MICROSCOPY Flashcards
What do you use upright microscopes for?
Large organisms and cells
What do you use inverted microscopes for?
Mostly single cells
- looking at a dish with thin layers
How do you find the magnification of modern microscopes?
Multiply the magnification of the ocular ( lens) by the magnification of the eyepiece
What is Snell’s Law?
The angle of reflection is equal to the angle of incidence (regardless of the surface material)
What is the difference between the refracted beam and reflected beam?
Refracted beam is the beam that is transmitted and bent when passing from one medium to another. Reflected is the beam that bounces back
How does the refracting beam enable the sample to emit light?
The refracted beam causes a dispersion of colours. The colours will heat the sample which will emit the light and highlight the sample/ specimen.
What is the refractive index?
How fast the light propagates through
What does the refractive index have to be to produce a perfect image compared to a blurry image?
The refractive index has to be the same throughout the apparatus and the medium/sample for the image being produced to not turn out blurry.
What is the numerical aperture (NA)?
it is a dimensionless number that characterises the range of angles over which the system can accept or emit light
- cone of light is wide or narrow dependent on the numerical aperture
How is resolving power related to numerical aperture (NA)?
Directly related
- the higher the NA the greater the resolution
How do you work out NA?
NA = n sin y n= lowest refractive index between the object and first objective element y= 1/2 the angular aperture of the objective
Define resolution.
Limit for the smallest resolvable distance between 2 points
How do you work out the smallest resolvable unit (d)?
d= wavelength of light / 2NA
What NA will you see 2 dots as 1 dot?
2NA
Define a Point Spread Function (PSF).
Describes 3D light distribution in an image of a point source
How does PSF relate to resolution?
If you have a v good clear PSF - you are at the highest resolution. If it is large, then it is a low resolution. The lower wavelength, better resolution.
What does PSF dependent on?
Numerical Aperture and the wavelength of fluorescent emission
What does bright field illumination show?
Different brightness intensities
What does phase contrast show?
Structural details emerge by staining of components
- black/white/greyscale contrast
- can see shape
What does DIC show?
Edge effects producing contrast and details (doesn’t need staining)
What does Fluorescent microscopy show?
Can identify cells and sub-microscopic cellular components
- add dyes/ fluorescent proteins or tracers that will emit light
- need to label the sample
How do you choose fluorophores?
- Use bright, stable fluorophores
- Shorter wavelength gives you better resolution
- Longer wavelength gives you less photo-toxicity/photobleaching
- Minimise the spectral overlap
What is the difference between Widefield vs Confocal vs TIRF Microscopy?
Widefield is the normal usual one
Confocal has a slicing effect used for more detail
TIRF is more used for cell movement and trafficking
How does fluoerescence microscopy work?
- sample gets excited by lights
- lights get de-fracted
- light is bent
- captured
What are some Pro’s of fluorescence microscopes?
- uses light sources of much lower power
- cheaper than confocal systems
- give high quality photographic images
What is Laser Scanning Confocal Microscopy (LCS) used for?
- localisation of proteins
- imaging multiple fluorescent stains
- 3D visualisation of stainings
- Quantification of protein expression`
What is the main difference between LCS and normal microscopy?
For LCS you select specific plane of sample and hide everything else
- only one layer
What is Total Internal Reflection Fluorescent (TIRF) microscopy?
Microscopy that you look at interface of the sample and the glass slide
How does TIRF microscopy work?
Laser will have a specific path that will bend
- creating a wave
- evanescent wave
What is an evanescent wave?
an optical phenomenon that can occur when light strikes the interface between two media of different refractive index
What are some TIRF microscopy applications?
- endocytosis / exocytosis
- dynamics of membrane-associated proteins
- protein arrangement
- focal adhesions
- growth cone migration
- receptor - ligand interactions
- single molecule behaviour
What is Single Plane Illumination Microscopy (SPIM)?
Microscope with 2 objective lenses on the sides - one is illuminating and one is capturing.
- embed sample into a glass capillary
- Rotating capillary will be present - containing our sample (360 view)
What is superresolution microscopy?
Group of microscopy techniques that allow these objects to be distinguished as separate identities
What techniques are part of Super Resolution microscopy?
STED, SIM and dSTORM
Which Super Resolution microscopy technique is used for live samples and which is used for fixed samples?
STED and SIM are used for live and fixed whereas dSTORM is used for just fixed
How does the SIM technique in Super Resolution Microscopy work?
Mixing the image with pattern grids that rotate to produce a cleaner image
What is the dStorm technique in Super resolution microscopy associated with?
Blinking events
How does the STED technique in Super Resolution Microscopy work?
De-activation of the fluorophore will allow highlighting of the particles inside the sample
How does electron microscopy work?
Beams of electrons are used to produce images.
- Wavelength of electron beam is much shorter than light therefore is a much higher resolution
How does Transmission Electron Microscopy (TEM) work?
Electrons scatter when they pass through thin sections of a specimen.
Transmitted electrons are used to produce image
Denser regions in specimen, scatter more electrons and appear darker
How does Scanning Electron Microscopy (SEM) work?
Uses electron reflected from the surface of a specimen to create image
Produces a 3D image of specimens surface features
- can avoid staining
What are some microscopy and Imaging Applications?
Live Cell Imaging Ionic Flux Determinations FRET FRAP DNA Paint Expansion Microscopy
What is FRET?
Interaction between two molecules
- One molecule can change its conformation to allow an open channel or to allow an interaction to another protein
- Energy of fluorophore will transfer energy from one protein to another to emit light
What is FRAP?
Bleach protein
- Waiting for recovery of the fluorophore over time
- Recovery due to behaviour or chemicals
What is DNA Paint?
Label (with dyes) DNA
- Image the DNA throughout time
Useful:
Can do SIM or STED microscopy
What is Expansion Microscopy?
Possibility to insert the cell into a polymer gel
- Gel will expand
- Can look at it in more detail
