Microscopy and Imaging Flashcards
How does light travel?
- as a wave, but it is thought of a distinct units (photons)
What is light refraction?
change in direction when moving from one medium to another duct to a change in refractive index (measure for the speed of light in a medium)
What is light diffraction?
Bending of light around an object
What is light reflection?
Bouncing of light off of an object
What is light absorption?
Interaction of a photon with an object such that the energy of the photon is taken up by the electrons in that object (usually generates heat)
What is light phase?
The occurrence of one whole wave within a specific time period (measured in degrees)
What is light wavelength?
The distance between the amplitude peaks of different waves
What is light amplitude?
The distance between the middle line and the peak of the wave
What is light polarisation?
- the orientation of oscillation of a wave
What are some imaging technologies?
- Brightfield
- Phase contrast
- Differential interference contrast (DIC)
- Darkfield
- WIdefield
- Laser scanning confocal microscopy (LSCM)
What are the different components of a microscope?
- eye piece
- objectives (different magnifications)
- stage
- condenser
- light source
- focus dial
What is Kohler illumination?
- Kohler, 1893
- process that provides optimum contrast and resolution by focusing and centring the light path and spreading it evenly over the field of view.
What is Brightfield microscopy
- the simplest of all the optical microscopy illumination techniques.
- sample illumination is transmitted white light, and contrast in the sample is caused by attenuation of the transmitted light in dense areas of the sample
- best suited to viewing stained or naturally pigmented specimens such as stained prepared slides of tissue sections or living photosynthetic organisms.
What is phase contrast?
- Zernike, 1934
- optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image
- Phase shifts themselves are invisible, but become visible when shown as brightness variations
What is the light path for phase contrast microscopy?
Condenser annulus -> condenser -> specimen -> objective -> 90 degree phase shift ring -> grey filter ring -> image plane
What is differential interference contrast (DIC)?
- Nomarski, 1952
- used for imaging live and unstained biological samples, such as a smear from a tissue culture or individual water borne single-celled organisms.
- enhances the contrast in unstained, transparent samples
- uses the interference between sheared beams and utilises polarised light
What is polarised light?
- light waves in which the vibrations occur in a single plane
- Plane polarized light consists of waves in which the direction of vibration is the same for all waves
- a plane polarized light vibrates on only one plane
What are fluorophores?
Molecules that accept a photon and emit a photon as a result
- can be expressed or attached to specific tapes via antibody affinity or via chemical affinity
- examples include green fluorescent protein/mCherry
What is fluorescent microscopy?
- instead of light being shone through the sample, light is shone on the sample
- it does this to look at the emission response
- the objective is both the sender and the receiver so no condenser is needed
How does fluorescent microscopy work?
- introduce fluorophores into the sample through staining or expression
- illuminate with the specific wavelength of the fluorophore(s) of choice
- filter for the expected emission wavelength
What is the airy disk?
- light coming from a fluorophore starts as a small point (point source) and expands in a sphere
- passing light through a lens attempts to focus it back to a small point
- the smallest focussed point is an airy disk which size is limited by diffraction
What is the Rayleigh criterion?
The minimum resolvable detail dictated by the overlap between the airy pattern of one point source and the airy disk of another
What is point spread function (PSF)?
- describes what a single point in the object looks like in the image
- presents an issue if imaging thicker objects as overlapping PSFs will get in the way of focussed points if they’re in front or behind them
What is a confocal microscope?
- uses a confocal pinhole to block out of focus light from reaching the detector
- only focussed light is let in
- varying the diameter of the pinhole allows thinner or thicker slices to be taken of the sample
What is the difference between confocal and widefield microscopy?
Without the optional sectioning of the confocal pinhole, it becomes a wide field microscope which captures out of focus light with focused light - which causes high background
What is green fluorescent protein (GFP)?
- discovered in jellyfish, 1962
- one of the most widely used tools in life sciences as they enable the majority of imaging done
- first expressed in living e.coli, 1994
- further work modified the protein to produce different colours
- development include bio sensors, eg calcium and pH indicators
What microscopy is used at St George’s?
- Confocal microscopy Nikon A1R = sample is illuminated by a scanned laser point, image is generated by high sensitivity photon directors
- Widefield microscopy Nikon Ni E = sample is illumined by a metal halide lamp/white light and image is generated by a camera
- Brightfield scanner Hamamatsu RS2.0 = sample is illuminated by a white light and image is generated by a camera and stitched together
