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College aantekeningen Imaging in Four Dimensions (NWI-BM016C) college 1 t/m 4
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L1: Introduction to microscopyDATE Jan 29, 2021
DONE
LECTURER Koen van den Dries
LEVEL Good
Revision 1
Revision 2
Revision date 09/02/21 - 05/03/21
Revision documents
SLIDES 20200129_CaputIntro_KoenvandenDries.pdf
WEEK WEEK 1
Word count 2589
Household regulations
Cellular imaging in Four Dimensions: Principles and applications
Werner Koopman and Jack Janssen (course coordinators)
An exam with 7 questions, written assay questions
One question for each lecture.
The questions will focus on which microscopical technique is best for which
problem.
Take home messages
Microscopy is fascinating
Microscopy is a rapidly evolving field with major recent breakthroughs
L1 Introduction to microscopy 1
, 💡 Resolution is not the same as magnification
💡 Wavelength and numerical aperture are the main determinants for
resolution
You can study cells without labels with phase contrast microscopy
Fluorescence microscopy allows to specifically label and visualise certain
certain cellular structures
In contrast to widefield, confocal allows 3D sectioning of samples
Lecture
A brief history of microscopy
Around 1600 the first microscopes were made. In the late 17th century
Anthony van Leeuwenhoek) the first real microscope was made which was
able to see bacteria.
Then for a long time, people did not want to believe that there was a limit
which was visible to the eye.
Therefore, microscopes were not looked at for a long time.
In the late 19th century, research started again, mainly in companies. This
speeded up the development a lot.
Around the 1930, the first electrons microscopes were made.
These microscopes deal with electrons, which are "clearer" than photons
(light)
In the last few years, a lot of noble prizes are rewarded to people who
developed microscopes and techniques.
The most important finding in 2008 is the nobel prize for implicating
fluorescent proteins which are found in nature for cell biology microscopy
techniques.
L1 Introduction to microscopy 2
, Resolutions and magnifications
Magnification is not the same as resolution
Magnification: how much bigger a sample appears to be under the microscope
than it is in real life
In practice: determined by objective magnification and eyepiece
magnification
The higher the magnification, the more light capturing ability the lens
has. Therefore, magnification often scales with resolution.
Resolution: the ability to distinguish between two points on an image (the
amount of detail)
In practice: determined by the numerical aperture of the objective and the
wavelength (λ) of the elementary particles used.
200 nm is the golden boundary of what is reachable with a light
microscope
Diffraction of light: the point spread function PSF
PSF the response of a microscope to a point source. An object can be
convoluted with PSF.
Diffraction will cause a spread on your camera.
Point spread function (airy disk)
The distance between the two point sources needs to be more than
200 nm, otherwise the point spread with cancel the image out.
Mostly there is an airy disk of 200 nm.
Object convoluted with PSF → image
Numerical aperture and airy disk size
💡 Higher numerical aperture → smaller airy disk size → better ability to
solve two points
L1 Introduction to microscopy 3
, 💡 Longer wavelength → bigger diffraction → the bigger the point
spread function.
Electrons have 1000 fold smaller wavelength than photons. Therefore it is
better.
Electron microscopy (intermezzo)
In electron microscopy you use magnets instead of lenses
Scanning or transmission
A problem: there is one type of electron. So there is only 1 "color".
Black and white image
There is a 1 or 2 nm resolution, therefore sub cellular structures can be
recognised such as organelles.
Light microscopy
Bright-field microscopy: you just pass light to the lens and it returns back
(normal cell culture).
Phase contrast-microscopy: you need to put phase-contrast rings in.
Based on the pattern of the ring, you get a different structure seen. You
can enhance the structure.
Nomarski differential-interference microscopy
Dark-field microscopy: also put phase-contrast rings in.
Fluorescence microscopy
How can we detect "transparant" biological structures?
Fluorescence!
What is fluorescence?
Chat: fluorescence is the emission of light by a substance that has
absorbed light
L1 Introduction to microscopy 4
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