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
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
