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College aantekeningen Cellular Imaging in Four Dimensions (NWI-BM016C) college 5 t/m 7
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College aantekeningen Cellular Imaging in Four Dimensions college 5 t/m 7
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L5: Dynamic imaging of cancerDATE Feb 26, 2021
DONE
LECTURER Katarina Wolf
LEVEL
Revision 1
Revision 2
Revision date
Revision documents
SLIDES
WEEK WEEK 5
Word count 2541
Table of contents
Table of contents
Summary
Confocal microscopy
Infrared multiphoton microscopy and window models
Summary future
Lecture
Brightfield microscopy
Confocal microscopy 1 photon microscopy)
Principle
Fluorescence vs. reflection microscopy
2D → 3D → 4D image processing
Single cell migration in collagen in vitro
Scientific questions
Multiphoton microscopy
The principle
Fluorescence/Second/Third harmonic generation
Advantages IR
Window models
L5 Dynamic imaging of cancer 1
, Limitations (pitfalls)
FLIM Fluorescence lifetime imaging microscopy)
Tumor growth, invasion (metastasis) and therapy: Connective tissue: mouse in
vivo, human ex vivo
Exam Questions:
Question 1
Question 2
Summary
3D and 4D imaging processing and depiction
Confocal microscopy
Fluorescence and reflection with virtual slicing
Infrared multiphoton microscopy and window models
Monitoring of cancer progression at cub-cellular resolution in vivo over
time.
Decreased animal numbers (because of window models)
Fluorescence from red-shifted fluorophores
SHG/THG to visualise tissue structures without labelling
80100% deeper tissue penetration
Reduced photobleaching and phototoxicity
FLIM to monitor environmental conditions.
Summary future
Access to deeper tissue areas, e.g. microendoscopes and adaptive optics
(i.e. bended mirrors to cope with scattering) or longer IR wavelengths.
Improved visualisation of biological complexity
New genetic mouse models
Incorporation of multiple biosensors
Characterisation of molecular processes such as intracellular signalling.
Automation of data extraction, quantification and visualisation
L5 Dynamic imaging of cancer 2
, Lecture
Invasion is the going of the tumor cells into tissues they would normally not be
present.
They often use the existing morphology to grow.
Collagen or blood
→ Intravasation: go into the neighbouring bloodstream
→ Extravasation: this will lead to metastasis
→ In vivo
A mouse is the most similar organism to perform research on.
Research is performed on connective tissues
Mice are expensive
It can be torture
Used as: window models, cancer endpoint models
→ A lot of in vitro models are used
3D collagen models.
You pour a liquid of tumor cells in the model
Or 2D models
→ Ex vivo
Skin slices
Brightfield microscopy
Tumor cells can migrate in a 3D collagen matrix, this can be seen with a
brightfield microscope.
Some cells are more focussed than others, this means that there are
multiple layers.
→ Confocal microscopy is able to have multiple layers in focus at once
L5 Dynamic imaging of cancer 3
, Confocal microscopy (1 photon microscopy)
Principle
The principle:
You have a laser that shoots in photons, you have a beam splitter 500 nm)→
this means that lights of smaller objectives will pass. The reflected light, goes
through a pinhole into the detector.
In focus = in focal plane.
Fluorescence is emitted in different layers of the object.
Only the light which is in the focal plane can go through in the pinhole.
Two most important characteristics
Pinhole
Why? It blocks out-of-focus light and only in focus images will be
collected.
The pinhole is a knife which can "slice" multiple images
The "knife" needs to move → next point
The object table is adjustable in height
Why? In order to catch multiple slides of the sample & more than one
layer of focal planes will be imaged.
→ the fact that you can image multiple layers is really conventional in contrast
to regular fluorescence microscopy.
Pinhole airy: only collects the in focus light → you can make a sharp image.
Pinhole open: collects all the light from all focal planes → you make a blurry
image, but you see more layers.
A migrating cell forms pseudopodia around its polar body.
Collagen fibers will lay in an angle.
L5 Dynamic imaging of cancer 4
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