TB4 - visualising molecules and processes in cells

What are the main fundamental problems with classical biochemistry and a geneticist approach?

they do not provide spatial or temporal information (so you dont see where something is in the cell, or when it is active)

How is microscopy better than classical biochemistry and a geneticist approaches?

Microscopy lets us visualise molecules in their cellular context, in space, in situ

How can microscopy be advanced with labelling?

• We can use labels etc to label specific components - this allows us to get spatial information

• This gives us a larger view of a system, which is an important complement to biochemical and genetic analysis

How does microscopy add time?

We can look at the dynamics of different processes - this gives us a larger view of the system

What is the scale of microscopy?

• Microscopy operates from 100um → 1/10 um

  • Covers a large range of size scales

• Only at the very molecular (sub nanometer) is where electron microscopy is used

• We can also enter the nanometer range with advanced light microscopy

What can you do with a microscope?

• Magnify things to visualise more detail

What do new, advanced microscopes have?

Now we have much more advanced microscopes - detectors can now be photon detectors, cameras, may not even have an ocular → all are built around an objective (the main imaging lens) → various different ways and sizes

What are the components of a digital microscopy system?

• Light source → Laser/LED

• Detector → CCD/sCMOS camera/photomultiplier tube (photon sensor)

• Control electronics

• Computer

Describe the light path of a transmission research microscope

• Light is sent through a condenser and focused into the image plane

• The sample transmits, blocks or deflects light - this is dependent on its structure

• Light that comes through is captured by the objective → this is then mirrored into the eyepiece or diverted to a camera via a mirror or switch

Describe the light path of a fluorescence research microscope

• Uses a specific excitation light source (laser or LED)

• Light is reflected by a dichroic beam splitter onto the specimen, exciting it

• Fluorophores are excited, and they emit at a longer wavelength

• Emitted light then passes back through the objective and beam splitter.

• The emitted light is then directed to a camera system or eye piece

Describe how images that we produce are not images in microscopy

• Images that we generate are not images - they are data (a matrix of numbers, with each value being the intensity at a pixel)

• This can be extended to 3D and time

• We digitilise spatial intensity information into the matrices, this allows us to extract quantitative information → spatial distributions, dynamics and intensities

What can we now do with microscopes?

• Image the spatial distribution of specific molecules inside cells

• Measure concentration, molecular weight and diffusion constant of molecules inside cells

• Determine distances between molecules and how strongly they bind

• Visualise and measure single molecules moving within cells → how they dynamically behave

→ this is in vivo biochemistry

What are the advantages of imaging?

• High specificity → labelling of fluorophores can be targeted to specifc molecules

• High sensitivity → visualise down to a single molecule

• Non invasive → dont need to destroy the cells (can do in situ)

• Multi dimensional data → rich data - spatial information, wavelengths (we can identify different targets) and other properties such as time

• Relative localisation and dynamics → we can see where things are relative to eachother, and how they move

• Single cell to high throughput → cell to cell variability → normal biochemistry gives averages, imaging allows us to assay what is happening in single cels and across all cels

Describe the dual nature of light

Light has a dual nature - particle (photons which are energy quanta) or as rays (waves with interference and polarisation) → in microscopy we think of it as waves

Describe the electromagnetic wave properties of light

electric and magnetic field perpendicular to eachother and to the direction of propagation

Describe the wavelength of light

Wavelength - difference between two maxima (measured in nm)

Describe the frequency of light waves

the number of wave cycles per second v = 1/period c = λv

Describe the amplitude of light waves

strength/height of wave

Describe the phase of light waves

position of a point in the wave cycle - where the maxima are along the direction of propagation

What do normal light sources produce?

A mix of many waves of different wavelengths, phases and directions

What are the different types of chromatic light?

• monochromatic light

• polychromatic light

What is monochromatic light?

All waves have the same wavelength

What is polychromatic light?

The waves have multiple different wavelengths → this is like white light

What is polarised light?

Amplitude of the electric and magnetic field are in the same orientiation → the electric field vectors oscillate in a single plane (or a well-defined pattern)

What is non-polarised light?

the electric field vectors oscillate in many random directions

What is coherent light?

• All maxima are at the same position → position of the phase is the same → waves have a fixed phase relationship, their maxima ae aligned in a stable way

Give an example of coherent light

laser light

What is non-coherent light?

Maxima are out of phase → the maxima and minima are not aligned

What is collimated light?

All the light waves travel in one direction (they are parrallel) → laser

What is divergent light?

All the light rays travel in different directions from a source

Describe the relationship between wavelengths and photons

Shorter wavelengths have a higher photon energy than lower ones

What are the different ways light can interact with matter?

• transmission

• reflection

• refraction

• diffraction

• absorption

• scattering

What is transmission of light?

• light enters a material and passes through

• the speed of light in the material depends on the refractive index

What is reflection of light?

light hits a boundary with a medium of a higher refractive index and bounces back - higher refractive indexes occur when the medium is denser → this angle of reflection will be the same as the incoming ray

What is refraction of light?

light changes direction when passing between different media with different refractive indexes → this kinks the path of light - this is used in lenses and focusing

What is diffraction of light?

when light encounters an object or moves through a small hole, it bends and spreads from a point

What is absorption of light?

the light energy is taken up by the material, and not transmitted or reflected

What is the scattering of light?

Light is re-emmited / reflected in many different directions

What is refraction?

• When light enters matter (with a higher density), at the interface, the speed of light changes

• This causes the light ray to bend - this is a kink in the direction of its path

• The magnitude of this change in path depends on the difference in the refractive index between the two media

What is snells law?

Snells Law - n - refractive index, 0 = angles to the normal in each medium

Describe the basics of how lenses work

• Light interacts with matter - this can alter the WL, polarisation, speed and pat of the light → this occurs because electrons in the material interact with the vibrations of the electromagnetic field

• In a lens, refraction at curved surfaces causes light rays to converge or diverge

• Lenses redirect light by controlling refraction

• Entering a medium slows light, and the curvature of the surfaces in a lens shapes how much these rays bend

what do converging lenses do?

focus parallel rays to a focal point

what do diverging lenses do?

spread rays out

What do prisms do?

• Prism: different wavelengths are refracted to different degrees → this splits up the components of polychromatic light into their different components

  • this is dispersion

What is the basic anatomy of a compound microscope?

• 2 lens components - objective and ocular

What does the objective lens do in a compound microscope?

objective (near the specimen) - does most of the magnification and image formation

What does the objective lens do in a compound microscope?

ocular - magnifies the intermediate image for the eye

Describe how a compound microscope works - the light path

• Light from an object is brought into focus at an image plane

• The eyepiece re-images the intermediate image, so rays enter the eye correctly and focus on the retina

  • the planes along the light path are conjugate planes

    • when the focus knob is adjusted, you move the position of the specimen relative to the planes, so light sharply focuses on the retina/camera

Describe how the objective and eyepiece form an image

• Convex (converging) lens focuses parrallel incoming rays into a point in its focal plane

• In a microscope the objective lens collects rays from a real point in the specimen, converging them to form a real, magnified intermediate image in an image plane in the microscope

• The eyepeice acts like a magnifying glass - taking the intermediate image and producing a virtaul image at infinity → the eye lens focuses this onto the retina

• The retina acts as a conjugate plane - light from the specimen is mapped to a point on the retina

Why does a point source of light in the specimen not appear as a perfect point in the image?

A point source of light in the specimen will not appear as a perfect point in the image → becuase of diffraction, the microscope forms a 3D pattern called the point spread function

How does the PSF look in 2D and 3D?

• in 2D this looks like a bright central spot (an airy disk) wit concentric rings

• in 3D, this looks like an elongated blob/cone of light

How do we find the diameter of an airy disk?

Diameter of the airy disk is measured as the full width at half maximum of the central peak

What does the PSF reflect?

The fundamental limit of resolution

Why does the PSF look different in an axial orientation?

• Out of focus light from above and below the focal plane can reduce contrast and resolution, making structures look blurred

• If you look from the size, the point is elongated along the optical axis, this is because the PSF is a 3D diffraction pattern - airy rings extend above and below the focal plane → this is why axial resolution is worse than lateral

What methods can we use to deal with out of focus light?

• Confocal microscopy

• Computational deconvulation

How does confocal microscopy work to deal with out of focus light?

this uses a pinhole to block out of focus light

How does computation deconvolution deal with out of focus light/

this uses the knowledge of the PSF to reassign blurred light back to its likley point of origin

What does the size of the PST depend on?

• The size of the PST (and so resolution) depends on the wavelength of light and the numerical apeture of the objective

  • a higher numerical apeture gives us better resolution

What is magnification?

How big the image appears

What is resolution?

smallest distance betwen two points that we can distinguish them as separable

How does increasing magnification relate to resolution?

Increasing the magnification beyond the resolution limit gives bigger pixels, but doesnt provide new detail

Describe the Rayleigh resolution limit

If we consider 2 point sources → if they are far apart, PSFs are clearly separated, producing 2 distinct peaks → as they move closer, PSFs overlap → at some point, the combined intensity profile no longer has a clear minima between them (as the maxima converge)- this is when they become unresolved

What is the Rayleigh criterion?

• Defines the minimum distance at which two points are just resolvable

• Condition where the central maximum of one PSF coincides the the first minimum of the other

• Distance depends on the wavelength and numerical apeture of the objective

• The minimum resolvable separation between the points is the radius of the airy disc. The equation is given by 0.61 * λ / NA. 

How are objective lenses complex?

• have many different lens elements inside them → engineered and expensive

• allow correction of different optical aberrations

What is the function of the objective lens?

allow correction of different optical aberrations

What are the numerical apertures and how do they relate to resolution?

• Higher NA (measures how much light the objective can collect) → this means they have a better resolution

How is resolution dependent on contrast?

• we need a good singa; to nouse ratio - this is the the tatio of the highest signals to the background

  • if the background is high/signal is weak, even high resolution cant be fully exploited

What is the SNR?

Signal to noise ratio - the ratio of the highest signals to the background

What techniques can be used tp increase contrast?

• Brightfield

• Darkfield

• Phase contrast

• Differential interference contrast

• Fluorescence widefiled

• Confocal

• Two-photon

Describe brightfieqld microscopy

• commonly used

• produces image on bright bacground

• use staining (cytological/hisrological) - this allow colouring of different structures differently

describe dark field microscopy?

• ncreases contrast without staining

• produces a bright image on a darker background

• good for live specimens

• only scattered light from the specimen enters the objective

describe phase contrast

• uses refraction and interference (phase shifts) caused by structures in the specimen to create high contrast and resolution images

  • light passes through different parts of the specimen and is delayed, this depends on the thickness and refractive index

• no staining

• useful for live specimens with little intrinsic contrast

Describe differential interference contrast

• interference patterns are used to enhance contrast between different features of a specimen

• high contrast of living organisms with 3d appearance

• useful for seeing structures in live, unstained specimens to see fine details

Describe fluorescence widefield

• fluorescent stains are used to produce an image

• uses in pathogen identification, species, distinguishing living from dead, determining location of specific molecules in a cell

• generate a darker background ad just label the object of interest

• creates contrast and specificity

• Can label different components of the cell and then produce a digital overlay

Describe confocal

• lasers scan multiple z-planes successively → produces multiple two-dimensional high res images at various depths

• construct 2-d into 3-d images → useful for examining thick specimens

Describe two-photon

scanning technique, fluorochromes and long wavelength light are used to penetrate deep into thick specimens

What are the two optical components in phase contrast?

annulus and phase plate

Describe the role of the annulus in phase contrast

produces a ring of light

Describe the role of the phase plate in phase contrast

this alters the phase and amplitude of the undiffracted light

Describe the principle behind phase contrast

• Light that mainly passes through the sample is diffracted and phase shifted

• Light that bypasses the specimen goes through the phase plate - his is attenuated and phase shifted

→ these components interfere, converting phase differences into intensity difference

• This results in a better visibility of cell shapes and internal structures, without the requirement for staining

What are the requirements of phase contrast?

well aligned - elements need to be in the correct conjugate planes and perfectly centred

What is fluorescence?

Physical property of fluorophores → they absorb light at one wavelength and re-emit at another (longer wavelength)

What are the emitted photons in fluorescence?

Emitted photons are of lower energy than those absorbed

Describe how conjugated ring systems can act as fluorophores

• light excites electrons to a higher electronic state

• some energy is lost by internal conversion

• the electrons then drop back to ground state, emitting a photon at a shifted wavelength

What sort of time scale is fluorescence?

Nano-second scale process

Describe the stokes shift?

• Difference between excitation maximum and emission maximum of a fluorophore

• Allows excitation and emission light to be separated using filter

Describe fluorescence widefield microscópy

• Uses fluorescent stains or labels to produce images

• Applications: pathogen identification/species detection, distinguishing live vs dead cells, determining location of specific molecules in the cell

• Background is mostly dark, only labelled objects light up → this allows high contrast and specificity

• Different celular components can be labelled with different fluorophores

• Digital overlays can be used to show multiple colours or targets

What are the applications of fluorescence wide field microscopy?

pathogen identification/species detection, distinguishing live vs dead cells, determining location of specific molecules in the cell

What is the Rayleigh criterion? (equation only)

he minimum resolvable separation between the points is the radius of the airy disc. The equation is given by 0.61 * λ / NA. 

Why is fluorescence imaging often used?

• important becuase it provides us with a higher signal to background ratio

  • this gives us contrast

What is autofluorescence?

A molecule being naturally fluorescent

Give an example of molecules with autofluorescence

Chlorophyll, collagen, elastin

What are auto fluorescent samples excited with?

typically excited with UV light (relatively low, short WLs)

When is autofluorescence useful?

Autofluorescence can be useful to determine the morphology of a tissue/organism

Why can autofluorescence in samples be an issue?

this increases background - this can obscure specimen detail

Where can autofluorescence come from?

one source is phenol red in tissue culture medium (used as an indicator)

What are the 3 classes of fluorophore used in microscopy?

• Organic dyes

• Green fluorescent protein

• Quantum dots

Describe organic dyes used as fluorophores

• consist of multi-ring structures with electron conjugated ring systems

  • antennas of photons

• bright and photostable

• small - 1nm

Describe GFP used as fluorophores

• genetically tag the protein/molecule to a fluorescent protein such as GFP → 3nm

• good for live cell imaging

Describe quantum dots used as fluorophores

• rystals with a certain property that they fluoresce at excitation with UV light

• excited by UV and emit at longer WLs

• doesnt bleach → photostable