Microscopy Flashcards
3 things microscope must do:
- Produce a magnifies image of the specimen
- Separate or resolve the details in the image
- Render the details visible to the eye
What does a microscope do?
Enhances ability to see objects by making them bigger
Magnification
- process of enlarging the physical appearance of something
- achieved by placing a biconvex lens into the light path
- light from object passes through lens and is bent (refracted) towards the eye
- objects seem bigger than they really are
Magnification that has no increase in detail is called?
Empty magnification
What is magnification a combination of?
All the lenses in the light path
Resolution
Shortens the distance between 2 separate pints in the microscopes field of view that can still be distinguished as distinct entities
What determines microscope resolution?
Representation of point spread function of single sources of light
Microscope resolution of airy disks
Resolvable: 2 pings separated by more than the radii of airy disks
Not resolvable: 2 points separate by less than the radii of airy disks
Wave length
Shorter lamda = better resolution
Numerical aperture
Higher NA = better resolution
Resolution equation
R = 0.61 lamda/ NA
What is the resolving power of a light microscope under optimal conditions?
200 nm
Increasing magnification means decrease of…?
FOV (field of view)
2 types of contrast
- Phase contrast
2. Differential interference contrast (DIC)
Phase contrast
- changes in refractive index causing light rays to shift
- causes a halo around cell
Differential interference contrast (DIC)
- light splits into 2 waves so light passes through different parts of sample
- gives a 3D Pseudo effect
- light and dark regions
Benefits of using DIC and Phase contrast
- method used living cells and they can be examined at natural state
- no harm, staining, or killing is necessary
DIC goods and bads
Goods
- gives 3D image, more detail
- important technique for imaging thick plants and animal tissues
Bads
- more expensive than phase contrast
- some plastics not suitable for DIC imaging
Staining
Strain different cellular components different colors to allow for identification of parts
Fluorescence Microscopy
Emission of light from a compound following adsorption of light of shorter wavelengths
- fluorescent proteins can be used to tag specific proteins in cells
Fluorescent dyes
Can be attached to wide range of antibiotics or phalliodin to allow specific structures in the cell to be visualized by microscopy
Epifluorescence microscopy (Epi)
- camera based technique
- fluorophores throughout sample are excited
- images contain interference from out of focus molecules
- good for fixed or live cell imaging and multicolor
- see structures throughout cell
2 types of confocal microscopy
- Spinning disc microscopy
2. Laser scanning confocal microscopy (LSCM)
Spinning disc microscopy
- camera based
- fast and suitable for photo sensitive samples
- imaging highly dynamic processes
Laser scanning confocal microscopy
LSCM
- point scanner
- slower
Advantages of confocal microscopy
- ability to control depth
- eliminates out of focus light using a pinhole
- optically section though 3D reconstruction
- image can be zoomed without loss of resolution
Disadvantages of confocal microscopy
- sample still illuminated causing phototoxicity
- slower than a camera
- resolution limited to 200 nm
Total internal reflection fluorescence (TIRF) microscopy
- uses camera to capture image(fast)
- reduces phototoxicity
- suitable for fixed and live cells
- showed skeleton of cell
- only see structure at adherent membrane
Example applications of TIRF
- time lapse in endocytosis
- focal adhesion dynamics
- exocytosis
Super resolution microscopy
- 2 objects seen as one in diffraction limited microscopy
- Nobel prize winning techniques
Direct stochastic optical reconstruction microscopy (dSTORM)
- uses fluorescent antibodies to label proteins
- used on dead samples
- increased resolution by x10
- randomly switching on and off causing “blinks” and nm precision
2 color dSTORM
- increased resolution allows finer detail
- proteins can be seen as distinct structures with no overlap
Advantage of dSTORM over EM
Preparation is easy and multi color 3D images can be taken
Electron Microscopy (EM)
- original super resolution microscopy
- objects as small as 1 atom can be seen
- uses a beam of electrons to illuminate sample as electrons have smaller wavelengths
- electromagnetic cools used to focus electron beam instead of glass lens
- can only be done on dead samples and preparation is complicated
What microscope has the highest resolution?
- EM
- behave it uses a beam of electrons instead of light
