Optical Imaging Flashcards
What is light?
Oscillating EM field that carries energy and can interact with matter
Visible spectrum wavelength range?
400-700nm
UV wavelength range?
100-400nm
IR wavelength range?
700nm-100um
Non-ionising range of the EM spectrum
Visible and IR (providing power levels are kept below safety limits)
What happens when visible light wave interacts with a medium?
- light slows down
light induces oscillation in the mediums electrons (polarisation field) –> creates change in phase of light field –> causes speed of light to decrease
Refractive index, n
n = speed of light in vacuum / speed of light in medium
Amount of energy in a wave
Etot = Nhv
N = #photons E=hv = min energy in a wave
Momentum of photons
p = hv/c
Molecular energy level structure
- electronic energy levels (electronic orbitals)
- vibrational and rotational modes of the nuclei
when may a molecule absorb a photon?
A molecule may absorb a photon if the energy of the photon corresponds to the difference between two of the energy levels
Boltzmann’s distribution
The distribution of energy within the energy states
For a large number of molecules, the ratio of #molecules in upper energy level to #molecules in lower energy level is given by…..
Nupper/Nlower = exp (ΔE/kT)
k = Boltzmann’s constant
What does a Jablonski diagram represent?
Molecular energy levels and various molecular transitions
Specular reflection
Reflection from a smooth surface
- output direction related to the input direction and surface normal
- often results in bright reflective artefacts
- does not contain much info about tissue
- can be used to detect the morphology of the tissue/sample surface
Scattering results from __________ and sources include _________:
- refractive index variation
2. collagen fibrils, organelles, nuclei, tissue bulk properties
Three types of scattering:
1 _______
2 _______
3 _______
- Rayleigh occurs if λ»_space; scattering particles (probability λ^-4)
- Mie occurs if λ similar to particle size (modelled using Maxwell’s equations)
- Raman
Diffuse reflection
Light is scattered in the tissue and then returns to the surface
Which is higher - scattering coefficient or absorption coefficient?
Scattering coefficient
Therefore, tissue is more likely to scatter light than absorb it - scattering has MFP of <100um
What is the anisotropy factor, g?
The mean cosine of the scattering angle
g = 1 --> light scattered completely in forwards direction g = 0 --> light scattering is completely isotropic
Mean free path, MFP =
MPF = 1 / μ
Reduced scattering coefficient, μs =
μs’ = (1-g)μs
Absorption coefficient varies with _____
Varies with wavelength
–> particularly strong absorption in the UV/Blue and IR regions
What is the optical window?
The 600-1100nm region where absorption is low and light can propagate deeper into tissue before being absorbed
Ballistic light
- non-scattering medium
- light travels undeviated in straight line
- intensity attenuated
- intensity distribution = localised peak
Snake-like light
- low scattering coefficient
- light deviates minimally from ballistic path
- intensity distribution = slightly broader
Can be used for OPT
Multiply-scattered light
- higher scattering coefficient
- no ballistic photons, few snake-like
- intensity distribution = broad
- not as much spatially resolved info - photons will have travelled through many different paths
Differential pathlength factor (DPF):
Average pathlength the scattered photons have travelled
Iz = Io exp(-μa z DPF)
Pulse Oximetry - how does it work?
Absorption varies depending on the conc of oxy- and deoxy- Hb –> monitor optical absorption at two wavelengths
- two light emitters at 650nm and 900nm
- records transmitted / backreflected light of the subjects finger
- different transmitted intensities at two wavelengths allows the relative amount of oxy- and deoxy-Hb to be calculated (relative absorption is between 650 and 900nm)
Breast imaging application
- breast surrounded by transparent refractive index matching medium
- sources and detectors around periphery
- high conc of Hb = tumour
Medical imaging limitations:
____ limits the resolution
Strong absorption by ____ limits the thickness of tissue through which tomographic imaging can be attempted
Scattering limits resolution
Strong absorption by blood
Optical imaging can obtain better _____ than other modalities for low tissue penetration depths (
better resolution for depths <100um
Scattering degrades _____ and absorption attenuates ____
Scattering degrades resolution and absorption attenuates signal
Lens formula
1/u + 1/v = 1/f
f = focal length u = object-lens distance v = lens-image distance
Image is INVERTED
Infinity corrected microscope
Short focal length OBJECTIVE lens + long focal length TUBE lens
- -> object magnified, M = f2/f1 (ratio of focal lengths)
- -> bundle of rays from each point on object are // between two lenses
–> back focal plane = fourier plane - info on the spatial frequencies
What is the benefit of the parallel rays between the object and tube lens in an infinity corrected microscope?
Allows further filters and slab optics to be inserted without causing significant deviations in the ray paths
What is the magnification in an infinity corrected microscope?
x4 - x100
An ocular can further magnify the primary image plane by x10
What does the wave equation describe?
How the electric field strength oscillates with time and spatial position
What does it mean by a ‘coherent’ wave?
Optical waves making up a light source maintain a constant phase
High coherence sources are monochromatic
Point spread function (PSF)
The intensity distribution in the image plane for a point-like object
- central peak
- side lobes = Airy pattern
PSF form is due to the diffraction limited performance of a lens
–> neighbouring objects may overlap in the image plane and not be resolved (dim objects may be hidden within the secondary lobes of neighbouring points)
Numerical Aperture is what?
NA = n.sin(a)
Measure of the light collecting power of the lens
- can increase this by improving the light collecting ability of a lens (e.g. collecting xrays from a larger angle)
How to improve the resolution of a microscope?
- decrease wavelength
- increase n
- increase max angle (at which waves can be collected)
- increase NA
Oil and water immersion
- increases value of n –> better resolution
What is missing from a non-infinity corrected microscope? How does it compensate?
Tube lens missing
Objective lens forms the real primary image plane without requiring a tube lens
Ocular with human eye lens then forms an image on the retina
Kohler illumination
Two lenses used together with a lamp source, the collector and condenser lenses:
- point on light source - light spread into a bundle of rays that pass through the sample
- -> removes spatial structure
- -> different points on source pass through at different angles - minimises glare
- condenser diaphragm controls amount of light reaching sample
What are conjugate planes?
Where do they exist in kohler illumination?
If an image exists in one plane, a similar image will exist at the other conjugate planes
retina, primary image plane and sample = conjugate planes
Brightfield microscopy is also known as reflected light microscopy. What is the set up arrangement?
Illumination optics are separated from the detection optics using a beamsplitter.
Objective lens is used as the condenser diaphragm
What is the mechanism of fluorescence?
What symbols are give for the radiative and non-radiative decay probabilities?
- molecule excited to a higher energy level through the absorption of a photon
- photon carries enough energy for molecule to reach one of the higher energy levels in E1
- molecule thermalises - loses energy through thermal interaction to lowest excited energy level
- decay back to ground by emitting fluorescence (radiative decay)
Γ = radiative decay probability k = non-radiative decay probability
Quantum yield
QY = Proportion of emitted photons to absorbed photons
Fluorescence lifetime
Average time after a photon is absorbed before a fluorescence photon is emitted
Stokes shift
Energy of emission is smaller than for excitation –> emitted photon is red shifted
Four types of fluorophore
Exogenous fluorescent chemical labels
- dyes can be added to tissues
Fluorescence proteins
- can be incorporated into organisms DNA
Endogenous fluorophores
- naturally occurring, present in animal tissues
Man made
-e.g. quantum dots
Fluorescence microscope
- Short excitation λ used with Kohler illumination
- Longer λ fluorescence emitted is imaged onto CCD camera
- Emission filter used to block illumination light
- Dichroic mirror used to separate excitation and emission paths –> reflects short λ and transmits long λ
- light source with excitation filter / laser
Dichroic mirror = interferomagnetic filters –> made of dielectric stacks of layers with different refractive indices
Modulation Transfer Function (MTF)
Described how an image of an object is degraded due to the imperfect transmission of the different spatial frequencies through an optical system
Frequency increases –> modulation of the imaged grating will decrease until contrast is zero (plain grey image)
Imaging systems with better resolution are able to transmit higher frequencies more efficiently than low resolution systems
Confocal scanning laser microscopy (CSLM)
- Laser / lamp focussed through pinhole
- light reflected from dichroic mirror and focussed onto sample
- fluorescence emitted passes through dichroic beamsplitter
- imaged through pinhole in front of detector
- fluorescence above and below the focal plane is not efficiently transmitted through detector pinhole –> microscope preferentially detects fluorescence from one plane
Out of focus light rejection –> better contrast and lateral resolution (decrease in PSF sidelobes)
Fluorescence emission spectra and often broad and overlap. How is a confocal microscope adapted to distinguish fluorescence spectra?
- Images are recorded at many different fluorescence emission wavelengths
- Spectrally dispersing the fluorescence onto a multi-element detector
Single photon excitation fluorescence vs two photon excitation fluorescence
Single:
- molecular energy difference from the ground state to first excited state is equal to the energy carried by one excitation photon
- transition by absorption of one excitation photon
Double:
- same molecular transition achieved by the absorption of two excitation photons at half the energy (2x λ)
- longer wavelength light less attenuated therefore can penetrate deeper into tissue
- high laser intensities used –> light focussed to single spot that can be scanned across the sample
OR - short pulse of instantaneous v high intensity
Two photon scanning fluorescence microscopy: Advantages and Disadvantages
Advantages:
- high signal collection efficiency
- long excitation λ –> deeper tissue penetration
- only focal plane excited –> minimises out-of-focus photobleaching/phototoxicity
Disadvantages:
- high intensity - pulsed lasers
- worse resolution (longer λ)
- high peak power –> damage/photobleaching
Flexible endoscopes use a fibre image guide. What features of the fibre image guide must be maintained during endoscopy?
- each fibre = one image pixel
- spatial organisation must be maintained from one end to the other –> bundle must be coherent
- illumination source = white xenon lamp (focussed into non-coherent bundle of optical fibres to be delivered to tissue
Hopkins rod lens endoscope
Light relayed through series of lenses housed in a rigid metal tube
- high image quality (high resolution), large FOV, large depth of field
Flexible wide field video endoscopes
Fibre image guide replaced with CCD chips (charge coupled device - photons on surface generate charge read by electronics)
–> better resolution + more flexible endoscope
Capsule endoscopy
‘Pill’ containing LED light source, camera and aerial
Narrow band imaging
Filters white light source to provide 3 30nm bandwidths of red, green and blue light
–> improves contrast of certain tissue features
Endoconfocal microscopy
- bundle of fibres –> laser focussed onto one fibre in bundle –> light exiting tip focussed onto sample
- fluorescent light passes through confocal pinhole and laser is scanned across proximal end of fibre bundle to scan across the sample
- scanning optics in external box
- fibre image guide limits resolution –> limited #fibres in bundle
