Light microscopy Flashcards
why are some things not visible?
Not enough photons
Photons outside the visible spectrum for our photoreceptors
Objects too small to resolve
Objects do not interact with or emit visible light
Objects and surrounding material interaction with light the same way (same refractive index)
Objects bend light in a way that hides them
how does a convex lens cause magnification?
refract light in a way that increases the angle between the top and bottom of the object, light spreads out from focal point, making object appear larger
what is magnification and how is it calculated?
size of object : size of image
objective lens x ocular lens
how do microscopes solve the problem of too little photos to view a sample
use of condenser lens to focus light onto the sample
solution to wrong kind of photons
use detectors or cameras sensitive to other wavelengths
solution to objects being too small for array of photoreceptors to resolve
compound lenses to magnify the sample
resolution
smallest distance at which two objects can be distinguished from one object
what determines resolution?
how much:
light is lost between specimen and lenses
light is gathered by lenses
wavelength used for illumination
refractive index and resolution relationship
match refractive index to give a higher resolution
refraction at interface between glass and air = less light enters objective lens
what is meant by the angular aperture of the objective lens
how much of the light is collected by the objective lens
higher angle = more light gathered
relationship between wavelength and resolution
shorter wavelength = higher resolution
limit of resolution for light vs electron microscopes
around 200nm vs 2nm
phase contrast
boosts contrast resulting from differences in refractivity between cells and cellular structures
DIC
uses polarised light to boost contrast from diffraction of light within the sample
solution to objects not interacting with visible light
stains or labels
fluorophores
molecules that emit energy in the form of photons of a longer wavelength when hit with specific shorter wavelength photons
eg quinine which is excited by UV light and emits blue light
mechanism of fluorescence
photons of a particular wavelength carrying a particular amount of energy excite electrons in the fluorophore. as electrons go back to ground state, energy is released as emitted photon. less energy is released as light than was absorbed so emitted photons have longer wavelengths
fluorescence microscopy
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 to detector
DAPI and Hoechst
small molecules that bind to DNA, fluorescent labels
emit fluorescence in UV/blue range
phalloidin
binds F actin
fluorophore-conjugated molecules
can be any colour
eg phalloidin
GFP
green fluorescent protein
derived from jellyfish
encodes fluorescent protein that can be incorporated into plasmids and genomes
hybrid tagged protein
adding fluorescent protein sequence at beginning or end of another protein
how gene expression can be monitored
adding FP gene after a promoter
when it is active, the fluorescent reporter fluoresces green
how to improve resolution in fluorescence microscopy?
reduce out-of-focus fluorophore excitation
reduce out-of-focus emitted light collection
confocal fluorescence microscopy
removes out of focus light
two photon confocal microscopy
improves resolution in z axis by only exciting fluorophores where bean of light cross paths
digital deconvolution
use of mathematical algorithms to improve images by reducing blurring effects caused by light diffraction
super resolution microscopy
higher resolution than diffraction limit
epitopes, monoclonal and polyclonal antibodies
part of antigen that the antibody recognises
monoclonal antibodies: bind one epitope
polyclonal antibodies: mixture of antibodies that recognise multiple epitopes on the same antigen
direct immuno-labelling
primary antibody with fluorescent label attached binds to antigen
indirect immuno-labelling
secondary antibody has the fluorescent label and binds to primary antibody
This allows for signal amplification as multiple secondary antibodies can bind to a single primary antibody, increasing the fluorescence intensity.
