Roeffaers & Hofkens
Exam 15/06/2023
Question 1 (8pt) Microplastics, minuscule fragments of plastic less than 5mm in size, are becoming
increasingly prevalent in our environment and subsequently, our bodies. (...) A recent study suggests that
the smallest microplastics exhibit pathogenic characteristics, particularly when their dimensions exceed
20 micrometers. Such particles cannot be readily engulfed by macrophages, resulting in a phenomenon
known as 'frustrated phagocytosis', potentially leading to inflammation and disease.
(a) Propose two optical microscopy-based techniques (note: prof zei dat ze alles buiten EM microscopy
bedoelen, source: de prof) that could enable the in vitro examination of the impact of these smallest
microplastics (5-20 micrometer diameter) on macrophages and other cells. Discuss the working
principles of the selected microscopy approaches, the contrast mechanisms of both methods
(including schematics if possible), the ability to visualise the plastics and the cells (shape, response,
...) and the strengths and weaknesses for this specific line of research. (note: ze hadden beter
toegevoegd 'lmao get fucked want ge krijg maar een halve pagina om alles te schrijven' ). (6pt)
Two optical microscopy-based techniques that could be used for in vitro examination of the impact of
microplastics on macrophages and other cells are confocal microscopy and super-resolution microscopy.
1) Confocal Microscopy:
- Working Principle: Confocal microscopy uses a pinhole to eliminate out-of-focus light, allowing for
optical sectioning of the sample.
- Contrast Mechanism: Fluorescently labeled microplastics and cells can be visualized using
specific fluorophores excited by laser light. The emitted fluorescence is detected by a
photomultiplier tube.
- Visualization: Provides detailed 3D images of the interaction between microplastics and cells,
showing their shape, distribution, and response.
- Strengths: Excellent optical sectioning capability, high resolution, and ability to visualize dynamic
processes in real-time.
- Weaknesses: Limited depth penetration, potential photobleaching of fluorophores, and the need
for fluorescent labeling.
2) Super-Resolution Microscopy:
- Working Principle: Super-resolution microscopy techniques, such as STED or PALM/STORM,
surpass the diffraction limit to achieve nanoscale resolution.
- Contrast Mechanism: By using specialized optics or photo-switchable fluorophores, super-
resolution microscopy can visualize structures at the nanometer scale.
- Visualization: Enables the visualization of individual microplastics and their interactions with cells
at a resolution beyond the diffraction limit.
- Strengths: Unprecedented resolution for studying nanoscale interactions, ability to resolve
individual molecules, and detailed imaging of cellular structures.
- Weaknesses: Complex instrumentation, specialized sample preparation, and potential limitations
in imaging speed and sample compatibility.
,(b) Could these techniques be applied to investigate how these microplastics interact with denser cell
clusters or even entire tissues? What considerations need to be made when studying such specimens,
and which methodologies would be most appropriate in these scenarios? (2pt)
These techniques could be applied to investigate how microplastics interact with denser cell clusters or
tissues. When studying such specimens, considerations include the penetration depth of the microscopy
technique, sample preparation methods, and the potential impact of tissue autofluorescence.
For denser cell clusters or tissues, confocal microscopy may be more suitable due to its deeper
penetration depth compared to super-resolution techniques. However, super-resolution microscopy can
provide detailed insights into nanoscale interactions within tissues if sample preparation and imaging
conditions are optimized. In conclusion, both confocal and super-resolution microscopy offer valuable
tools for studying the impact of microplastics on cells and tissues, each with its own strengths and
limitations that should be considered based on the specific research objectives and sample
characteristics.
Question 2 (4pt) These false colour images (colour scale at right bottom), are a time series (time stamp
indicated on each image in seconds) were recorded of a fluorescently stained axon. Rationalise which
microscopy techniques were used to record the images of the upper row (Technique A) and lower row
(Technique B).
(Ik doe m'n uiterste best om de twee rijen van 6 foto's te beschrijven hieronder) A: in elke foto zie je ongeveer
6 intensiteitspieken onder elkaar (denk aan de vorm van een axon dus), maar vrij slecht geresolveerd, met
timestamps tussen 0.036s-0.218. B: in elke foto zie je een stuk of 40-50 kleine piekjes (dus al veel beter
geresolveerd dan techniek A) met timestamps tussen 8.667-8.849.
• Based on the description provided for the two sets of images:
• For the upper row (Technique A) with approximately 6 poorly resolved intensity peaks resembling the
shape of an axon and timestamps between 0.036s-0.218s, the microscopy technique used is likely
**wide-field microscopy**. Wide-field microscopy provides a broad field of view but lacks optical
sectioning, resulting in lower resolution images with overlapping signals from different focal planes.
• For the lower row (Technique B) with 40-50 well-resolved small peaks and timestamps between
8.667s-8.849s, the microscopy technique used is likely **confocal microscopy**. Confocal
microscopy offers optical sectioning capabilities by using a pinhole to eliminate out-of-focus light,
resulting in higher resolution images with improved contrast and clarity compared to wide-field
microscopy.
,Exam 20/08/2021
Vraag 1
Je hebt asbest nanotubes van 100nm dik, 10 µm lang in uw cel. Stel 2 optische microscopie methoden voor
om die nanotubes te gaan visualiseren in uw cel.
Geef bij elk het principe schematisch weer. Geef voor en nadelen van elke microscoop. Welke zou je zelf
gebruiken? (4,5 punten). Als we in tissue of dikke cellagen asbest nanotubes willen visualiseren, welke
technieken kan je hier dan het beste voor gebruiken? (1 punt). Zou je een molecule van 3000 cm^-1 Raman
scattering kunnen detecteren samen met fluorescentie als je een ook een fluorescentie doet met een laser
van 463 nm golflengte. Spectrum van de fluorofoor is gegeven. (! Raman scattering is een zeer zwak signaal
dus een beetje overlap met het emissiespectrum betekent dat je het niet kan waarnemen) (2 punten).
• To visualize the asbestos nanotubes of 100 nm thick and 10 µm long in a cell, the following optical
microscopy techniques can be used:
• Fluorescence Microscopy:
- Principle: Fluorescence microscopy utilizes fluorescent labels that can be attached to the
nanotubes. When these labels are illuminated with a specific wavelength of light, they will emit
light that can be detected to visualize the location of the nanotubes.
- Advantages: High sensitivity, specific labeling of nanotubes, possibility of three-dimensional
imaging.
- Disadvantages: Potential photobleaching of fluorophores, limited penetration depth in thicker
tissues.
- Personal Choice: I would use fluorescence microscopy due to the specific labeling and high
sensitivity.
• Stimulated Emission Depletion (STED) Microscopy:
- Principle: STED microscopy utilizes an excitation and depletion laser to enhance resolution by
turning off fluorophores around the focal point, allowing the nanotubes to be observed with higher
resolution.
- Advantages: Superior resolution, possibility of high-resolution three-dimensional imaging.
- Disadvantages: More complex setup, higher costs.
- Personal Choice: For visualizing asbestos nanotubes in cells, I would choose STED microscopy for
its superior resolution.
• For visualizing asbestos nanotubes in tissues or thick cell layers, techniques such as confocal
microscopy or multiphoton microscopy can be used due to their optical sectioning capabilities and
depth penetration in thicker samples.
• Regarding the detection of a molecule with a Raman scattering frequency of 3000 cm^-1 along with
fluorescence when using a laser of 463 nm wavelength, it may be challenging to detect the Raman
signal if there is overlap with the emission spectrum of the fluorophores. Raman scattering is indeed a
weak signal, and overlap with the emission spectrum can cause interference, making it difficult to
accurately detect the Raman signal. It is important to carefully consider the spectral properties of both
Raman scattering and fluorescence to minimize any interference.
, Vraag 2
For the two situations of time-lapsed imaging of axons, one with lower resolution and the other with better
resolution, different fluorescence techniques can be used, depending on the description of the images
and the time intervals between them.
• Lower resolution time-lapsed imaging:
o For lower resolution time-lapsed imaging of axons, where the images are less sharp and may
exhibit more blurriness, wide-field fluorescence microscopy can be used.
o Explanation: Wide-field fluorescence microscopy illuminates the entire sample at once,
providing a wide field of view but with limited optical sectioning capabilities. This can result in
images with lower resolution and more blurriness, especially for three-dimensional structures
like axons.
• Better resolution time-lapsed imaging:
o For better resolution time-lapsed imaging of axons, where the images are sharper and more
details can be observed, confocal fluorescence microscopy can be used.
o Explanation: Confocal fluorescence microscopy utilizes a pinhole to detect only light from the
focal plane, allowing for optical sectioning images with improved resolution and contrast. This is
particularly useful for visualizing structures like axons with more detail and clarity.
• Based on the description of the images and the improved resolution in the second situation, it is likely
that confocal fluorescence microscopy was used due to its ability to obtain optical sections and
capture better resolution images of axons.
Exam 10/06/2021
Vraag 1
Photos of the same actin structure in a cell: The photos were taken using fluorescence microscopy,
where actin filaments were labeled with a fluorescent dye that fluoresces under specific illumination.
• Photo 1 (lower resolution, black background):
- The first photo was most likely captured using wide-field fluorescence microscopy, where the
entire sample is illuminated and captured at once, resulting in an image with lower resolution
and a black background signal.
• Photo 2 (higher resolution, black background):
- The second photo is likely obtained using confocal fluorescence microscopy, which uses a
pinhole to detect only light from the focal plane, resulting in higher resolution and a sharper
image of the actin structure with a black background.
