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Samenvatting CLB-30306 Advanced cellular imaging techniques (CLB-30306) $3.74
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Samenvatting CLB-30306 Advanced cellular imaging techniques (CLB-30306)

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Samenvatting van ACIT

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  • January 1, 2021
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  • 2020/2021
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ADVANCED CELLULAR IMAGING TECHNIQUES CLB-30306

Cell Biology part

Live cell imaging is powerful for our understanding of cells by the dynamics of a system, not just
seeing a structure but seeing how it changes over time and the dynamics of molecular interactions
show us how cells work.

Microscope construction
Early microscopes
 glass beads used as
lenses  van
Leeuwenhoek
o 1 lens
 Later compound
microscope
designed uses 2
lenses

Standard items in a microscope:
 Objective and eyepiece magnify the sample
 Condenser lens illuminates the sample

Working of a lens: refraction
 Lenses in cameras and microscopes act similarly
 Light rays originating from a point on the subject are redirected towards a point in the image
plane
 A lens redirects light by refraction (bending) on the glass-air surfaces
 Refraction occurs at a transition in refractive index (optical density); air 1; glass 1.

Lens equation
1/f = 1/s + 1/s’ (f = f’)
Magnification= s’ /s

Object close to focal plane: magnification (microscope)
Object far from focal plane: size reduction (photo camera
lens)

Conclusion: placing an object near the
focal plane of the objective will generate an enlarged image far away
Moving it even closer to the focal plane will generate an image at infinity
Objectives with short focal lengths (several mm) are used in microscopy
Object is close to the objective lens for large magnification and efficient light collection

Position a camera/image sensor (CCD or CMOS chip) at the image plane and you will acquire an
enlarged digital image

Compound microscope
 Two-step magnification to achieve a greater overall magnification
 Enables advanced illumination/detection techniques like DIC
 The objective lens forms a greatly enlarged intermediate image

,  This image is then further enlarged with a second lens (eyepiece) to form a
virtual image that can be viewed with your eye lens (or a camera lens)
 Total magnification = Mobjective x Meyepiece (typical up to 100 x)




Infinity optical system
 There are two lenses but no intermediate image
 Object is placed in the focal plane of the objective (s=f; s’=∞). Image at f tube
 Each point of the object forms a parallel bundle of light under a shallow angle α
 A tube lens forms a real image on a camera sensor. Magnification = f tube/ fobjective
 An additional eyepiece lens can be used for viewing the image with your eyes
 Advantage: Infinity optical systems allow introduction of auxiliary optical components (e.g.
filters) into the optical path with minimal effect on focus position and image quality. Because
there is no intermediate image it is more difficult to
disturb image formation.

Lens aberrations
 An ideal lens will image light that originates from a
single point on the sample onto a single point in the
image plane
 Lens aberrations cause image blur and image
distortion
 Spherical aberration: Parallel light rays that travel
close and far away from the optical axis are not
focused to the same point - as a consequence, the
image appears blurred
 Plan lenses are corrected for spherical aberrations
 Lenses are designed to work optimal for a specific
configuration

Chromatic aberrations: Lenses refract different colours
differently (chromatic dispersion)
 Chromatic dispersion: refractive index of a material is not constant but dependent on light
colour
 Achromats are corrected such that blue and red light are imaged into a single common focal
point
 Plan achromats are also corrected for spherical aberrations
 Plan apochromats are the most highly corrected lenses

, Numerical aperture
The numerical aperture of a microscope objective is a
measure of its ability to gather light and to resolve fine
detail in a specimen
 High NA lenses collect more light and form
brighter (less noisy) images
 High NA allow you to see more detail (spatial
resolution)
 Numerical aperture: NA= sin μ.
o μ: half angle of the collection cone
 Best lenses: NA=0.95. Maximal possible: NA=1
 Image brightness ~ NA2. And decreases with image magnification

Immersion lenses
 Rays coming from the object may become reflected at the interface between the coverslip
and air. They are not collected for image formation
 A film of oil with the same refraction index as glass prevents loss of light rays
 Oil immersion increases the numerical aperture: NA= n sin μ
o n is the refractive index of the medium (air, oil, or water immersion)
o Oil: n=1.516 - NA for best oil lenses 0.95*1.516 = 1.45
 Oil immersion increases the light collection of a lens and the resolution of a microscope

Resolution and contrast generation
Microscope resolution is the minimal distance at which two items are distinguishable from each
other and is limited by diffraction and can be improved by optimizing sample illumination.
Image contrast can be generated by techniques other than light absorption alone

Diffraction: light is not propagating in a straight manner. So a parallel beam is not focused by a lens
to an infinitely small spot, but forms a diffraction-limited Airy pattern (also termed point spread
function). Radius of the first dark ring in the Airy patterns equals: R airy = 1,22 *λ /(NAcondensor + NAobjective)

Rayleigh criterion: two items can be distinguished if the distance between them is equal to the radius
of the airy pattern.
Minimal resolvable resolution = microscope resolution: d = R airy, which makes the minimal size of an
item to be observed through light microscopy 200 nm.

Optimal size of pixels are needed to record the spatial information in an image

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