Fluorescence Microscopy

Question 0

Using the Jablonski diagram and the principles of the Stokes shift, explain fluorescence.

Answer 0

Fluorescence is a natural property of certain molecules, where they emit light of certain wavelength after absorbing light energy of a lower wavelength (and thus higher frequency and energy). This emitted light can be observed using fluorescence microscopy. The jablonski dg shows the ground state of electrons (S0), and as it absorbs the excitation (light) energy, the electrons are excited to a higher level temporarily. It loses some energy (in forms other than light), known as the non-radiative energy, before falling back into the ground state. As the electron falls back into ground state, the energy it has is released in the form of light, but since some energy is lost, the light has a lower frequency (and thus higher wavelength). On microscopy, this shows up as a different colour of higher wavelength (and the difference in wavelength is the Stokes shift).

Question 1

What is the Stokes shift?

Answer 1

It is the distance between the peaks of the absorbance and the emission spectrum of the light wavelength

Question 2

What is a common structure of fluorescent molecules (fluorophores)?

Answer 2

Aromatic rings

Question 3

Outline the parts of the fluorescent microscope.

Answer 3

Light source (Arc lamp): high intensity, short wavelength
Excitation diaphragm: controls the amount of light passing through
Excitation filter: only allows light of a certain wavelength through, making it monochromatic
dichroic filter: allows light to pass through to the Objective, but reflects the returning light towards the emission filter and Ocular
Objective
Emission Filter: only allows light of certain wavelength through
Ocular

Question 4

Outline the common problems and challenges experienced in fluorescence MS.

Answer 4

• Bleedthrough: when a fluorescent molecule emits with a broad emission peak (or when there is broad collection parameters), the peak can cross over into another peak, and 1 fluorophore can be picked up by 2 channels.
• Blur: out-of-focus light decreases resolution
• Bleaching: excited fluorophores react to become non-fluorescent
• Phototoxicity: light can harm cells
• Background/autofluorescence: intrinsic fluorescence of cells

Question 5

How do you counter fluorescence bleedthrough?

Answer 5

By labelling with only 1 fluorophore and check to see how bad the bleedthrough is in the 2nd channel

Question 6

Define photobleaching and outline the methods to counter this effect.

Answer 6

It is the irreversible destruction of an excited fluorophore.
Counter by: (think how to minimise damage by the light)
Scan for shorter time
Use high resolution, ie high NA (numerical aperture) objective: high NA allows more focused light, therefore smaller area illuminated leading to higher focus and resolution, and therefore less damage
Use wide emission filters (filters out more light?)
Reduce excitation intensity
Use anti-fade reagents (not compatible with viable cells)

Question 7

Outline the 4 approaches in removing Blurs

Answer 7

• Optically: mechanically, basically using better equipment: confocal, 2-photon fluorescence excitation etc
• Computationally: using computer to process stack; Deconvolution (using mathematics)
• Hybrid: Require both approach
• Combination: combine optical and computational approaches

Question 8

Explain the differences between a normal (wide-field) fluorescence ms and a confocal ms by contrasting the parts on the equipments.

Answer 8

Wide-field fluorescence ms floods the entire specimen evenly with the light source, and all parts of the specimen are excited, causing fluorescence (FL), and the resulting FL picked up by the photodetector/camera includes a large unfocused background. However, the confocal ms uses a laser (inst of arc lamp), an Excitation Pinhole instead of a Diaphragm to create a point illumination, has an Emission Pinhole along with the filter (only filter in widefield), to filter out non-specific wavelengths, and has a PMT instead of an Ocular. Therefore only FL close to the focal plane can be detected, and the optical resolution is therefore high. To create a 3D image, it needs to take various sections (optical sectioning). The downside is that it blocks out much of the light at the pinhole, therefore FL signal is decreased, therefore longer exposure time needed.

Question 9

List the uses of fluorophores.

Answer 9

Conjugated Ab
Organelle labelling
DNA/RNA labelling and quantification
Physiological measurements
Analysis of fast dynamic processes
Molecular interaction studies (FRET)

Question 10

What is the excitation and emission spectra of the GFP?

Answer 10

Excitation = 395 and 470 nm
Emission = 509nm (peak)

Question 11

Explain FRAP.

Answer 11

Fluorescence Recovery After Photobleaching.
It’s a technique that measures cellular kinetics, ie the rate at which cellular mechanisms work.
It uses a laser to bleach an area of the cell, and measures the rate at which fluorescence recurs (due to redistribution of fluorescent molecules). Measures the diffusion coefficient and the mobile fraction

Question 12

Explain the concept of FRET.

Answer 12

Fluorescence/Forster Resonance Energy Transfer.
It’s a method used to analyse molecular interactions.
Requires that the molecules to be very close together 10-100 Angstroms.
The absorbance peak of acceptor must match emission peak of donor, ie there has to be significance spectral overlap
We basically use specific wavelength light to excite donor molecule. If there is interaction between donor and acceptor molecules, the emission energy of the donor will be absorbed by the acceptor, and in turn emitted as a higher wavelength (donor transfers non-radiatively to the acceptor). If this wavelength light is seen, then we can confirm interaction between the molecules.

Question 13

Outline the uses of confocal/2-photon microscopy in neuroscience

Answer 13

Axon pathfinding in embryogenesis and regeneration
Signal transduction in growing axons
Analysis of brain cytoarchitecture
Intravital imaging of neuronal signalling

Question 14

Explain the principle of Two-photon Excitation.

Answer 14

Using normal excitation wavelengths (high intensity, low wavelength) eg UV, it may cause damage to live cells. We therefore use Two-photon excitation to create the same effect, by using 2 photons of half the excitation energy needed (therefore double the wavelength). We illuminate sample with light of twice the wavelength, and only if the two photons are absorbed simultaneously will it excite the fluorophore. Emission of 2nd harmonic is at half the wavelength of excitation.

Question 15

Explain the advantages of using 2-photon confocal microscopy

Answer 15

It will only occur at the point of focus (where the 2 photons are taken up by the molecule), therefore no out-of-focus emission
Longer wavelength light (usually infrared) means deeper tissue penetration), less phototoxicity in live cells
2nd harmonic or CARS imaging of unlabelled specimens possible (ie. don’t need to label the cells with fluorophores)

Question 16

Explain the Limits of Resolution (LOR) using Abbe’s Law.

Answer 16

LOR explains that the maximum resolution of light microscopes is limited by certain factors. Abbe’s law (and equation) suggest that d = wavelength/2n sin(theta), where d = minimum resolving distance and n = numerical aperture. The smaller wavelength of light used, the smaller the d and the higher the resolution. The greater the n, which is the determined by the microscope, the smaller the d.

Question 17

Explain the Airy Disc relating to the numerical aperture.

Answer 17

The Airy disc is a disc illustrating the size of the focal spot produced with a certain Objective, determined by the N. If the n is larger, then the Airy disc is smaller.

Question 18

Explain TIRF.

Answer 18

Total Internal Reflection Fluorescence.
It’s when the incident light is reflected using TIR, but there is an evanescent wave (energy wave) that goes across the cover-glass and excites the specimen for 100nm.

Question 19

What are the 3 categories of Super-resolution methods?

Answer 19

1) . Structured Illumination: SR-SIM
2) . Spatially Patterned Excitation: STED; RESOLFT
3) . Localisation methods: STORM; PALM

Question 20

Briefly the concept of the Structured illumination.

Answer 20

SR-SIM: Structured illumination Superresolution microscopy
Diffractive grating in light path, grating is slowly rotated, and the light waves superimposed with specimen, which generates more resolution.
Slow technique, therefore can’t use on live, fast-moving cells

Question 21

Briefly explain the concept of the STED.

Answer 21

Stimulated Emission Depletion
Uses 2 laser sources, the excitation laser beam (subject to Abbe’s law), and the STED laser (hollow in middle). The STED is superimposed on the Excitation laser, and it quenches the activation of the excitation laser where they overlap (depleting its energy- basically driving down the electrons back to ground state before the molecules can fluoresce), allowing only the centred excitation beam to pass through. As a result, the effector spot (Airy Disc) is far smaller (as if the numerical aperture is larger), thus maximizing resolution.

Question 22

Outline the concept behind the Localization Methods.

Answer 22

-STORM: Stochastic Optical Reconstruction Microscopy
-PALM: Photoactivated Localization Microscopy
Basically, these techniques involve fluorophores that could be turned on and off, and imaged separately, and combined together to get entire image. Since there is no overlapping of diffraction (as in conventional FL ms, where we cannot actually see the individual fluorophores as they are blurred), the resolution is much higher. (Think pixels. This techniques creates greater no of “pixels” as we can see the individual fluorophores, whereas we couldn’t in conventional technique as they are blurred)