Imaging and Microscopy

Question 1

What does the term Resolution mean in microscopy

Answer 1

Resolution is used to describe the ability of a microscope to distinguish details of a specimen/sample. AKA the minimum distance between 2 distinct points of a specimen where they can still be seen

Question 2

What are the limitations of a normal wide wield microscopy

Answer 2

• The resolution is around 200 nm
• Have to use sections –> if you aim to visualise on a deeper level, wide field microscopes can only view cut sections/part of a tissue instead of the whole depth
• EX-vivo biopsy samples

Question 3

What is the differences between Confocal microscopy and wide field microscopy

Answer 3

• Focal plane: Direct the view to the only selected part and blur out other parts that are not on the same plane
• Pinhole: Allows only focused reflected light coming from focal plane to reach the detector, thus other parts that are unable to go through the pinhole will appear dark

Question 4

Is the resolution better in confocal compared to the wide field?

Answer 4

No, the images are better however the resolution might be slightly better

Question 5

What is STORM microscopy?

Answer 5

STORM: STochastic Optical Reconstruction Microscopy

Takes control over which fluorephore is turned on. So switchable fluorophores that, when beamed by light, can change its confirmation between bright and dark state (ON/OFF) at different time points

Question 6

What does SIM targets?

Answer 6

SIM= Structured Illumination Microscopy

SIM targets the lower energy spots to compute a more detailed version of it. Low- and High energy patterns can be combined, directed at an angle that creates a strong, clearer signals of an image

Question 7

When do you apply STORM and SIM

Answer 7

For STORM and SIM special advanced microscopes: expertise are needed.
STORM is mainly used for specific research
SIM can also be used for specific research, but can also be used for more routine diagnostics as an alternative for Electron microscopy

Question 8

Can we have super resolution images with a standard microscope

Answer 8

YES! We can expand 2 viewpoints further apart by EXM (Expansion Microscopy)

Question 9

What are the steps for EXM

Answer 9

1. Fix sample
2. Anchor + polymerise: Spot 1 wants to view crosslinked, and will NOT be affected spatially during digestion and expansion
3. Digestion –> labelling
4. Expand with water

Question 10

How can you make a tissue transparent

Answer 10

You have to avoid differences in refractive index. Most lights are blocked by lipids. Removal of lipids and add solvents to match the refractive index (RI)

Question 11

What can you study with in vivo imaging and microscopy

Answer 11

Study biological processes and structures

Question 12

What reasons do drug treatment fail

Answer 12

• Incomplete tissue penetration: the mAb cannot penetrate the tumor or there is limited uptake. It will only bind to to the first few tumorcells. In 3D/monolayer, this won’t be an issue
• Heterogenous tumor population: mAb reaches target, however there is a mixed expression of drug targett. The drug will be taken up in the targeted tumor cell. Tumor cells are all different so the target may not be present in all cells
• Off target uptake: the drug targeting the nucleus ends up in cytosol, the drug is incorporated in the lysosome or completely degraded. The drug did enter the cell, however did not end up where it should be. Only in cytosol is staining