protein expression
  • Higher contrast
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13
Q

Summarise EM microscopes

A
  • Electron beam
  • High res
  • 3D sample structure (SEM)
  • Intracellular structures (TEM)
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14
Q

What are the 5 main stains?

A
  • Hematoxylin and Eosin (H&E)
  • Nissl
  • Golgi
  • Osmic acid
  • Immunohistochemistry
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15
Q

What are the stages of tissue preperation?

A
  1. Tissue harvested & preserved
  2. Tissue is cut
  3. Mounted into glass slide
  4. Staining
  5. Coverslip
  6. Image
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16
Q

How are tissues harvested & preserved during preparation?

A

Snap frozen or chemically fixed (PFA)

Keeps it as close to normal as possible

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17
Q

How are tissues cut during preparation?

A
  • Frozen or embedded in wax or resin
  • Microtome of cryostat
  • um thick
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18
Q

How is tissue mounted into a glass slide during preperation?

A

Snap frozen tissue is chemically fixed

19
Q

What colour is H&E?

A

Dark blue/violet

20
Q

What charge does H&E have?

A

Positively charged / basic

21
Q

How does H&E work to make a stain?

A

Binds to -ively charged DNA & RNA in the nucleus

Binds to -ively charged RNA in the RER - neurons have a lot more RER than other cells in the NS

22
Q

What colour does eosin stain?

A

Pink

23
Q

What charge does eosin have?

A

-ively charged

24
Q

How does eosin stain?

A

Binds to +ively charged amino acid side chains in the cytoplasm

Not specific to cell types

25
Q

What colour is nissl stain?

A

Dark blue / violet

26
Q

What is nissl stain also known as?

A
  • Aniline
  • Thonine
  • Cresyl violet
27
Q

What charge does nissl stain have?

A

+ively charged / basic

28
Q

How does nissle stain stain?

A
  • Binds to -ively charged DNA & RNA in the nucleus
  • Binds to -ively charged RNA in the RER - neurones have more RER than other cells
  • Not specific to cell types
29
Q

What colour is golgi stain?

A

Black

30
Q

How does golgi stain stain?

A
  • Impregnates tissues wiht potassium dichromate & silver nitrate
  • Only stain cells randomly - will be different every time
31
Q

What colour does osmic acid stain?

A

Black

32
Q

How does osmic acid stain?

A
  • Binds to lipids - myelin is a fatty sheath
  • Also fixative for tissue used in EM
33
Q

What stain is similar to osmic acid?

A

Weigert stain

34
Q

What are antibodies like in immunohistochemistry?

A
  • Fluorescent
  • Enzyme complex that will produce a dye precipitate in the presence of its substrate
35
Q

How do antibodies bind to the protein of interest in immunohistochemistry?

A
  • Cell markers e.g. filaments
  • Protein expression e.g. receptors
36
Q

How are the antibodies for immunohistochemistry produced?

A
  • Antibody raised against antigen of interest
  • Antigen introduces to animal
  • Stimulates immune response & antibody production
  • Collect & purify antibodies
37
Q

What are the teo types of immunohistochemistry?

A
  • Direct immunohistochemistry
  • Indirect immunohistochemistry
38
Q

How does direct immunohistochemistry work?

A
  • Single antibody
  • Binds directly to the antigen
  • Tagged w flurophore or enzyme complex
39
Q

How does indirect immunohistochemistry work?

A
  • 2 antibodies –> primary & secondary
  • Primary antibody –> binds directly to antigen
  • Secondary antibody –> binds to primary antibody, tagged w flurophore or enzyme complex
  • Signal amplification
40
Q

What are the 4 types of immunohistochemistry images?

A
  • NeuN
  • Glial fibrillary acidic protein (GFAP)
  • IBa1
  • TH (Tyrosine Hydroxylase) enzyme
41
Q

What does NeuN look like?

A
  • Protein express in neuronal nuclei
  • Does not stain the cytoplasm
  • Stains most neuronal cell types
42
Q

What does GFAP look like?

A
  • Intermediate filament
  • Helps astrocytes to maintain their shape
43
Q

What does IBa1 look like?

A
  • Specifically expressed in marcophages/microglia
  • It is up regulated during the activation of these cells
44
Q

What does TH enzyme look like?

A
  • Catalyses the conversion of the L-tyrosine to L-DOPA
  • Expressed in the neurones that produces catecholamines: Dopamine, NA and A

Fundamentals of neuroscience (38 decks)

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