Methods In Neuroscience 2 Flashcards
What is the key principle of a fluorescent microscope?
Blue light is shone at a GFP through a microscope. The GFP then emits a green light which is detected..
In detail how is blue light shone at the GFP via the fluorescent microscrope?
Blue light is first turned on.
It then passes through an excitation filter (which blocks other wavelengths of light getting through)
This blue light is then reflected off a dichroic mirror and shone through an objective lens onto the GFP
Excitation filter is on exam
How is green light detected from GFP?
Green light comes off the GFP and goes back through the microscopes objective lens
This then passes up the microscope and goes THROUGH the dichroic mirror (it isnt reflected)
The light then passes up through an emissions filter which doesnt let any other light through
This is then detected and seen through a tube lens
Emissions filter is on exam
What are GCaMP - GFP based calcium indicators?
These are proteins based on GFP which help us to detect calcium
An example = cpEGFP
In this example there are two proteins bounds M13 and CaM
When these proteins bind to calcium in solution the GFP shine
This can help us detect calcium in the brain, especially when neurones become active.
The higher the resolution of a microscope, the better
Which is better a wide field or confocal microscope?
One exam!
Wide field collects light from below and above the focal plane. This is saying it collects light outside of the focal plane. Whereas confocal only uses light from the focal plane
This reduces their area of excitation
To excite the whole sample you use a laser to scan all parts of the sample.
However:
Confocal microscopy rejects light not coming from the focal plane and decreases the area of excitation.
This produces images at a much better resolution than the wide field microscope
Problems with using GFP microscopy to study the brain
The animal is usually sedated using sodium channel blockers
Thus the animal becomes stressed
Also these channel blockers make neurones less excitable. The animals neurones dont perform usual behaviours..
Solutions to the disadvantages of GFP microscopes? (Problems of sedating animals)
You could not sedate the animal
Make the animal believe it is acting in a certain way i.e. via virtual reality
Or using a free moving animal
How to use virtual reality to study the brain?
Do with mice
Put animal on a bowl that moves in different directions . Allow animals to turn
Keep head of animal fixed
Usually train animal to do this voluntarily
Free moving methods of testing an animals neurones?
Fit a tiny fluorescent microscope to an animal like a mouses head
A image can be made by a light guide . I.e. a light is shone which actives neurones in the brain
How to do neuronal stimulation by light? Using channelrhodopsin.
Either stimulate a neurone and observe the behaviour
Or inhibit a neurone and then see if there is an absence of behaviour
You can use blockers in the brain which can excite or inhibit neurones
You can also induce mutations which can make neurones become stimulated more or less
One example of using channel rhodopsin to study neuronal stimulation by light
Insert artificial channels into an organisms brain which respond to light
This can be a sodium potassium channel and doesnt have to be extremely selective
This can open the neurone
This will lead to the membrane being polarised.
How can we use Halorhodopsin = chloride channel to study neuronal stimulation by light?
This halorhodpsin channel usually hyperpolarises the membrane
We can study this by shining a light and waiting for activation
This channel helps us to understand certain conditions, like epilepsy.
