Lecture 8: Optics 1 Flashcards
Describe what light may be. how it can act, properties.
Light behaves as both a particle and a wave
Can bounce (reflect) and bend (diffract or refract)
Has wave properties – Amplitude – Wavelength: visible is between 400-700 nm (White light carries all visible wavelengths) – Frequency – Direction of travel – Direction of vibration
What is diffraction and what is the product of diffraction?
- Due to its wave properties, light undergoes diffraction (deviation of the light wave direction)
- Light wavefronts diffract when they travel through narrow slits e.g. apertures
- Wavefronts spread and interact, causing constructive and destructive interference
What causes the airy disk formation?
The shape of the diffraction pattern varies according to wavelength and size of the aperture.
Thus varying the shape of the airy ring which is a property of constructive and destructive interference.
What generates an airy disk?
Diffraction through a circular aperture creates an Airy Disk
What determines the distance to the first dark ring?
The radius of the Airy disk is the distance r from the centre to the first dark ring
What is a PSF?
PSF
– intensity distribution of Airy Disk in 3D
- describes diffraction pattern of a point source of light in x, y, and z
What is an image?
An image is produced by an instrument. It is not the same as the object but is some ‘facsimile’ of the object e.g. it may be only 2D.
What does the PSF describe and the implication?
The ‘point spread function’ (PSF) describes what the instrument does to information from the object to produce an image.The image is the object convolved by the PSF.
Describe the convolution in the microscope
Image = Object X PSF
(For each point, take the object and multiply it by the entire PSF centred at that point in theimage and sum the results of the multiplication)
Describe the relationship of the PSF to the image;
The object is a large number of points arranged in space
The image is the superposition of a large number of points convolved with the PSF.
Is PSF constant?
No, it can suffer from spherical abberation
What does airy disk vary with?
NA
Size of Airy diffraction pattern determined by resolving power of objective lens
Resolving power: Determined by NA
What is NA?
- NA = ability of lens to capture light rays
- Determines ability of lens to gather light and resolve detail
- Typically: large NA ~ small WD
What is the equation for NA?
NA = n x sin(α)
n = refractive index alpha = one-half of the objective's opening angle (determined by focal length of objective lens)
What are some refractive indices?
Refractive index n is limiting (air 1.0, water 1.33,oil ~1.51)
What is the criterion for being able to resolve between two points?
Raleighs criteria
When the intensity in the valley is reduced by ~25% compared with the two maxima, the two points are discrete (this is just sufficient for the human eye to see two separate points)
How does resolution relate to nyquist?
Nyquist sampling of an image of two points separated by the Rayleigh resolution
Nyquist sampling - sampling interval twice the highest specimen spatial frequency to preserve the spatial resolution in the resulting digital image
Describe the two plains of resolution;
Lateral (X and Y) and axial resolution
What is the equation for lateral resolution;
R(lat) = 1.22 wavelenght / (Na objective + Na cond)
if Na (ob) = Na (cond) (Epiflourescence)
then R(lat) = 0.61 wavelenght / NA
structures that lie closer than this distance cannot be resolved in the lateral plane using a light microscope
What is the equation for axial resolution?
R(axial) = 2.(wavelength).n / (Na)^2
Whats a van Leeuwenhoek microscope?
Focal nob
What is the property of thin lenses?
1/p + 1/q = 1.f
p = distance from object to lens q = distance from lens to image f = focal distance
q/p = magnification
How many lenses does a a compound microscope x10 objective have?
Microscopes/objectives contain a combination of these lenses that focus, magnify or correct aberrations
i.e heaps / tricky question
How is magnification of a microscope determined?
Objective x lens = total
What are some types of obberations?
Spherical
Chromatic
Field curvature
What is spherical abberation
Spherical aberration occurs due to the increased refraction of light rays when they strike a lens near its edge, in comparison with those that strike nearer the centre.
What is chromatic abberation?
Chromatic aberration is a failure of a lens to focus all colours to the same convergence point. Lenses have a different refractive index for different wavelengths of light
What is field curvature?
a flat object normal to the optical axis cannot be brought into focus on a flat image plane.
Check the slide in that demonstrates how to read a lens and interpret it
Slide 43
What is a correction for the slides in a system?
- In non-immersion lenses, chromatic and spherical aberration can be introduced by the coverslip – design with correction for standard coverslip thickness
- In oil immersion lenses, coverslip thickness less important if mounting medium RI is similar
- However, mounting in e.g. aqueous will introduce aberrations and alter PSF
- Therefore, immersion and mounting medium must be chosen carefully
What are some important parts of a microscope?
Condenser
Turret
Stage
Diaphragm
What is Kohler Illumination?
Achieves even illumination with non-uniform lightsource
Principal components:
–Collector lens – collects lamp light
–Field diaphragm – controls amount of light entering sample
–Aperture diaphragm – alters sample contrast and condenser NA (match to objective NA for maximum resolution)
Why is even illumination critical?
Even illumination critical for image formation in brightfieldand other contrast mechanisms
What are the two types of objects in terms of microscope imaging?
Phase and amplitude objects
Amplitude objects can be visualised by the human eye. Phase cannot.
Phase objects primarily make up tissue therefore we must adopt to this.
What are some contrast mechanisms?
Stains i.e H&E
DIC
HMC
- Stained samples – not for live cells
- Colourimetric stains e.g. Haematoxylin and Eosin
- Brightfield illumination
- Belong to Amplitude objects
What is H and E stain and what is it used fir?
Principal histological stain – e.g. pathology
Haematoxylin – labels e.g. nuclei blue
Counterstain with Eosin Y red/pink- Eosinophilic structures stained e.g. proteins/red blood cells/cytoplasm
How does phase imaging work and how do microscopes utilise this?
Biological samples are mostly phase objects
Phase shifts occur because of the different refraction indices of the specimen and the surrounding medium
Contrast mechanisms designed to convert phase differences into intensity differences
No stains, therefore useful for live cell imaging
Describe the phase shift physics of the microscope;
- Total phase shift of ½ λ – destructive interference occurs •Non-diffracted light hits the phase plate (dimming and phase shift ¼ λ) •Phase shift of ¼ λ - bypasses phase plate •Portions of ring diffracted by specimen (plasma membrane/organelles)
- Focused on specimen by condenser
- Ring shaped light formed by condenser annulus
- Condenser setting and phase objective must match!
- Only possible with phase objectives
What is a unique contrast that utilises interference?
Differential interference contrast (DIC)
Images appear 3D under unique oblique illumination
Explain the physics of DIC;
- Plane polarized light (at 45°) is split into two rays (prism 1 – separates light into components polarised 90° to each other)
- Rays pass through condenser and travel parallel through specimen (in very close proximity ~0.2um)
- The thickness/refractive index of the specimen changes the path length of the two rays
- Rays are focused by the objective on the second prism and recombined into one polarised at 135°
- The optical path differences of the two rays leads to interference
- Gives rise to “shadows” and a pseudo three-dimensional appearance
What is the hoffman modulation contrast? (HFC)
Modulation contrast visualizes transparent, low-contrast specimens by converting phase gradients into brightness gradients
How does HFC work?
•Off axis slit plate = generates oblique illumination onto the specimen which has a phase gradient
Light then goes through the Hoffman modulator at back focal plane
Accentuates contrast differences
•Three neutral density regions – 100% transmission, 15% and 1%
•Slit is aligned with 15% region
Basically =
•Light passes through sample – refracted to different degrees
•Passes through different modulator regions
= Accentuates contrast differences
What is fluorescence?
Absorption of light at one wavelength and prompt re-emission at a longer wavelength
What can fluorescence be compared with?
–Luminescence: Light given off by a chemical process
–Phosphorescence: Like fluorescence, but light emitted after much longer time
Why use fluorescence for microscopy?
- Specificity
- Sensitivity
- High Contrast Images
Describe the fluorescence physics;
Absorption of energy by fluorochromes occurs between the closely spaced vibrational energy levels of the excited states in different molecular orbits
i.e incidence of light causes electrons to jump up excited states and their return between rings releases energy depending on the size of the jump as within each ring is a number of vibrational energy states (sub rings)
Do all relaxations of electrons release light?
no they can internally convert.
Describe the absorption of a typical flurophore;
For a typical fluorophore, broad λ excitation generate e excitations to various vibrational energy levels of excited states
Some transitions have higher probabilities than others
When combined, they constitute the absorption spectrum of a molecule
Describe flourescence;
Loss of energy through internal conversion leads to Stokes Shift(difference between maximum Abs and Em)
Emission spectrum is also a spreadof wavelengths – relaxation of e’s to G0 produces different λ s dueto multiple vibrational energy levelsof G0
Fluorophores can undergo this Abs/Em cycle hundreds to thousands of times
What is 2 photon excitation?
Using a laser to excite
Describe 2 photon excitation advantages and disadvantages
Two photons of half energy (double the wavelength) can arrive simultaneously
Advantages: Better light penetration/minimises scatter, reduced phototoxicity
Disadvantage: Expensive laser, lower resolution than single photon Exc
What are some typical flurophores that are conjugated
FITC and Texas Red often conjugated to antibodies/dextrans
What are some important flurophore properties?
Extinction Coefficient:How much light the dye absorbs
Quantum Yield:Fraction of absorbed light re-emitted as fluorescence
Common flurophores?
CFP EGFP mRFP Cerulean mCherry
All used to label specific structures in tissue, and detected by fluorescence microscope…
What are the potential arc lamps for fluorescence?
- Typically Xenon or Mercury lamp
- Arc needs to be started (igniter)
- Kohler illumination alignment
- Produces a wide spectrum of light for fluorescence excitation
How is excitation and emission separated?
Filter cube
Describe the characteristics of the filter cube;
3 filters
- Excitation filter (removes unwanted wavelengths from white light)
- Dichroic mirror (reflects excitation wavelength and allows emission to pass through)
- Excitation filter ( processes emitted light)
Typed of filters;
Short pass
Long pass
Band pass
What is bleed through?
Emission spectral overlap
What is photo bleaching?
Not all the energy goes into producing fluorescence
• Some leads to photobleaching and other photodamage
Does more excitation = more fluorescence?
No saturation can occur
What happens as we increase excitation really high?
Saturation (all available fluorophores in excited state)
• Increased damage
• Reduced z resolution
What can we use to detect emission?
Photodetector
- Charge Coupled Device (CCD)
- Matrix of photodiodes
- Stores and transfers light information
- High efficiency
What makes a photodetector optimal?
- Cooled (reduce noise)
- No color filters
- Intensified (iCCD) or electron multiplying (emCCD) for low light applications
What is confocal microscopy?
- shallow depth of field
- Pinhole aperture - elimination of out-of-focus glare
- Enhanced contrast over widefield fluorescence
- Point scanning using diffraction-limited laser spot
- serial optical sections from thick specimens – 3D reconstruction
