Lecture 10 Microscopy Techniques Flashcards
Compound microscopes
Magnify both the objective and ocular
The eyepiece/ocular picks up light from specimen
Resolving Power
The ability of an objective to resolve two distinct objects that are very close together
Numerical aperture (NA)
- Higher the NA, the greater the resolution
- How much light is let in
Both magnification and resolution is important
Upright vs Inverted microscope
Upright is the one typically used and it observes the surface of the specimen
The inverted microscope looks at it from below with the light source on top and it can look at tissue culture or live cells
Stage micrometer
Used to calibrate the ocular micrometer
Each division is 10 um
Petroff Hausser
Counting chamber grid on the slide to count the number of cells with the microscope (does not allow to size)
Types of staining technqiues
- Simple staining: uses a single staining agent
- Generally uses alkaline dye (+ charged) that binds to (-) charges on the cell surface - Differential staining: uses multiple types of stain
Cells are fixed before applying stain
Cell staining
Use of staining agent to stain cells
Chromogen works as a dye to colour the cells for easy visualization
Light Microscopy Variations
- Dark Field: light source partially blocked off, scattering
- Phase Contrast: phase shift in wavelength of light depending on scattering and absorption of light
- Differential Interference Contrast: based on light refraction by different parts of the cell
- Confocal Scanning: eliminates out of focus light, allows for 3D images
- Fluorescence: looks at fluorescence
Types of fluorescent microscopes
- Upright
- Inverted (objective on the bottom)
- Confocal
How do fluorescent microscopes work?
- Shine laser at specific wavelength
- Excite fluorophore to higher energy state
- Fall back to ground state and emit light
Components of fluorescent microscopes (emission filter, excitation filter, etc.)
Emission filter - filter out wavelengths, except specific one and eliminate other sources of light
Excitation filter - select specific wavelength in spectrum, filter out other light
Fluorescent labelled cell (antibody, stains)
What is confocal scanning? How does it work?
3D reconstruction of specimen
Higher resolution that regular fluorescence microscope
Takes a picture every few micrometeres of the cell and combines the images to create 3D image
Sub types of electron microscopes (EM)
EM are ultra high resolution and magnification that can resolve cell structures <1 nm and 10^6 magnification
- Transmission (TEM) - electron transmitted through an ultra-thin specimen
- Scanning (SEM) - focused beam of electrons “scan” the specimen, doesn’t go through (only surface) and uses e- instead of light (only takes pics in black and white)
