1
Q
What is the correlation between the length of the wavelength and the energy of an individual photon?
A
The shorter the wavelength, the higher the energy of an individual photon
2
Q
What is the Jablonski Diagram?
A
- Shows energy states of a hypothetical molecule
- If photon of light absorbed, energy state of electron raised
3
Q
How quick is phosphorescence in comparrison to fluorescence? What is it?
A
- Energy from the sunlight can charge and then emit light later
- Very slow manner 10^-1, 10^-2s
4
Q
What is the speed of fluorescence emission upon excitation?
A
- Very quick (nearly instant)
- <10^-6 usually of the order of 10^-9
5
Q
What are the two peaks when looking at a graph detailing intensity (Y) and wavelength (X) of a fluor?
A
- Area of excitation
- Area of emission
6
Q
What are 4 different terms for molecules that exhibit fluorescence?
A
- Fluorochromes
- Fluorophores
- Fluorescent probes
- Colloquially flours
7
Q
How many features of fluorescence are there?
A
3
8
Q
What is the first feature of fluorescence?
A
Emitted light always has less energy than the absorbed loght (some energy is lost in the process)
9
Q
What is Stoke’s Law?
A
Emitted light is of a longer wavelength, the shift in wavelength is known as Stoke’s shift
10
Q
In what direction along the spectrum does emitted light always move from excited light?
A
Shifts to the right
11
Q
What is the second feature of fluorescence?
A
The emission spectrum is characteristic of that substance
12
Q
Is the plot for each fluorochrome the same?
A
NO
Absorbtion (excitation) and emission (fluorescence) spectra can be plotted for each fluorochrome
13
Q
Where is fluorescein’s peak excitation and what does this mean?
A
Light at 480 is the most effective to cause an excitation
14
Q
What is the third feature of fluorescence?
A
- The whole process is inefficient
- A lot of energy has to be used in order to see detectable fluorescence
15
Q
What is the Energy yield of fluorescence?
A
- 0.1-1%
- Light source causing the excitation has to be 100-1000 times bigger in order to produce a visual yield
16
Q
What are two different methods to establish a fluorescent specimen?
A
- Primary fluorescence
- Secondary fluorescence
17
Q
What is primary fluorescence and what does it mean when using it?
A
- An untreated specimen that is naturally fluorescent
- Autofluorescence
- Can be useful such as certain blood components as do not need to add a dye
- However, must be aware of it if adding other fluorochromes as they may interact
18
Q
What are secondary fluorescents?
A
Fluorophores/chromes used
19
Q
What are 5 essential components of a fluorescent microscope?
A
- Light source
- Primary filter
- Fluorescing specimen
- Secondary filter
- Objective
20
Q
What are two essential factors when considering what light source to use in fluorescent imaging?
A
- The light source has to emit photons with the right energy (lots of photons in the area of peak excitation)
- Light has to have high intensity due to low yield of fluorescence
21
Q
What light is normally used in fluorescent imaging?
A
Broad band white light that is then filtered
22
Q
What is a short-pass filter?
A
- Allow light at shorter wavelengths to pass through while blocking longer ones
- The labels added (e.g. SP440) means that after this wavelength light will no longer pass through- it is a maximum
23
Q
What is a long pass filter?
A
- Filter that transmits light with wavelengths longer than a specific cut-off value while blocking shorter wavelengths
- The labels added (e.g. LP40) means that nothing below this wavelength can pass through- it is a minumim
24
Q
What is a band pass filter?
A
- A combination of a short pass filter and a long pass filter
- Is critical for selecting specific emission wavelengths
- Contains a second number (e.g. BP405/14). This tells us how wide the filter is, the smaller the width, the more specific the filter as we reduce the light going through the gap
25
How can the two intensities from one fluorochrome be controlled in order for visability?
- Can add long pass and short pass filters to the excitation wave and a much larger long pass alone to the emission wave
- Means the excitation peak is contained and the emission light is free to pass through to visability
26
Is it possible to use two fluorochromes in one instance?
- Yes, filters can be chosen so that two fluors can be seperated
- Have to make sure that the fluorescence of the two excitation wavelengths are far enough away that the emission does not overlapp
27
What is the difference between a transmitted light fluorescence microscope and a incident light fluorescence microscope (epifluorescence)?
**TLF**: Light passes through the sample, with a second filter in the objective.
**Epifluorescence**: Light is directed onto the sample, using a dichroic mirror to separate excitation and emission light.
28
How does a chromatic beam splitter work?
* A chromatic beam splitter separates excitation and emission wavelengths in fluorescence microscopy.
* It reflects shorter excitation wavelengths toward the sample and allows longer emission wavelengths to pass through to the detector.
* Paired with excitation and emission filters, it ensures efficient excitation and accurate detection by blocking unwanted light.
29
What is a benefit of a chromatic beam splitter over a second filter?
Chromatic beam splitters efficiently separate light by wavelength with minimal signal loss, enabling simultaneous wavelength separation, reducing optical complexity and improving speed and alignment compared to using a second filter which requires and extra step and loses signal.
30
What are 3 important aspects of fluorescent objectives?
- Objective should have high numerical aperture
- Immersion objectives important
- The fewer the lenses the better (apochromatic lens helps with broadband light but here we only have a single wavelength and no fringe, having multiple lenses would make the light less intense)
31
What are 2 important applications of fluorescence microscopy?
- Study of structural and function of living systems at the cellular level
- Relationship and movement of cellular components and cells within tissues
32
What are 4 limitations of the fluorescence microscopy method?
| Not of fluorescence
- Obtaining a specific marker (the dye istelf has a size as it is a protein, so would this change how the system behaves)
- Visualising the marker deep in tissues
- Maintaining the fluorescence (We use up the fluorescence every time it is read out so do not want to bleach or use it up before applying it to our sample)
- Making sure specimen is not damaged and normal behaviour occurs
33
What are 3 developments that are underpinned by advances in fluorescence miscroscopy?
- Fluorescent probe chemistry
- Molecular biology
- Lasers (high intensity at a speciic wavelength) and fast computers
34
What are 5 practical issues with fluorecence microscopy?
- Autofluorescence
- SPectral cross-talk
- Photobleaching
- Blur
- Quenching
35
What is quenching in fluorescence microscopy?
Quenching in fluorescence imaging refers to any process that decreases the intensity of fluorescence emitted by a fluorophore.
It reduces the amount of detectable signal, which can affect the quality and interpretability of fluorescence imaging experiments.
EXAMPLES:
• Collisional quenching: The excited fluorophore interacts with another molecule (a quencher), causing the energy to dissipate non-radiatively instead of being emitted as light.
• Static quenching: A quencher forms a stable complex with the fluorophore, preventing fluorescence emission.
• Resonance energy transfer (e.g., Förster Resonance Energy Transfer or FRET): Energy from the excited fluorophore is transferred to another nearby molecule instead of being emitted as fluorescence.
• Photobleaching: Over time, exposure to light can chemically degrade the fluorophore, permanently reducing fluorescence.
36
What is an example of a fluorescent DNA dye?
- Hoescht33258
- Taken up by live cells
- Allows myotubes to be identified
36
What is immunofluorescence?
Coupling a fluorochrome to an antibody to study an antigen
Having a specific epitope means we will only have fluorescence in the specifiic area
37
Who came up with immunofluorescence?
Coons, 1940
38
Read GFP paper
2008, Shimomura, Chalfie, Y.Tsien
39
What is fluorescence resonance energy transfer?
- 2 fluorochromes whose excitation and emission spectra overlapp (emission wavelength of the first is chosen so that it is the excitation of the next)
- If near enough, can transfer electrions and cause emission
40
What are two issues with conventional fluorescence microscopy?
- Bleaching over the sample
- In thick specimens there is a mixture of in focus and out of focus fluorescence blur surroundong the focal point
41
Who and in what year was the confocal miscroscope invented?
- Mavin Minsky
- 1955
42
When did development of the confocal microscope occur and why wasn't it earlier?
- 1980s
- There wasn't the technical availability earlier
43
Name 7 advances that allowed the development of the confocal microscope.
- Fast digital computers
- Elegant software
- Lasers to provide bright light sources
- Better mirrors
- Sensitive charge-coupled imaging devices
- High resolution displays
- New fluors
44
What is the confocal principle?
* The confocal principle relies on a pinhole placed conjugate to the focal point of the lens (for the detector) to block out-of-focus light, ensuring only light from the focal plane is detected.
* A small volume of the specimen is illuminated and imaged at a time, and scanning is required to construct the full image, enabling high-resolution, optical sectioning and 3D imaging.
45
Can you visualise a confocal image instantly?
- No
- Have to use a computer to reconstruct later
46
What is a confocal microscope reliant on for later scanning?
- A detector
- Highly sensitive which detects the intensity of the fluorecence emitted
47
In confocal microscopy, what does a smaller pinhole equate to?
- More precise image
- But less light coming through
- Have to compromise
48
What do pinholes in confocal microscopy ensure about the entry of light?
Light only comes from the focal plane (all the other light hits outisde the focal point)
49
What are two ways in which we can image a larger sample in confocal microscopy?
- Move the stage in small steps to build a matrix of intensity (not very fast)
- Use a dichroic smart mirror to precisely tilt the mirror to scan the beam of excitiation and emission along the sample
50
Where are the two pinholes located in confocal microscopy?
- One for the laser
- One for the detector
- Ensures the focus planes are aligned with each other
51
What are 4 experiments that White et al 1987 did using confocal fluorescence?
1 - Tissue culture cell labelled with microtubule antibody
2- Individual microtubules resolved with confocal (way higher resolution)
3 - Additionally fertilised egg of a sea urchin stained with anti-tubulin
- Could see the goings on inside the cell
4 - Spherical plasmacytoma cell stained with an antibody against endoplasmin
- The spherical structure could be dissected
52
What are 7 factors that make up a commercial confocal microscope?
- Detector
- Detector aperture
- Beam splitter
- Argon laser (tuneable lasers and filters for different wavelengths for the detector and light)
- Moveable galvanic mirrors
- Eyepiece
- Objective
53
What is used to place the sample in a confocal microscope?
Bright field microscope
54
What controls movement and image acquisition in a confocal microscope?
- A computer
- Can even select the fluo used, the computer then selects the correct filters
55
What is the relationship between lasers and fluors in confocal microscopy?
- Can create lasers that match the excitation spectrum of the fluors
- Different lasers emit different wavelengths which need to be matched to the fluor
- Can select a laster to excite two fluors if their excitation spectrum overlaps
56
What is a benefit of using on laser to excite two fluors and a benefit of using two seperate lasers in confocal microscopy?
**One laser**: Simultaneous imaging with reduced photobleaching, suitable for dynamic studies (looking for changes).
**Two lasers**: Optimised excitation for dim signals, better spectral separation, and improved image clarity.
57
What are two essential imaging considerations when constructing a 3D reconstruction of a data set in confocal microscopy?
- The thickness of the slice selected by the size of the pinhole- the smaller, the thinner (reduce the intensity of the light coming out)
- The step size between the spectrums- how big of a step do we need through the sample before making the next slice?
58
What is a single maximum projection in confocal microscopy?
- Put different imaged sections on top of each other
- Look down the stack and select the pixel of the highest intensity
- Then move onto the next pixel etc
- Get the highest intensity from all the sections
- Software does this now
59
What are 3 problems with confocal microscopy that 2 photon miscroscopy overcomes?
- Still some photobleaching outside the focal plane (due to hourglass illumination)
- Phototoxicity- some chromophores create phototoxicity in the excitation and emission process which damage living cells
- Poor penetration
60
Who developed the 2-photon excited fluorescence microscopy?
Denk and Webb
61
How does 2-photon microscopy work?
- For Fluorescence Microscopy.
- Uses two photons at the same time to deliver the same energy as a single photon
- Each photon uses double the wavelength (half the energy)- combined has the same effect of a single photon
- Need to have the energy close to the focal point (if gap is too big, there will not be the density to cause excitation)- need enough photons at the same time to interact
62
Why does 2 photon microscopy cause less bleaching?
Excitation only at a very small spot, only this area can be bleached
63
What are 6 advantages of 2 photon miscroscopy?
- Less photodamage and photobleaching
- IR light penetrates deeper into the specimen
- No out of focus fluorescence
- Can excite UV dye without UV laser (damaging to sample)
- Can excite autofluorescent components
- No pinhole alignment
64
What are 5 disadvantages of 2 photon miscroscopy?
- Needs titanium-sapphire laser (mai tai)
- Cost
- Complicated to run and operate
- Safety issue dur to laser power
- Cannot use on pigmented cells as they explode
65
What is a barrier that all light imaging modalities have?
The diffraction limit
66
What is the diffraction limit?
The diffraction limit defines the smallest structures resolvable with light microscopy, approximately 200 nm for visible light.
It arises from the wave nature of light, which causes light to spread as it passes through an aperture (e.g., a lens or objective).
67
What is the consequence of the diffraction limit for biological research?
Some biological structures are smaller than the diffraction limit such as organelles (Huang et al 2020)
68
What is the numerical diffraction limit?
0.2-0.25 micrometers
(200-250nm).
69
What are three different approaches to overcome the diffraction limit?
- Near field imaging
- Patterned illumination
- Stochastic switching
70
Explain Near-field microscopy.
- Get as close to the sample as possible so the diffraction spreading out does not cause too much blur
- The damage done by the diffraction is therefore not significant
71
What is the resolution of near field microscopy?
<20nanometers
72
What are 2 limitations of near field microscopy?
- Not considered a real super resolution microscopy (you're literally just getting close asf).
- Sample has to be extremely flat- unlike a lot of biological proteins
73
What are two categories of super resolution patterened illumnation microscopy?
- Negative
- Positive
74
What are two types of negative super resolution patterened illumnation microscopy?
- STED (Stimulated Emission Depletion Microscopy)
- RESOLFT (Reversible Saturable Optical Fluorescence Transitions)
75
What are two types of positive super resolution patterened illumnation microscopy?
- SIM (Structured Illumination Microscopy)
- SIMM (Saturated Structured Illumination Microscopy)
76
What does STED stand for (negative patterning)?
STimulated Emission Depletion
77
Outline how STED (negative patterning) works?
- Within the same light path, have two lasers (exciting laser and depletion laser)
- Both of which are bound by the diffraction limit (have a certain patch size)
- Cause excitation in one area, then draw around this area with the depletion laser
- Therefore the drawing means the diameter is smaller than the diffraction limit
78
What are 2 benefits of STED (negative patterning)?
- Prevents fluorophores from emitting light (stimulated emission)
- No additional processing required
79
What is the moire effect?
- Overlapping net patterns have well defines symmetries
- When they move, they create a methmatically defined pattern
80
How is the moire effect used in positive patterning?
- Use the interference of the sample underneath the pattern to calculate from the pattern we know
- Calculate the change and therefore the underlying pattern
81
What does SIM (positive patterning) stand for?
Structured Illumination Microscopy
82
Outline SIM (positive patterning)
- Sinusoidal pattern created by combining two light beams
- Images multiple snapshots of the sample structure and excitation pattern taken and computationally reconstructed.
- Enhances resolution (~100 nm), overcoming the diffraction limit.
- Light pattern used is limited by the diffraction limit
83
What is the lateral dimensions (resolution) of SIM (positive patterning)?
100nm
84
What is SSIM (positive patterning)?
Saturated Structured Illumination Microscopy.
85
Outline SSIM (positive patterning)
- Instead of a single pattern, use a depletion light to make the borders of the pattern sharper- makes the bars slightly narrower than diffraction
- Sufficiently strong excitation, fluorescence emission from a fluoreophore will saturate
86
What is the resolution of SSIM (positive patterning)?
50nm
87
What method of super resolution achieves temporal control over emissions?
- Stochastic Switching - used by STORM and PALM
- Altering the wavelength of light turning fluorescence off and on.
88
Outline how stochastic switching super resolution microscopy works?
- Lower intensity beam than normal (inefficient) fluorescence
- Only excite some of the molecules
- Push the readout beam to read which molecules are active
- Active molecules become bleached
- Re-excite sample with beam
- Some fluors excite (but we can be certain that they are not the same ones as before)
- Repeat
89
What are 2 issues with super-resolution stochastic switching microscopy?
- Very time consuming
- If want to go back to rescan- we cannot as the sample is bleached
90
How can we rescan a stochastic switching sample?
- Use a special dye that goes into rest state once activated
- Therefore, we can return later when it is no longer in its rest state to rescan
91
What are STORM and PALM?
- Stochastic optical reconstruction microscopy
- Photo-activated localisation microscopy
92
How do STORM and PALM work?
- Once an image for each individual fluorophore has been generated their respective locations can be mapped to produce a combined image
- The image is no longer limited by interference from other fluorophores as the density of fluorescence in each image is kept low, but by how precisely each fluorophore is localised
93
What is the lateral resolution of STORM and PALM?
20nm
Imaging in Biomedical Research
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