Lecture 7: Microscopy Flashcards
A microscope is a…
…lens, a light source (not necessarily visible light) & a camera.
This can be manifested in different ways meaning there are many forms of microscopes
Light can…
be refracted, reflected, absorbed and emitted or altered in other ways and microscopes use this property of light in different ways
Types & Examples of microscopy include:
Bright-field | Phase contracts | DIC | Laser-focussed microscopy
Brightfield microscopy is useful for…
Tissue - that can be fixed in sections & then stained.
The theory behind this is that the space around the sample is light and something is needed that can attenuate the light as it runs through the sample.
👎 It’s limitations include the fact the sample needs to be stained which means it cannot be used to look at live cells.
Phase contrast microscopy uses…
the property of the sample and the way it interacts with the light that passes through to create a contrast.
It utilises how different parts of light changes as it passes through different parts of the sample.
DIC utilises the same concept…
phase contrast uses, but takes advantage of the polarisation of light.
This differs from phase contrast in that it is a different type of contrast.
(Phase contrast concept:)
The property of the sample and the way it interacts with the light that passes through to create a contrast.
It utilises how different parts of light changes as it passes through different parts of the sample.
The Rayleigh Criterion is
Background/Context:
There is a mathematical limit to how small one can go when analysing a sample.
Whenever light passes through a sample, it is diffracted.
This means that the best-focused spot of light that can be produced by a lens with a circular aperture in known as an airy disk.
The issue is 2 airy disks interacting and affecting the image.
…the minimum resolvable detail dictated by the overlap of the airy pattern of one point source with the airy disk of another.
Fluorescent microscopy utilises the…
{Fluorescent Microscopy}
…emission properties of the sample
Fluorophores are chemical compounds that accept a photon and emit a photon as a result.
Fluorophores can be expressed or attached to specific targets via antibody affinity.
This allows for the highlighting of specific structures within a sample and also to detect something that is too small to see with other techniques (due to diffraction expanded the light being emitted).
The absorption emission graph explains…
{Fluorescent Microscopy}
…how fluorescence occurs at a higher wave length following absorption of a photon. The bigger the difference between absorption and emission spectrum, the better the results.
Widefield microscopes use an…
…LED or metal halide lamp to excite fluorophores in a sample.
The emission is filtered and captured using a camera.
Anything out of focus is still captured as well (no optical sectioning).
Confocal microscopes use…
…lasers to scan across a sample to excite fluorophores with the emission filtered and passed through a pinhole before detection by a photomultiplier tube (more sensitive than a camera)
The pinhole allows for optical sectioning
SPIM is a…
…sheet of laser light that is passed through the sample perpendicular to the axis of observation
The emission is filtered and captured using a camera.
Optical sectioning occurs through the illuminating sheet.
The point spread function describes…
…how the perfect point of focus within a source changes across the z axis.
- The point looks smaller and smaller as you move through the z axis -
The issue is the sample looked at is always 3D, meaning a lot of detail is lost
Blocking the out of focus light helps with limiting this (widefield vs confocal)
