light microscopy

why are things visible?

• source of illumination emits photons that act on objects

• eye lense focuses light emitted by / bouncing off from objects

• photoreceptor cells in retina activated by photons of light with certian wavelengths

6 ways light reacts with matter

• transmission

• reflection

• refraction

• diffraction

• absorption

• scattering

why are things not visible? (6)

• not enough photons - eg turn lights off

• wrong kind of photons - only ultraviolet to infared

• objects too small for our array of photoreceptors to resolve -

• objects do not interact with or emit visible light

• objects interact with light the same as surrounding material

• materials or lenses bend light to cloak the object

as an object gets further away from the detector, the angle between the object’s top and bottom…

decreases

convex lenses

• refract / bend light, increasing the angle of the top and bottom of the object

• spreads out the light hitting detector, making object appear larger

magnifying lens

refract light coming from an object

object appears larger and further way

compound lens parts and roles (5)

• light from specimen hits detector

• eyepiece (ocular) lens magnifies the image from the objective lens

• objective lens magnifies light from specimen

• condenser lens focuses light onto the specimen

• virtual image appears to observer as enlarged specimen

magnification equation

product of objective lens x ocular lens

‘not enough photons” fix on microscope

use condenser lens to focus light on sample

“wrong kind of photon” fix on microscope

use detectors or cameras sensitive to other wavelengths

“object too small for array of photoreceptors to resolve” fix on microscope

use compound lenses to magnify sample

resolution

smallest distance at which two objects can be distinguished from one object

what determines resolution? (3)

• how much light is lost between specimen and lenses

• how much light is gathered by lenses

• wavelength used for iillumination

smaller / shorter wavelength…

higher resolution

mis match between elements / refractive indexes in light path result in..

loss of signal between sample, lenses and detector

match refractive index =

higher resolution

refraction at interface between glass and air =

less light enters objective lens

immersion oil role

• refractive index = glass

• prevents loss from refraction and gathers all light

angular aperture of the objective lens

• how much of the light is collected by the objective lens

• cone of light coming from sample

• higher angle means more light gathered

numerical aperture

0 number that describes the light gathering ability and resolving power of an objective lens

light is information

• more information, better resolution so:

• limited refraction between specimens and lens

• shorter wavelength of illumination (more energy)

limit of resolutions between microscopes

• ~200nm for visible light microscopes (ideal, ~500nm without super-resolution techniques)

• ~2 nm for electron microscopes

brightfield or transmitted light

• some structures within cells are visible because they interact with light differently from surrounding medium (water, air)

• many things in cells are hard to see without stains or labels

Phase contrast

boosts contrast resulting from differences in refractivity between cells and cellular structures

DIC (differential interference contrast)

uses polarised light to boost contrast from diffraction of light within the sample

2 typs of enhanced contrast in label-free microscopy

• dic (differential interference contrast)

• phase-contrast

“objects interact with light the same as surrounding medium`’ fix on microscope

use optics that boost contrast between components with different material properties

“objects do not interact with or emit any visible light”

• use stains or labels that can be detected by eyes or camera

fluorophores

molecules that emit energy in the form of photons of a longer wavelength when hit with specific shorter wavelength photons.

• e.g. Ub to blue, green to red, red to infared

attach fluorescent molecules to molecules or proteins that label cellular structures

quinine

fluorescent molecule, excited by UV light, emitting blue light

mechanism of fluorescence (3)

• photons of a particular wavelength carrying a particular amount of energy excite the electrons in the fluorophore

• as electrons go back to ground state (unexcited) , energy is released as emitted photon

• less energy is released as light than as absorbed, so emitted photons have longer wavelengths

fluorescence microscopy

• light passes through the objective lens to and from sample

• goes through excitation and emission filters

• dichroic (2-colour) mirror: shorter wavelengths reflected onto sample, longer wavelengths pass through detector

A visible light

B excitation filter
C ocular lens
D emission filter
E dichroic mirror
F objective lens

Commonly used fluorescent labels

• DAPI anf Hoechst - small molecules that bind to DNA, UV/ blue

• Fluorophore-conjugated molecules, e.g. phalloidin, from toxic mushrooms, binds to F-actin

• fluorescent molecules that localises to cells or organelles

GFP - green fluorescent protein (2)

• derived from jellyfish A. victoria

• encodes fluorescent protein that can be incorporated into plasmids and genomes

how to use a fluorescent protein sequence (2)

• FP sequence at beginning or end of another protein to make a hybrid tagged protein

• FP gene after promoter to monitor gene expression. green = promoter active

how to increase resolution in fluorescence microscopy (2)

• reduce out-of-focus fluorophore excitation

• reduce out-of-focus emitted light collection

confocal fluorescence microscopy (3)

• improves resolution by removing out of focus light

• excitation source = laser

• pinholes focus light in x,y,z direction onto specimen

two-photon confocal microscopy (2)

• improves resolution in z by only exciting fluorophore where beams of light / photons cross paths

• 2 longer photon beams energy adds up to that of one shorter photon

digital deconvolution

• mathematically improve images using information about the diffraction of light, removing artefacts

super resolution microscopy (4)

• technical and mathematical methods

• resolution better than the diffraction limit

• 200 nm in x,y

• 500 in z

epitope

the part of the antigen that the antibody recognises

monoclonal antibodies

bind to one epitope

polyclonal antibodies

mixture of antibodies that recognise epitopes on the same antigen

antibodies

defences against pathogens

bind tightly to target molecules through their variable regions

direct and indirect immuno-labelling

• direct: primary Ab binds to antigen epitope, with label attached

• indirect: primary Ab doesn’t have label, secondary AB binds to first AB.

• 2nd Ab’s antigen is the primary’s constant region

immunohistochemistry

antibou + enzyme → colour change in sample

immunofluorescence

multiplexed fluorescent labelling

• combining multiple dyes / antibodies

• multiple fluorescent labels in one image

• labelled in sequence, then put together in composite image