Hoofdstuk 1
Resolution limit The smallest distance to separate 2 maxima from
each other
Objective can not capture all maxima and minima
Defined by λ and NA !!
Optical resolution limit (formula)
Dispersion Refraction index depends on λ
Refraction index n = ratio speed of light in medium
Bending of the light to the normal – fish example
n ↑ more bending, less diffraction, more
resolution
Snell’s law n1*sinα1=n2*sinα2
Critical angle Angle light will reflect not refract
Reflection Wave reflects, same λ, angle
Diffraction Light spreads out when passing narrow opening
Opening width ~bending :
Big opening – more bend – larger DP – maxima closer
togheter
Diffraction pattern 1 slit: Undeviated, deviated – interferance light
(DP) maxima – minima (example interference)
Interference 2 slits: Interferance between waves – constructive
and destructive
Lens Focuses light in focal point (f) – focal distance
Condensor Focuses light on the sample
Objective Magnifying lens, capturing as much as possible - NA
Ocular Focuses light from objective before detector
Field diaphragm Controls how much of the field is illuminated
Infinity optics Object positioned exactly at the focal plane of the
objective
Image at infinity
Solution: Tube lens: Focus light before focal plane of
eye piece
Conjugate planes Points in focus
Illumination path Conjugate plane where you see lamp filament in
focus ☹ – you want parallel rays through sample
Köhler illumination Focus of the light on the sample, only illuminate
what you need
1) focus light on sample (with condensor)
2) Field diaphragm (narrow, focus (3rd conj.pl.)
3) Field diaphragm (put in center, open diaphragm)
Airy pattern Diffraction pattern, resolution, blurres point
Central disc + concentric rings
Numerical Aperture Angle objective can make, how strongly it can focus
NA light
Tells something about the opening of the lens
NA ↑ WD ↓ , DOF ↓ , M↑
Working distance Distance between sample and lens
(WD)
Depth Of Field Sharpness image
(DOF)
Contrast See difference between intensity (black/white)
, Contrast ~ Resolution (~ Diffraction)
Convex lens Light on the sides bends differently than in the
middle = aberration
, PLAN APO lens Lens corrected for cervical aberration (so that
colours have the same focal point)
Zernike phase A phase plate will shift the fase of not refracted light
contrast to allow interference
A condenser annulus let lights through a ring
See structures better + white ring around cell
Hoofdstuk 2
Fluorescence Absorption of light, followed by emission with
higher λ
Jablonski diagram Different states, Part of energy is lost higher λ
Extinction Capacity to absorb light
coëfficiënt
Triple state = Forbidden state
Fluorochrome can not go back to ground state
(eventually loss of energy)
Life time = Half time, intensity has reduced with 50%
Zie ander document (soorten microscopen)
Resolution limit The smallest distance to separate 2 maxima from
each other
Objective can not capture all maxima and minima
Defined by λ and NA !!
Optical resolution limit (formula)
Dispersion Refraction index depends on λ
Refraction index n = ratio speed of light in medium
Bending of the light to the normal – fish example
n ↑ more bending, less diffraction, more
resolution
Snell’s law n1*sinα1=n2*sinα2
Critical angle Angle light will reflect not refract
Reflection Wave reflects, same λ, angle
Diffraction Light spreads out when passing narrow opening
Opening width ~bending :
Big opening – more bend – larger DP – maxima closer
togheter
Diffraction pattern 1 slit: Undeviated, deviated – interferance light
(DP) maxima – minima (example interference)
Interference 2 slits: Interferance between waves – constructive
and destructive
Lens Focuses light in focal point (f) – focal distance
Condensor Focuses light on the sample
Objective Magnifying lens, capturing as much as possible - NA
Ocular Focuses light from objective before detector
Field diaphragm Controls how much of the field is illuminated
Infinity optics Object positioned exactly at the focal plane of the
objective
Image at infinity
Solution: Tube lens: Focus light before focal plane of
eye piece
Conjugate planes Points in focus
Illumination path Conjugate plane where you see lamp filament in
focus ☹ – you want parallel rays through sample
Köhler illumination Focus of the light on the sample, only illuminate
what you need
1) focus light on sample (with condensor)
2) Field diaphragm (narrow, focus (3rd conj.pl.)
3) Field diaphragm (put in center, open diaphragm)
Airy pattern Diffraction pattern, resolution, blurres point
Central disc + concentric rings
Numerical Aperture Angle objective can make, how strongly it can focus
NA light
Tells something about the opening of the lens
NA ↑ WD ↓ , DOF ↓ , M↑
Working distance Distance between sample and lens
(WD)
Depth Of Field Sharpness image
(DOF)
Contrast See difference between intensity (black/white)
, Contrast ~ Resolution (~ Diffraction)
Convex lens Light on the sides bends differently than in the
middle = aberration
, PLAN APO lens Lens corrected for cervical aberration (so that
colours have the same focal point)
Zernike phase A phase plate will shift the fase of not refracted light
contrast to allow interference
A condenser annulus let lights through a ring
See structures better + white ring around cell
Hoofdstuk 2
Fluorescence Absorption of light, followed by emission with
higher λ
Jablonski diagram Different states, Part of energy is lost higher λ
Extinction Capacity to absorb light
coëfficiënt
Triple state = Forbidden state
Fluorochrome can not go back to ground state
(eventually loss of energy)
Life time = Half time, intensity has reduced with 50%
Zie ander document (soorten microscopen)
