microscopy Flashcards
what is microscopy?
Using microscopes to view objects/specimens that are not visible to the naked eye
name the parts of a microscope
- detector
- objective
- specimen
- light conditioning system
- light source
where does light travel through the microscope?
goes through the objectives and reaches the detector
describe the light microscopic specimen
- cover glass
- sample surrounded by embedding medium (might contain anti-bleaching agent)
- glass slide
role of the incubator box?
prevents focus instability
how is co2 atmosphere maintained?
controller allows adjustment of air flow and % co2
more complexes images…
take longer to capture
If something moves very fast, you need to make sure you capture the sample not the surrounding
what is the triangle of frustration?
about signal detection
- temporal resolution
- spatial resolution
- sensitivity
pixel area and resolution?
small pixel area = high resolution
in order to capture a fast moving image what do you have to sacrifice?
resolution
what are some of the markings on objectives and what do they mean?
magnification - how big you can see the sample
application - modify light in order to see different things, some objectives are suitables for certain applications over others
immersion medium – not all objectives can see samples in air, they may need to be immersed in water or a type of oil – e.g. won’t be able to focus if you use on objective that works in air and you immerse it in oil
Numerical aperature - nothing to do with magnification
a higher numerical aperture means what?
a better, crispier image of the sample
what is meant by light microscopy?
Brightfield: condensing the amount of light that goes through, which gives some 3D properties. Observing a sample
3 parts to light microscopy
1) Histology
2) Phase contrast – cell morphology
3) Time-lapse (heart cell differentiation, cell migration)
what is histology?
immunohistochemistry
-where is my protein of interest found? find it using antibodies
chemicals that react with different parts of the tissue as they have affinity for basic and acidic components, allowing you to distinguish the different parts of a tissue
PROS: allows you to distinguish the different parts of a tissue
CON: less detail of what the cell is doing
what is meant by phase contrast – cell morphology?
a technique used for gaining contrast in a translucent specimen without staining the specimen
what is meant by time-lapse?
the method involving live cell imaging from a single observation intime
can show the relevance of movement if for example you find the gene in a virus, remove it and the cells don’t migrate as much without the gene
explain what happens in electron microscopy?
2 types:
Transmission EM
Scanning EM
- source of light is a beam of electrons - electrons go through the specimen and find it more difficult to go through certain denser regions and so appear darker
- electrons move faster and more easily through less dense regions, appear lighter
what is meant by the fluorescent mode in light microscope?
there is a surge of light, and the light source is a particular wavelength of light
-the light is modified through different filters and eventually reaches the sample, and the sample reflects some type of fluorescence
-transformed to digital data and transmitted to computer
flourscence - absorption and emission?
certain types of molecules absorb light – they are excited by a certain wavelength and generate energy (they are at their peak)
at some point the energy is lost (they lose their peak). the excited molecule will then meet another wavelength and reach their peak again (and so on). when they release the energy they emit light
what is the stokes shift?
Stokes shift – gain and loss in energy
Different between the wavelength at which the molecule of excited, and the wavelength of emission when the molecule loses its energy
what is photobleaching and when does it occur?
bleaching of flurochromes: due to high intensity illumination the flurochromes might lose their ability to permanently emit light
-when gain of energy cannot be released anymore - there’s bleaching. instead of releasing energy and having fluorescence, the fluorescence is lost, and you lose the ability to see the fluroform in detail
role of fluorescent proteins?
these proteins can be fused with other proteins and introduced into cells via transection
-this allows the live study of fluorescent tags in organisms/cells
confocal vs widespread?
confocal microscopescans a sample with a focused beam of light
only a small volume can be visualised by confocal microscopes at once - bigger volumes need time consuming sampling
role of lens?
illuminate and magnify
