Advanced Imaging Technologies

Advanced imaging technologies
Summary
1. Imaging experiment workflow:

a. Overview of steps in imaging experiments, including sorting
samples, live cell imaging, and whole organ imaging.
b. Importance of choosing the right microscope and accessories
for specific imaging tasks.
2. Live cell imaging:
a. Parameters to consider:
i. speed
ii. resolution
iii. sample environment (temperature, CO2, humidity)
iv. dyes
v. sample movement
vi. focus stability
vii. imaging interval
viii. phototoxicity/ photobleaching.
b. Suitable microscope:
i. Widefield
ii. Multipoint
iii. lightsheet.
3. Widefield and spinning disk microscopy:
a. Widefield microscopy involves fluorescence excitation and
focal plane imaging.
b. Spinning disk microscopy offers high speed, lower
photobleaching, but more out-of-focus signals compared to
laser scanning confocal microscopy (LSCM)
4. Whole brain and organ imaging
a. Challenges:
i. light scattering by tissue
ii. limited penetration depth of short wavelengths
iii. the need for large working distance.
b. Suitable microscopes:
i. One-photon
ii. Two-photon
iii. Spinning disk
iv. Lightsheet microscopy (depending on organ size and
transparency)
5. Two- photon microscopy
a. Advantages
i. Reduced phototoxicity
ii. Reduced dye bleaching
iii. Greater imaging depth
iv. Better signal-to-noise ratio
b. Disadvantages
i. Expensive
ii. Large
iii. Complex laser
iv. Not suitable for infrared-absorbing cells
6. Optical tissue clearing
a. Enhances imaging by reducing light scattering in tissues,
making them more transparent for deeper imaging.
7. Separating fluorophores with similar spectra
a. Techniques for separating similar dyes using specific
microscopes.
b. Example: autofluorescence in tissues, and its separation from
specific fluorescents markers like GFP.
8. Fluorescence lifetime imaging (FLIM)
a. Measures the characteristic ‘lifetime’ of fluorescence events to
distinguish between different fluorophores based on their
decay time.
Recurring ideas
1. Optimization and planning:

a. Emphasis on thorough planning and understanding of the
sample and imaging requirements.
b. Importance of pilot experiments and control measures.
2. Challenges and solutions:
a. Continuous focus on addressing challenges such as
phototoxicity, light scattering, and dye separation.
b. Solutions include choosing the right microscope and
optimizing imaging parameters.
3. Technological integration:
a. Integration of advanced technologies like FLIM, spinning disk
microscopy, and optical clearing to enhance imaging
capabilities.
Questions
What parameters must be considered when performing live cell imaging,
and why are they important?
- Speed:
o determines how quickly images can be captured, which is
crucial for observing dynamic processes.
- Resolution:
o affects the clarity and detail of the images.
- Sample environment:
o Conditions like temperature, CO2, and humidity must be
controlled to keep cells alive and functioning normally.
- Dyes:
o Selection of appropriate dyes is important for specific labeling
and minimizing phototoxicity.
- Sample movement:
o Needs to be minimized to avoid blurring.
- Focus stability:
o Ensures consistent imaging over time.
- Imaging interval:
o The frequency of image capture, balancing temporal
resolution and phototoxicity.
- Phototoxicity/ photobleaching: reducing these effects preserves
sample integrity and fluorescence signal over time.
- Data storage: efficient storage solutions are necessary for managing
large volumes of imaging data.
How does spinning disk microscopy differ from laser scanning confocal
microscopy (LSCM), and what are its advantages?
- Spinning disk microscopy:

o Uses a disk with multiple pinholes to scan the sample,
providing high-speed imaging with reduced photobleaching.
- Advantages:
o High speed: faster image acquisition compared to point
scanning in LSCM.
o Lower photobleaching: less damage to the sample due to
lower light exposure.
o Drawbacks: lower Z resolution and more out-of-focus signals
compared to LSCM.
What are the benefits and drawbacks to two-photon microscopy for deep
tissue imaging?
- Benefits:
o Reduced phototoxicity: less damage to the sample due to
longer excitation wavelengths.
o Reduced dye bleaching: longer wavelengths reduce
photobleaching of fluorescent dyes.
o Greater imaging depth: can penetrate deeper into tissues.
o Enhanced signal-to-noise ratio: better separation of excitation
and emission light.
- Drawbacks:
o Expensive and complex lasers: requires specialized, costly,
and complex laser system.
o Limited to fluorescence imaging: mainly applicable for
fluorescence-based techniques.
o Not suitable for infrared-absorbing cells: ineffective for
pigmented cells that absorb infrared light.
How does fluorescence lifetime imaging (FLIM) differentiate between

fluorophores, and what is its key advantages?
- Differentiation mechanism:
o FLIM measures the average time a fluorophore spends in the
excited state before returning to the ground state.
o Each fluorophore has a characteristic fluorescence lifetime,
which allows FLIM to distinguish between different
fluorophores based on their decay times rather than just their
spectral properties.
- Key advantage:
o FLIM can differentiate between fluorophores with overlapping
emission spectra, providing more precise and accurate
imaging of multiple targets within the same sample.
What challenges are associated with whole organ imaging, and how can
they be addressed?
- Challenges
o Light scattering: tissue scatters light, reducing image clarity
and depth.
o Penetration depth: short wavelength light cannot penetrate
deeply into thick tissues.
o Large working distance: imaging entire organs requires
microscopes with large working distances.
- Solutions
o Optical clearing: reduces light scattering by making tissues
more transparent.
o Two-photo microscopy: uses longer wavelengths for deeper
penetration and reduced scattering.
o Optimized microscopes: choosing suitable microscopes (e.g.,
spinning disk, lightsheet) depending on the organ size and
transparency.

Advanced Imaging Technologies

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Advanced imaging technologies

1. Imaging experiment workflow:

1. Optimization and planning:

- Spinning disk microscopy:

How does fluorescence lifetime imaging (FLIM) differentiate between

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