Graham's Flashcards
(113 cards)
1
Q
What length scales does geometrical optics refer to? What are the basic assumptions?
A
Length scales larger than the wavelength
The particle picture of light, can describe reflection, refraction etc.
Can ignore wave effects such as diffraction.
2
Q
What is the Huygens-Fresnel principle
A
Can deconstruct the wavefront into spherical secondary wavelets.
Amplitude of the optical field at any point later on is the superposition of these
3
Q
What is the paraxial approximation?
A
All angles are small; i.e sin(theta) = theta
4
Q
What does a positive radius of curvature refer to?
A
Convex face of lens
5
Q
What does a negative radius of curvature refer to?
A
A concave face of a lens
6
Q
What does a positive focal length mean for a lens and the image created by it?
A
The lens is converging; will create a real image behind the lens
7
Q
What does a negative focal length mean for a lens and the image created by it?
A
Diverging lens; a virtual image is formed in front of the lens
8
Q
Write the ray vector, what does each variable mean?
A
[P] - Distance from optical axis (lateral)
[theta] - angle between ray and optical axis
9
Q
How many ray transfer matrices would you need for a thick lens?
A
4:
1) pre-interface
2) post-first-interface (in-lens)
3) pre-second-interface (in lens)
4) Post-second-interface (out of lens)
10
Q
When thinking about ray transfer matrices, what is a thick lens the same as?
A
Two thin lenses
11
Q
What is a principal ray?
A
The rays which denote the FOV
12
Q
What are marginal rays?
A
The rays which leave the object with the widest angle but still pass through the aperture stop (imagine as coming from object at optical axis with big angle towards lens)
13
Q
What is the entrance pupil?
A
An image plane of the aperture stop, on the object side of the instrument
14
Q
What is the entrance window?
A
An image plane of the field stop, on the object side of the instrument
15
Q
What is the aperture stop?
A
The apertire which limits the angle of rays that can pass through an instrument . Placed on a plane where the principal ray crosses the optical axis. Controls the image brightnesss.
16
Q
What is the field stop?
A
The aperture which limits the field of view of the instrument. It is placed on an image plane.
17
Q
What is the exit window?
A
An image plane of the field stop, on the exit side of the instrument
18
Q
What is the exit pupil?
A
An image plane of the aperture stop on the exit side of the instrument.
19
Q
Draw and label a diagram for two lenses, including all of the apertures in the image.
A
See notes (L3)
20
Q
Draw the propogation of the marginal and principal rays in a 4f system
A
See notes (L3)
21
Q
What is an aberration?
A
A property of an optical system that causes light to be spread out over a region of space rather than focused to a point.
22
Q
Explain why chromatic aberration happens
A
The refractive index of a material is wavelength dependant; this means that dispersion through a lens is different for different wavelengths of light. And white light is constructed of many wavelengths
23
Q
What is the circle of least confusion?
A
The axial location with the best compromise for the focal point for different wavelengths of light.
24
Q
How can chromatic aberration be overcome?
A
Two lenses with different focal lengths can be used to form an achromatic doublet to compernsate for the deviations for different wavelengths.
25
What is a disadvantage of the achromatic doublet? How can this be overcome?
Need the refractive index not to change with wavelength which is only usually valid for small range of wavelengths.
Introduce an apochromatic lens instead; at least 3 lenses for correcting the aberration.
26
Describe spherical aberrations and how the can be overcome
A third order aberration that arises when the paraxial approximation is no longer used. Light near the edge focuses closer to the lens than that near the optical axis.
Overcome with an aspheric lens; reverse of spherical.
27
Describe Astigmatism as an aberration
There are two types; optical and visual.
Visual - non-symmetric lens
Optical - Off-axis illumination; lens appears to be non-symmetric.
28
Describe coma aberration
Parallel rays not parallel to the optical axis are focused in different places; get a comet-like tail
29
Give the equation for the numerical aperture, what is it?
NA = n sin(theta); n is the immersion medium.
The range of angles over which the system can accept or emit light.
The angle is equal to half the beam.
30
How can the numerical aperture be improved?
Make the lens aperture wider
Reduce the working distance
Increase the RI of the immersion medium
31
How can a lens be used to map it's aperture in real life
For a lens with finite positive focal length, the far-feld diffraction pattern from the light is a an FT of the front focal plane at the back focal plane
32
What is a conjugate plane?
The FT(FT(P)) i.e FT of the FT of a plane
33
Where do low-frequencies appear in the Fourier spectrum of an image?
Near to the centre of the spectrum
34
Describe the Rayleigh criterion for image resolution
This is the diffraction limit for being able to resolve two separate light sources. Images are resolved when the centre of one airy disk overlaps with the first dark line of the next disk. d = wavelength / 2NA
35
How can the resolution in a microscope be improved?
Largest possible NA
Shortest possible wavelengths
36
What is the PSF? what relation does it have to an image formed by an instrument?
The response of the optical system to a point source.
The image formed is a convolution of the PSF and the object being imaged
37
What causes the resolution of an instrument to be worse than the diffraction limit?
Aberrations
38
What is used to describe aberrations?
Zernicke polynomial basis
39
What is the Zernicke polynomial basis?
An orthogonal basis described using a Radial function and angles.
Used to find the relative contribution of different aberrations present in the optical system
40
Describe how a Shack-Hartmann Wavefront sensor works
The wavefront is focused by lenslets onto a sensor. Depending on the phase of the wavefront; the signal will appear in different places on the sensor. Can reconstruct the whole wavefront. Convert phase info to spatial info
41
Explain how a deformable mirror works
A mirror that with electronically controllable pistons can be used to alter it's shape so that upon reflection, a conjugate phase is applied to the incident wavefront; correcting the aberration.
42
Describe how a spatial light modulator works
A deformable mirror except it has a uniform surface and liquid crystlals with individual electrodes for altering the phasefront of the light.
Liquid crystals orientation is controlled with an electric field from the electrodes.
43
Describe the difference between S, D and P waves in microscopy
S - Surround wave; passes through samble without interacting
D - Diffracted; Scattered by the object
P - Particle, sum of P and D
44
Whats is widefield and bright field microscopy?
Widefield collect a large FOV
Brightfield collects S and D light
45
Describe Dark-field microscopy
Only the D wave is collected by placing a focussing ring at the condenser annulus. A high NA condenser is used to focus light onto specimen.
Low NA objective doesn't collect the S wave, which is diffracted at a large angle
46
2 issues and advantage of dark-field microscopy
Need a lot of light
Resolution sacrificed by low NA objective
Get background free images
47
Describe phase-contrast microscopy
Interferometrically maximising the difference in amplitude between the objective and background in the image plane by advancing the phase of the surround light.
Place a phase-plate at the conjugate plane to the condenser annulus; still have a field stop there.
48
Describe fluorescence microscopy
Excitation -> non-radiative decay - > fluorescent emission.
Fluorescent emission has a lower energy than absoprtion; can block out each with filters if want to separately
49
What is an endogenous fluorophore?
Fluorescent molecule already present in the sample, just need to excite
50
What is an exogenous fluorophore?
Tagging or staining a sample with a fluorescent molecule
51
Why are exogenous fluorophores useful?
can be designed to bind to only specific chemicals; have a higher quantum yield than endogenous
Can be sensitive to parameters such as pH or temp.
52
What is a stokes shift?
The shift in wavelength between the excitation and emission spectrum
53
Draw the setup of a fluorescent microscope and label each part
See lecture notes L7
54
What are the radiative processes in Fluorescence microscopy?
Absorption: High energy, short wavelength, ground to S2 state.
Fluorescence: S1 to ground state de-excitation
Phospohorescence: Excited triplet state to ground state (technically forbidden)
55
What are the non-radiative processes in microscopy?
Vibrational relaxation - from most to least excited S2 state, very quick
Internal conversion - from a higher S state to lower S state because energy levels of the two overlap
Intersystem crossing - the electron changes spin multiplicity from singlet to triplet state. Forbidden by theory.
Photodegeneration: rupture of chemical, causing degradation of molcule; photobleaching.
56
Describe Raman Imaging
Probing of the vibrational spectrum of molecules, through Raman scattering probes. Narrow peaks are produced corresponding to each vibrational transition. Spectrum acts as a fingerprint for the molecule, without any label needing to be added.
57
Describe on-axis holography
An object is illuminated with coherent light, generating a scattered light beam.
The scattered + reference interference pattern is recorded on holographic film.
Film transmission is proportional to incident intensity during recording.
Illuminating the film with the reference beam generates a hologram.
58
What is a limitation of on-axis holography?
A second twin-image is generated alongside the reconstructed image.
59
Describe off-axis holography
The beam is split into two parts; a reference arm which bypasses the sample and an object arm which scatters off it. Interfere at holographic film. Signal depends on the phase between the two beams.
60
What is an advantage of holography?
Every point of recording contains infro about every point of the object due to the interference.
Cutting a hologram reduces the angular range; i.e ability to record high k-vectors. Majority of info still contained in the image. Loss-resilient way of transfering info.
61
What is the difference in digital holographic imaging?
Hologram mixes phase and amplitude info. Local phase difference between object and reference can be extracted. FT can be performed to extract phase only, and only wanted features. Can iDFT to get spatial basis and thus the wanted info. Phase differences can tell about RI variations in transparent samples.
62
Advantages of Digital Holography and a disadvantage
Quantitative phase info
Can volumetrically reconstruct from single image
Use very low optical powers, no staining
Samples need to hav high transmission at wavelength of choice
63
What is depth of field?
The axial distance over which the object remains in focus. i.e axial resolution
64
Give the equation for axial resolution of a diffraction limited PSF in a microscope or telescope
r_axial = (1.4 llambda n) / (NA)^2
65
What is scanning microscopy?
Only collecting light from one point at a time; or only illuminating over one point at a time.
The PSF is given by the scanned element
66
Describe confocal scanning microscopy
Pinholes placed in front of the laser and the detector. Source and detector are scanned; PSF of illumination scanned by PSF of detector. Resolution is improved by PSF^2 .
67
What is epi-illumination?
The same objective is used to focus the excitation and the emission.
68
Give the lateral resolution of a scanning confocal microscope
r_lateral = 0.4 llambda n / NA
69
What are D and d in the out-of-plane light rejection ratio equation?
D is the pinhole diameter, d is the Airy disk diameter.
70
How can confocal microscopy be made quicker?
By using a spinning disk
71
How does MPM work?
Two photons with a longer wavelength behave as a single one with twice the energy; is very improbable. Need high intensity, so use ultrashort pulses.
72
How does MPM lead to optical sectioning?
MPM fluorescence in non-linear, emitted signal depends on sq. of intensity so generation is localised to close to focus. Damage to ample restricted to just the focal region.
73
What is the optical window?
600-1300nm, where scattering is minimal and absorbance is quite low
74
What are the advantages of MPM?
Can use light which penetrates deeper in living tissue for excitation
Less absorption away from focus
Excitation wavelength well matched to many common fluorophores
High quality localisation due to NL effects
Low tosicity for exciting wavelength
75
Describe light sheet microscopy
Sample illuminated by a sheet of light propogating perpendicular to the detection objective. CCD camera used to detect the widefield.
76
How is the light sheet produced for microscopy, what is the compromise?
Made with cylindrical lens or sweeping a focused Gaussian beam. Need to compromise between axial resolution and FOV
77
Where is a Bessel beam used, why?
In MPM; Diffraction is slower under propogation, but have equal power in each ring, meaning that PSF is blurred. NL interaction means the signal is reduced significantly from outer rings.
78
What scales does super-resolution imaging refer to?
Sub-wavelength
79
What are the two key scattering types in tissue, describe each. What does scattering highly depend on?
Rayleigh - Forward and back scateering equal. For sizes much smaller than the wavelength.
Mie - Scattering is mostly forward-directed. For sizes much bigger than wavelength.
Both depend on medium RI
80
What is attenuation in tissues? What is th optical window?
Attenuation = Absorption + Scattering
Optical window where attenuation is lowest 600 - 1300nm
81
How does scattering vary with wavelength?
Decreases with wavelength
82
What is the issues with wanting to image deeper into sample?
Need longer wavelength which lowers resolution
83
Describe OCT
Have a Broadband source and a reference arm. Combine in sample arm. Changing path length of reference beam allows axial scanning. Changing angle of mirror to sample allows traverse scanning. Essentially Michelson interferometer.
84
What is the transverse resolution in OCT?
Set by the focusing optics, so have optical diffraction limit PSF; llambda / 2NA
85
How can spectrum be found using OCT?
Broadband light source for spectrometer based; simultaneous spectral detection but non-scanning.
Swept-source with laser wavelength sweep; more compact.
86
What is speckle, why does it happen in OCT?
Some of the light is back-scattered so get superpositions; giving distorted signal.
87
Describe SNOM
Near-field imaging before diffraction takes place. Nanoscale aperture very close to sample collects light giving a tiny PSF.
88
Advantages and disadvantages of SNOM
Limited sample prep
Asymmetric fibres can be used for polarization sensitivity
Slow - fragile tip
Uniformly flat sample required
89
Describe STED
Excitation beam excites fluorophores
After a delay (fast compared to spontaneous emission) STED beam introduced causing emission from all regions covered and depletion from fluorescent molecules. Spontaneous fluorescence observed only from area not covered by STED beam.
90
Describe the effective PSF of the STED beam
Excitation beam illuminates sample as a gaussian beam
STED beam is annular beam with very small hole, causing saturated depletion of fluorescence everywhere.
Effective PSF given by the dimension of the hole
91
How is the STED beam made?
Spatial light modulator or spiral phase plate in Fourier plane.
Laguerre-Gaussian laser beam used; circularly symmetric transverse modes. Size of hole not diffraction limited; intensity of rings can go beyond saturation limit.
92
Describe FIONA
Have a single emitter so the diffraction limit does not apply and can resolve position to a high accuracy (one nanometer). Resolution proportional to 1/ sqrt(N)
93
Describe PALM
Photoactivatable Fluorophores (same as FIONA)
94
describe STORM
Photoswitchable fluorescent dyes (same as FIONA but can switch on/off)
95
Describe stochastic Multiple Localization for STORM and PALM
Sample labelled densly with photoswitchable probes
Only some molecules switched at a time; so PSFs don't overlap and minimum diffraction is achieved with very low light intensity.
Localization of emitter determined by intensity of PSF i.e resolution = 1/ sqrt(N)
Iterate process
96
How can Stochastic multiple localization be done in 3D
Cylindrical lens for astigmatism. Ellipticity of individual fluorophores will depend on the z-position in sample.
97
Describe Moire fringes for Structured illumination micrscopy
Two superimposed grating patternns (get diagonal fringes)
By illuminating a sample with a grating; get moire fringes from the superposition of grating and sample.
Low spatial frequency Moire fringes can be collected by objective.
98
Describe Structured Illumination Microscopy
Objective collects spatial frequency of k_0 = 2NA / llambda . Observe convolution of grating illumination FT and sample FT. Spatial frequency distribution overfills objective, but since observing repeating pattern, the overfilled part will appear as part of a shifted copy by k_0. If used 3 orientations of grating, can completely reconstruct image with doubled resolving power
99
Describe a basic camera
Digital cameras have a semiconductor pixel array, which convert photons to electrons; temporarily stored in a potential well before reading.
100
What is exposure time?
Time that charge accumulates before reading out
101
What is Frame rate?
Number of images read out per unit time
102
What is Quantum efficiency
Rate of conversion of photon strikes into electrical signal
103
What are 3 types of noise?
Read - Inherent in circuitry
Dark - Thermally induced noise, when no light
Shot - Arises from Poisson stats of light arrival time at the camera. Propto the sqrt(photons)
104
What is Full well capacity?
Amount of charge that can accumulate on single pixel before saturation
105
What is Dynamic range
Ratio of maximum to minimum intensity that can be detected by a single frame
106
Describe CCD
Charge-coupled device; array of pixels.
107
Describe rolling shutter
In CMOS cameras, as one row is read, another is still being exposed.
108
Describe SPAD arrays
Avalanche photodiode has large reverse bias, produces internal multiplication process from photon striking semiconductor.
Extremely sensitive, and very quick
109
Describe event-based imaging
Only recording parts of a scene that are changing in time for faster acquisition. Set a thershold value for intensity change
110
Describe ghost imaging
Pair of correlated photons. Each sent down a different arm. Bucket arm detects if photon passed by object. Camera triggers to measure position of other photon if bucket detects one. Exposure only when detected photon so noise is low. Detecting photon that never interacted with sample. Take one photon per pixel
111
Describe a DMD
Digital micromirror device used for detecting portions of image that strike the photodetector. Changing pattern means recording intensity that hits different portions of object. Can build image quickly by choosing good pattern sequence. Called compressive sensing
112
Describe how RGB cameras work
Bayer filter and 4 pixels per image point (2 green, a red and a blue).
113
Describe hyperspectral imaging
2D image with spectral info
Light from a point is focused onto grating and linearly dispersed along linear detector. Can scan spot-to-spot.
Can also do push-broom spectral with an image per wavelength line.
Can also image whole sample with sequential colour filters
