optical microscopy

equations for light microscopy

$wavelength = \frac{speed~of~light}{frequency}$ $energy~of~light=h*f$

where h is plank’s constant = $6.626×10^{-34}~JHz^{-1}$

what is the maximum resolution for conventional light microscopy

200-300nm

what are the different ways light can interact with biological matter

1. absorption

2. reflection: specular and diffuse

3. scattering: elastic and inelastic

4. fluorescence

what is the main way contrast is produced in optical imgaging?

absorption → allows us to distinguish different biological structures producing the contrast necessary to resolve details from the background

what is absorption dependent on?

1. the absorption coefficient of the material

2. the wavelength/frequency of light → high frequency/ short wavelength (blue) light is more easily absorbed

what is scattering?

types?

interaction of light photon with molecules causes a deviation from it’s original path

1. elastic → energy is conserved

2. inelastic/Raman scattering → energy is lost to the molecule which caused the interaction

what determines noise in optical imaging?

how is it related to frequency/wavelength?

1. inelastic Raman scattering → determines how deep you can penetrate tissue

2. low frequency/short wavelength red to infrared light scatter less and can therefore penetrate deeper into the tissue

fluorescence

absorbed energy from the photon is converted into another photon type

magnification equation

$$magnification=\frac{image~size}{actual~object~size}$$

resolution definition

smallest detail your microscope can distinguish from the background or other closely related details

resolution equation

$resolution=\frac{0.61~\lambda}{numerical~aperture}$ where numerical aperture = $nsin(\theta)$

n: refractive index of the material between the sample and the lens ex: air = 1, oil =1.5

• NA is analogous to the cone of light coming from the specimen that reaches the lens

what is the rayleigh criterion

it defines the minimum angular resolution (minimum distance at which two objects need to be, to be seen as distinct object and not a blurred blob)

1. when light passes through a circular aperture ex: camera lens → light isn’t focused into a perfect point → produces an airy disk

2. Airy disk → central bright spot surrounded by concentric faint rings

3. criterion states that → two points are resolved when the center of the diffraction disk of one image falls on the 1st minimum (dark ring) of the other diffraction disk

angular separation formular (no need to memorise?)

$$\theta=1.22\frac{\lambda}{D}$$

D :diameter of the aperture

this equation basically tells you

1. the larger the aperture the better your resolution will be

2. the smaller your wavelength (blue or UV light) → the better your resolution → but you lose penetrative power

types of light microscopes

1. upright transmittance/transillumination microscope

2. fluorescence microscopy

3. epifluorescence microscopy

4. confocal microscopy

5. multiphoton microscopy

6. light sheet microscopy

7. super resolution microscopy

8. label-free microscopy

9. raman spectroscopy

transillumination microscope

transmittance mode:

• light passes from → lamp bellow the sample → condenser → through sample → into objective lens

• contrast derived from absorption of light and a bit from scattering → absorption described using beer lambert’s law

reflectance mode:

• light source is located above the sample → light reflects on the surface of the sample → returns to the observer’s objective lens

• used for opaque samples that does not let light through

what are the limitations of transmittance microscopy → how do we overcome them?

1. in thin samples: intrinsic differences in absorption are low compared with the large transmitted light from background → endogenous contrast is low

2. in thick samples: cells and structures are stacked on top of each other → increases scattering → increases blur

for thin samples contrast is improved using exogenous contrast agents to increase absorption ex: haematoxylin and eosin

• haematoxylin: stains nuclei ribosomes and chromatin purple blue

• eosin: stains cytoplasm, collagen and connective tissue pick

what is the basis of fluorescence microscopy

1. fluorophore absorbs a light photon → probability of absorption is described by the extinction coefficient

2. energy excites electrons to a higher energy level → when then fall back to stable level they produce visible light

   1. quantum yield → ratio between number of emitted fluorescent photons vs no. of absorbed photons

   2. lifetime: average time a fluorophore is in the excited state

3. stoke shift: difference in emission and excitation wavelength → allows us to filter for the emitted wavelength → improves SNR

stoke shift

$$stoke~shift = \lambda_{emission} - \lambda_{excitation}$$

typically emission wavelength is larger than the excitation due to the loss of energy

what is multiplexing

adding different fluorophores to a single sample to distinguish different molecules and biological processes

difference between fluorescence and phosphorescence

fluorescence → instantaneous production of excitation light which stops immediatly when light source is removed

phosphorescence → delayed emission of excitation light which persists after source is removed (glow in the dark effect)

how are fluorophores tagged?

1. fluorophores are linked to an antibody specific to a cellular protein/antigen

2. naturally fluorescent proteins can be genetically engineered to be produced in an animal cell ex: green fluorescence protein

what is epi-fluorescence microscopy

1. light source produced above the samples if filtered → only excitation wavelength is let through

2. light hits sample and both excitation and emitted wavelengths are reflected back

3. dichroic mirror filters out emittance wavelength

produces a black background where only the emittance light makes it to the observer

limitations of fluorescence microscopy

1. limited resolution → 200-300

2. out of focus noise: some of the emitted light is scattered within the sample

   1. when an objective focuses on a point it captures 2 cones of light → the focal plane and “wings” which are out of focus

   2. light beam excites a central area but also some unintended areas around it due to it being a cone

   3. produces a halo around the actual central beam which decreases SNR

   4. this issues becomes worse with depth

3. photobleaching: repeated illumination of fluorophores damage them

4. phototoxicity: interaction between excitation light and fluorescent light can damage cells (especially short wavelength/high energy)

5. low number of clinically available probes

how are out of focus noise decreased?

1. confocal fluorescent microscopy

2. light sheet microscopy

3. multiphoton microscopy

confocal microscopy

1. a lazer is used to decrease the volume of the light beam → reduces the cone of out of focus light → increases resolution

2. a pinhole is placed in front of the detector → reduces the amount of out of focus light → improves imaging depth as well

3. lazer is scanned across the imaging plane

4. stage can be moved in the z axis → samples is moved thought the focal zone → allows for optical sectioning → visualise tissue at specific depths → 3D image (confocal laser scanning microscopy) → only possible due to good out of focus rejection

   1. increases acquisition time

   2. depth is still limited to 500 microns due to light loss in tissue

multiphoton microscopy principles

1. instead of a single high energy/frequency (blue) photon → 2 or more photons at half the energy (infrared) are used to excite the sample → double the wavelength → less attenuation and scattering

2. if both photons hit the fluorophore at the same time their energy is summed up → emites the same photons as the single high energy photon would produce

3. due to non-linear absorption → multiphoton absorption is extremely rare and would only occur at the centre of the focal point due to it’s high photon density

multiphoton microscopy pros and cons

1. since longer wavelength/low frequency IR light is used → less scattering and absorption → increased imaging depths (1,000$\mu$m compared to 50-100$\mu$m)

2. large stoke shift between IR and visible light → easier filtering of excitation light from emission light

3. due to non-linear absorption → nearly all of emitted light must come from the focal spot centre (out of focus light suppressed) → increased spatial selectivity and sensitivity

4. lasers can be pulsed and has higher flux → increased probability of 2 photon absorption

cons → high acquisition time

light sheet microscopy

1. aims to reduce acquisition time by using a light sheet instead of a point source → produces plane by plane scanning (2D)

2. The laser sheet can be physical/optical (shaping the laser beam) or digital (integrating a line scanning of points in time that moves much faster than the acquisition time of the detector

3. this reduces photobleaching as there is a smaller irradiated area

4. can be used in conjunction with multiphoton excitation

disadvantage of light sheet microscopy

striping artifacts

1.  sample features that scatter, absorb, or otherwise perturb the incident beam upstream result in weaker downstream illumination, visible as dark “stripes”.

   • Change in beam intensity the deeper it travels through the tissue

   • Corrected by modulating the light or post image processing

what is super resolution microscopy and what are the types

fluorescent imaging techniques that have a resolution bellow classical diffraction limits (<200-300nm)

1. stimulated emission depletion microscopy

2. structured illumination microscopy

3. stochastic optical reconstruction microscopy

stimulated emission depletion

2 lasers are focused at the focal place:

1. excitation laser

2. depletion laser: suppresses the excited fluorophores near the excitation focal point via stimulated emission (basically photobleaches the fluorophores)

produces a donut shaped ring of suppressed fluorophores causing only a very small centre dot to be fluorescent

structured illumination microscopy

1. diffraction grating is used to illuminate sample (unknown pattern) with a known light pattern → patterns overlap

2. overlapping of patterns produces an interference pattern (beat pattern) called moire fringes

3. These patterns contain information about the sample's detail that would be invisible in a normal microscope

4. Computational method are then used to reconstruct the information from the pattern into a high-resolution image

stochastic optical reconstruction microscopy

1. photo switchable fluorophores are used → their fluorescence can be controlled to emit a sufficient fraction of light for only a limited amount of time before bleaching to a dark state

2. fluorophores are excited using a laser and are trapped in a dark triplet state by a chemical buffer

3. a weak activation laser then illuminates the sample → only provides enough energy for a random percentage of molecules to exit dark state to glowing state

4. produces a stochastic rate of random fluorophores blinking

5. several images are taken over time → since only a few molecules are visible in each frame → system treats them as a single point → increases resolution (20nm)

what is label free microscopy + what are the types

instead of just tracking absorption we track chromophores → they absorb light differently and different wavelengths (intrinsic optical signal) → this change can be tracked to infer molecular composition or physiology

1. multispectral and hyperspectral imaging

2. raman imaging

multispectral imaging

1. multiple images are acquired using different continuous wavelengths of light across the whole electromagnetic spectrum

2. captures the difference in chromophore light absorption at that wavelength

3. assembles data into a 3D hypercube → x,y axis denotes positional information + z axis denotes wavelength absorption spectrum

hyperspectral imaging pros and cons

pros:

1. quantitative information on the chemical composition of chromophores

cons:

1. heavy computational loads → more wavelength + more pixels

2. requires complex modelling of light pathlengths into tissue for quantification

3. poor depth resolution as it is very susceptible to the effects of scattering on spatial resolution → superficial layers or thin samples only

raman spectroscopy

1. detects inelastic raman scattering → incident light hits molecule and deviates from its path → some of the energy is absorbed by the molecules → produces a raman scattered wave with lower energy

2. since scattered wavelength has longer wavelength than original it produces a similar effect to the stoke shift

3. however this interaction has a low occurence therefore → high-intensity source (lasers) and long acquisition times (minutes to hours) are needed

4. the wavelength of the scattered light is dependent on chemical structure of the sample → each peak corresponds to a specific molecular bond vibration

why is tissue clearing important

1. scattering is the main limiting factor for depth resolution → mainly caused by lipids in cellular membrane → needs to be removed whilst preserving fluorophores and cellular matrices

2. need to match tissue refractive index with that of the suspending medium

what are the types of tissue clearing techniques

1. organic solvent method

2. hydrophilic method

3. hydrogel-based methods

organic solvent tissue clearing

1. sample stained with antibody

2. sample completely dehydrated → causes sample to shrink (may destroy certain structures)

3. lipids removed using organic solvents

4. sample immersed in refractive index matching organic solvent

hydrophilic tissue clearing method

1. sample is decolourised

2. detergent is used to remove lipids

3. samples is then stained

4. placed in RI matching water soluble reagent

no dehydration step but can cause tissue enlargement

hydrogel based tissue clearing method

1. synthetic gel is made from monomers and polyepoxide → acts as a structural matrix to support tissue after delipidation

2. delipidation performed using detergents

3. sample is stained

4. sample placed in RI matching solution

requires longer and more complex preparation process