Biomedical Imaging Flashcards
Describe the three parts of effective microscopy:
- Magnification (ratio of the size of the original object)
- Resolution (ability to separate two neighbouring points of information)
- Contrast (visibility against background, needed for 1. and 2., black and white is the perfect contrast)
Describe magnification:
An object can be focused generally no closer than 250mm from the eye (age-dependent)
Describe resolution:
The most important part of microscopy
Allows for resolution of two points (e.g. 2 proteins interacting inside the nucleus)
A greater NA=more resolution
Decreased wavelength=smaller distance=larger resolution
Theoretical resolutions - 200nm (light microscope) and 0.2nm (electron microscope)
Defined Abbe’s equation:
dmin=1.22wavelength/2NA
dmin=resolution (minimal distance to resolve)
List the components of the standard microscope:
3 lenses: Eyepiece Objective Condensor And light source
Describe numerical aperture (NA):
The ability to distinguish two observes close together
Directly relates to the resolving power
The higher the NA, the wider the angle (cone) of light, and the higher the resolution
However, the higher the NA, the volume of the sample in focus is smaller (payoff)
NA=n sin u (n=refractive index, u=angle)
Describe the refractive index and its importance in microscopy:
Refers to how light travels or refracted through a material
Refractive index matching needs to occur so light isn’t lost
Describe the speed of light in microscopy:
The electrical interaction between light and the charges within the specimen determines its speed
List the basic types of microscopy:
Brightfield (requires some degree of staining)
Don't need stains: Phase contrast Darkfield Differential interference Contrast DIC Polarised Hoffmann
Fluorescence
Brightfield, phase and fluorescence micropscopy are everyday types; the others are more specialised.
Describe brightfield microscopy:
Brightfield illumination - no contrast
Details occur via phase differences and by staining of components
Stained region absorbs light, reducing the amplitude of the signal
Edge effects (diffraction, refraction, reflection) produce contrast and detail
Describe Koehler illumination in light microscopy:
Spreads light just enough to fill field of view (overcome shadowing on edge of specimen)
Ensures illumination is centred and even
Matches NA of condensor with objective to achieve maximal
Improper set-up results in shadows, artifacts and incorrect colours
Prerequisite for contrast-enhancing methods
Describe phase contrast microscopy:
Used in tissue culture
Used to image transparent unstained specimens (living cells)
Unstained regions with higher refractive indexes, slow movement of light producing a shift in phase that results in scattering of light
Describe differential interference microscopy (DIC):
Used for thicker specimens (>10um)
Unstained specimens
3D effect
Describe fluorescence microscopy:
Uses light as a particle
Excitation and emission depend on the probe as to which colours occur
Fluorescence is the absorption of light which results in the emission (release) of light of a different wavelength (usually longer)
Some tissues are naturally fluorescent (e.g. collagen)
Describe the emission and excitation spectra in fluorescence microscopy:
Represent the wavelength at which the probe is most excieted (although it can be excited along the spectrum with a lower intensity)
Short wavelengths in and longer wavelengths out
Describe the excitation –> emission wavelengths colours:
UV –> Blue
Blue –> Green
Green –> Red
Red –> Far red
Describe the components of a fluorescent microscope:
Eyepiece
Filter block
Objective
Epi-illumination light source (LED, mercury bulb)
The objective acts as a condensor
Describe dichroic filters in fluorescence microscopy:
Have a special filter to absorb certain wavelengths of light
Filters ca be either long pass or band pass (important to know for double labelling)
Describe the advantage of widefields in fluorescence microscopy:
Collect light emitted from the entire depth of specimen, acquisition is fast
Provide a highly flexible system for live cell imaging, at low cost
BUT photobleaching occurs so not practical
Describe the the problems with fluorescence microscopy:
In thicker specimens, you get out-of-focus light (above and below focal plane)
Full illumination of specimen leads to photobleaching
Describe confocal microscopy:
Removes out of focus light from above and below the focal plane (remove blur)
Scanning back and forth to produce an image from slices made up of layers of pixels
Describe the laser scanning type of confocal microscopy:
Scans laser spot across specimen
Uses a pinhole (to achieve optical slicing)
Good for live cell imaging, expensive, less photobleaching?
Describe the multi-photon type of confocal microscopy:
Far-red pulsed laser - out of focus light is removed by laser intensity as only enough is used to excite the focal plane (no pinhole)
Live animal imaging and deep tissue imaging (good penetration)
Pollen grains used to calibrate the machine
Describe optical sectioning (3D information):
‘z series’ axial resolution is less
Extended focus - images are stacked on top of each other
Series of projections from different angles - can be rotated and spun around
3D stereo images - need 3D glasses
List the advantages of confocal microscopy:
Optical sectioning 3D reconstruction Improved resolution Use of specific wavelengths (multi-labelling) Very high sensitivity Digital images Computer controlled systems Advanced imaging techniques
List the applications of confocal microscopy (what it can be used to image):
Immunolabelling - single and multiple labels Protein trafficking - fluorescent proteins Organelle identification Live cell imagine Subcellular function Ion concentrations Molecular mobility - FRAP Protein-protein interaction - FRET e.g. intracellular calcium
Name the limitations of confocal microscopy:
Laser light penetration is limited
Laser light can be damaging
Describe the two types of electron microscopy:
Transmission (TEM) - analogous to brightfield light microscopy, electron tomography
Scanning (SEM) - standard, environmental SEM
Allows for the study of intracellular structure and pore sizes (e.g. in pharmacy, less resolution)
List the applications of TEM:
Internal cellular structures (organelles)
Micro-organisms
Viruses, phages, DNA
Protein structure, membrane interfaces
Macromolecular organisation
Energy dispersive x-ray detector - detect x-rays from specimen
Describe the uses of:
Light microscopy
TEM
SEM
Surface morphology/sections
Sections,small particles, thin membranes
Surface morphology
Describe the illumination method of:
Light microscopy
TEM
SEM
Visible light
High speed electrons
High speed electrons
Describe the resolution of:
Light microscopy
TEM
SEM
200nm
0.2nm
3-6nm
Describe the magnification of:
Light microscopy
TEM
SEM
10-1000x
500-500,000x
20-150,000x
Describe the depth of field (and NA) of:
Light microscopy
TEM
SEM
- 002-0.05nm (NA 1.5)
- 004-0.006nm (NA 10-3)
- 003-1mm
Describe the lens of:
Light microscopy
TEM
SEM
Glass
Electromagnetic
Electromagnetic
Describe the image formation of:
Light microscopy
TEM
SEM
On eye by lenses
On phosphorescent plate by lenses
On cathode tube by scanning device
