Lecture 8: Optics 1

Question 1

Describe what light may be. how it can act, properties.

Answer 1

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

Question 2

What is diffraction and what is the product of diffraction?

Answer 2

• 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

Question 3

What causes the airy disk formation?

Answer 3

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.

Question 4

What generates an airy disk?

Answer 4

Diffraction through a circular aperture creates an Airy Disk

Question 5

What determines the distance to the first dark ring?

Answer 5

The radius of the Airy disk is the distance r from the centre to the first dark ring

Question 6

What is a PSF?

Answer 6

PSF
– intensity distribution of Airy Disk in 3D
- describes diffraction pattern of a point source of light in x, y, and z

Question 7

What is an image?

Answer 7

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.

Question 8

What does the PSF describe and the implication?

Answer 8

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.

Question 9

Describe the convolution in the microscope

Answer 9

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)

Question 10

Describe the relationship of the PSF to the image;

Answer 10

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.

Question 11

Is PSF constant?

Answer 11

No, it can suffer from spherical abberation

Question 12

What does airy disk vary with?

Answer 12

NA

Size of Airy diffraction pattern determined by resolving power of objective lens

Resolving power: Determined by NA

Question 13

What is NA?

Answer 13

• NA = ability of lens to capture light rays
• Determines ability of lens to gather light and resolve detail
• Typically: large NA ~ small WD

Question 14

What is the equation for NA?

Answer 14

NA = n x sin(α)

n = refractive index
alpha = one-half of the objective's opening angle (determined by focal length  of objective lens)

Question 15

What are some refractive indices?

Answer 15

Refractive index n is limiting (air 1.0, water 1.33,oil ~1.51)

Question 16

What is the criterion for being able to resolve between two points?

Answer 16

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)

Question 17

How does resolution relate to nyquist?

Answer 17

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

Question 18

Describe the two plains of resolution;

Answer 18

Lateral (X and Y) and axial resolution

Question 19

What is the equation for lateral resolution;

Answer 19

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

Question 20

What is the equation for axial resolution?

Answer 20

R(axial) = 2.(wavelength).n / (Na)^2

Question 21

Whats a van Leeuwenhoek microscope?

Answer 21

Focal nob

Question 22

What is the property of thin lenses?

Answer 22

1/p + 1/q = 1.f

p = distance from object to lens
q = distance from lens to image
f = focal distance

q/p = magnification

Question 23

How many lenses does a a compound microscope x10 objective have?

Answer 23

Microscopes/objectives contain a combination of these lenses that focus, magnify or correct aberrations

i.e heaps / tricky question

Question 24

How is magnification of a microscope determined?

Answer 24

Objective x lens = total

Question 25

What are some types of obberations?

Answer 25

Spherical Chromatic Field curvature

Question 26

What is spherical abberation

Answer 26

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.

Question 27

What is chromatic abberation?

Answer 27

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

Question 28

What is field curvature?

Answer 28

a flat object normal to the optical axis cannot be brought into focus on a flat image plane.

Question 29

Check the slide in that demonstrates how to read a lens and interpret it

Answer 29

Slide 43

Question 30

What is a correction for the slides in a system?

Answer 30

* 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

Question 31

What are some important parts of a microscope?

Answer 31

Condenser Turret Stage Diaphragm

Question 32

What is Kohler Illumination?

Answer 32

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)

Question 33

Why is even illumination critical?

Answer 33

Even illumination critical for image formation in brightfieldand other contrast mechanisms

Question 34

What are the two types of objects in terms of microscope imaging?

Answer 34

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.

Question 35

What are some contrast mechanisms?

Answer 35

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

Question 36

What is H and E stain and what is it used fir?

Answer 36

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

Question 37

How does phase imaging work and how do microscopes utilise this?

Answer 37

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

Question 38

Describe the phase shift physics of the microscope;

Answer 38

* 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

Question 39

What is a unique contrast that utilises interference?

Answer 39

Differential interference contrast (DIC) Images appear 3D under unique oblique illumination

Question 40

Explain the physics of DIC;

Answer 40

* 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

Question 41

What is the hoffman modulation contrast? (HFC)

Answer 41

Modulation contrast visualizes transparent, low-contrast specimens by converting phase gradients into brightness gradients

Question 42

How does HFC work?

Answer 42

•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

Question 43

What is fluorescence?

Answer 43

Absorption of light at one wavelength and prompt re-emission at a longer wavelength

Question 44

What can fluorescence be compared with?

Answer 44

–Luminescence: Light given off by a chemical process | –Phosphorescence: Like fluorescence, but light emitted after much longer time

Question 45

Why use fluorescence for microscopy?

Answer 45

* Specificity * Sensitivity * High Contrast Images

Question 46

Describe the fluorescence physics;

Answer 46

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)

Question 47

Do all relaxations of electrons release light?

Answer 47

no they can internally convert.

Question 48

Describe the absorption of a typical flurophore;

Answer 48

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

Question 49

Describe flourescence;

Answer 49

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

Question 50

What is 2 photon excitation?

Answer 50

Using a laser to excite

Question 51

Describe 2 photon excitation advantages and disadvantages

Answer 51

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

Question 52

What are some typical flurophores that are conjugated

Answer 52

FITC and Texas Red often conjugated to antibodies/dextrans

Question 53

What are some important flurophore properties?

Answer 53

Extinction Coefficient:How much light the dye absorbs Quantum Yield:Fraction of absorbed light re-emitted as fluorescence

Question 54

Common flurophores?

Answer 54

``` CFP EGFP mRFP Cerulean mCherry ``` All used to label specific structures in tissue, and detected by fluorescence microscope...

Question 55

What are the potential arc lamps for fluorescence?

Answer 55

* Typically Xenon or Mercury lamp * Arc needs to be started (igniter) * Kohler illumination alignment * Produces a wide spectrum of light for fluorescence excitation

Question 56

How is excitation and emission separated?

Answer 56

Filter cube

Question 57

Describe the characteristics of the filter cube;

Answer 57

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)

Question 58

Typed of filters;

Answer 58

Short pass Long pass Band pass

Question 59

What is bleed through?

Answer 59

Emission spectral overlap

Question 60

What is photo bleaching?

Answer 60

Not all the energy goes into producing fluorescence • Some leads to photobleaching and other photodamage

Question 61

Does more excitation = more fluorescence?

Answer 61

No saturation can occur

Question 62

What happens as we increase excitation really high?

Answer 62

Saturation (all available fluorophores in excited state) • Increased damage • Reduced z resolution

Question 63

What can we use to detect emission?

Answer 63

Photodetector - Charge Coupled Device (CCD) - Matrix of photodiodes - Stores and transfers light information - High efficiency

Question 64

What makes a photodetector optimal?

Answer 64

* Cooled (reduce noise) * No color filters * Intensified (iCCD) or electron multiplying (emCCD) for low light applications

Question 65

What is confocal microscopy?

Answer 65

* 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