Lecture 1 - Light microscope Flashcards
Why can we see things?
Photoreceptor cells in retina activated by photons of light with certain wavelengths
Lens focuses light emitted or bounced off an object
Why are some things not visible
Not enough photons
Wrong kind of photons (outside of visible spectrum
Objects too small of photoreceptors to resolve
Objects dont interact with or emit visible light
Objects interact with light the same as surrounding medium
Materials or lenses bend light to cloak object
If an object is not visible due to not enough photons, what can be done to make it visible?
Use a condenser
If an object is not visible due to the wrong kind of photons, what can be done to make it visible?
Use a detector to detect wavelengths outside of visible spectrum
If an object is not visible due to objects being too small to resolve, what can be done to make it visible?
use a compound lens
If an object is not visible due to an object not interacting with light, what can be done to make it visible?
Use optics
If an object is not visible due to objects interacting with light the same as the surrounding medium, what can be done to make it visible?
Use stains or labels
What do convex lenses do
Refract or bend light to increase the angle which spreads out light making an object larger (slide 9)
How does a compound microscope work?
Light from specimen hit detector
Eyepiece (ocular) lens magnifies image from objective
Condenser lens focuses incoming light
Virtual image that appears to observer as enlarged specimen
(slide 12)
How is magnification on a microscope calculated
Eyepiece (ocular) x objective
What is resolution
Smallest distance at which 2 objects can clearly be distinguished from one object
What determines resolution
How much light is lost between specimen and lenses
How much light is gathered by lenses
Wavelength used for illumination
What is a refractive index
Mismatch between elements resulting in loss of signal between sample, lenses and detector
How can refraction be minimised
Properties of lenses used in air can be adjusted to compensate
Special oil which is the same refractive index as glass to prevent loss from refraction
What is angular aperture of a lens
The angular aperture of a lens is the angular size of the lens aperture as seen from the focal point
(slide 20)
What affect does angular aperture have on microscopy
Higher number = more light = more resolution
What is numerical aperture
number that describes light gathering ability and resolving power of an objective lens
What wavelengths have the highest energy
Shorter wavelengths (also equals higher resolution
What is the resolution for light microscopes
200nm
500nm without super-res techniques
What is the resolution for electron microscopes
2nm
How are specimen on a microscope slide visible
Structures are visible because they interact with light differently from surrounding medium
Many things in cells hard to see without stains or labels
How can you boost resolution
Phase-contrast boost resulting from differences in refracting between cells and cellular structures
DIC uses polarised light to boost contrast from diffraction of light without the sample
What does hematoxylin stain
Nuclei
What does Eosin stain
Eosin
What does Oil Red O stain
Fat droplets
What does Alizann red stain
Bone
What does alcian blue stain
cartilage
What does light blue stain
Collagen fibres
What are fluorophores
Molecules that emit energy in the form of photons of a long wavelength, when hit with specific shorter photons
(slide 30)
(emit longer wavelength than they absorb)
What is does quinine do
Excited by UV light, emits blue light (example fluorophore) (slide 29)
Anti-malarial
Describe the mechanics of fluorescence
Photon of a particular wavelength, carrying a particular amount of energy excite electrons in a fluorophore
As electrons go back to ground state energy is released as photons
Less energy is released as light than absorbed, so emitted photons have longer wavelengths
What are some commonly used fluorescent labels
DAPI and Hoescht - molecules bind to DNA - blue
Fluorophore-conjugated molecule - any colour eg. Phalloidin
Fluorescent Protein (GFP) used to incorporate into plasmids and genomes (2008 Nobel prize)
(can localise to cells or organelles)
What can Fluorescent proteins be used to study
Monitor gene expression
How does Fluorescent microscopy work
Light passes through objective lens to and from the sample
Goes through excitation and emission filters
Dichroic (two colour) mirror - shorter wavelengths reflected onto the sample, longer wavelengths pass through detector
(slide 32)
How can you get better resolution of fluorescent microscope
Reduce out of focus fluorophore excitation
Reduce out-of-focus emitted light collection
How is a confocal fl. microscope better (2-photon)
Removes out of focus light
improves res in z by only exciting fluorophore where beams of light cross paths
What is digital deconvolution
Improves res using information about light defraxtion
What are antibodies
Defences against pathogens that bind tightly to target molecules through variable regions
What is an antigen
What an antibody binds to (on the outside of a pathogen usually)
What is an epitope
Part of an antigen that antibody recognises
What are monoclonal antibodies
bind to one epitope
What are polyclonal antibodies
Mix of antibodies that recognise epitopes on same antigen (slide 42)
