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2. Contrast modes in light microscopy: Bright field 2.1 Bright field transmission (absorption = imaginary part of refractive index) An object, keeping the phase of an incoming wave constant and decreasing the amplitude is called amplitude object. Contrast is A0 –A1,2 Bright filed microscopy is the most simple and basic light microscopy method Sample is illuminated from below by a light cone In case there is no sample in the optical path a uniform bright image is generated An amplitude object absorbs light at certain wavelengths and therefore reduces the amplitude of the light passing through the object Amplitude difference Wavelength l Uniform bright field image Bright field image of Moss reeds 2
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Literature Exam: Monday the 7th of Febraury Prev week's lecture:
Thursday lectures: Room 250 in the Hauptgebude" (main building) of
the University. They will start this Thursday!Literature:S.G.
Lipson, H. Lipson, and D.S. Tannhauser, "Optical Physics", 3rd
edition ISBN X (hard back), I (paper back)Jerome Mertz,
"Introduction to Optical Microscopy" Roberts & Company
Publishers, 2010, ISBN , Greenfield Sluder "Digital microscopy",
vol 81 of "Methods in cell biology" eds: Greenfield Sluder, David
E. Wolf, 3rd edition, Elsevier Academic Press, 2007 ISBN , , 608
pagesand for more advanced coverage of some topics:Pawley (ed),
"Handbook of Biological Confocal Microscopy", 3rd edition, Springer
(2006) ISBN-10: X, ISBN-13: 1 2. Contrast modes in light
microscopy: Bright field
2.1 Bright field transmission (absorption = imaginary part of
refractive index) An object, keeping the phase of an incoming wave
constant and decreasing theamplitude is called amplitude object.
Contrast is A0 A1,2 Bright filed microscopy is the most simpleand
basic light microscopy method Sample is illuminated from belowby a
light cone In case there is no sample in the opticalpath a uniform
bright image is generated An amplitude object absorbs light at
certain wavelengths and therefore reduces theamplitude of the light
passing through the object Amplitude difference Wavelength l
Uniform bright field image Bright field image of Moss reeds 2 2.
Contrast modes in light microscopy: Bright field
2.1 Bright field (absorption = imaginary part of refractive index)
very little absorption: impractical for thin objects Increase
contrast by staining = chemical contrasting: dyes to mark cell- and
tissue structures Most dyes selectively accumulate within cells
(e.g. lipophilic, hydrophilic) Dyes are often present as ions:
positive charge: cationic or basic dye anion: anionic or acidic dye
Staining often requires fixation 3 2. Contrast modes in light
microscopy: Bright field
2.1 Bright field (absorption = imaginary part of refractive index)
Bright field staining: common for histological cross sections: E.g.
hematoxylin and eosin stain:Popular in histology for morphological
inspection of biopsy specimen toidentify malignant changes The
basic dye hematoxylin colors (blue- purple) basophilic structures
which areusually the ones containing nucleicacids: ribosomes
chromatin-rich cell nucleus RNA in cytoplasm Eosin colors (bright
pink) eosinophilicstructures which are generallycomposed of
protein. hematoxylin and eosinstaining of cancer cells 4 2.
Contrast modes in light microscopy: Bright field
2.1 Bright field (absorption = imaginary part of refractive index)
Gram-staining (crystal violet, alcohol wash, safranin or fuchsin
counterstain):Method of differentiating bacterial species into two
large groups based on high amount of peptidoglycan in cell walls.:
Gram-positive: bacteria appear after staining dark blue
Gram-negative: crystal violet is washed out. Stained red afterwards
byfuchsine or safranin. Bacillus cereus: Gram-positive Pseudomonas
aeruginosa: Gram-negative 5 Geometric Optics of a Microscope
2. Contrast modes in light microscopy: Bright field Blackboard
exercise: Geometric Optics of a Microscope Image Planes and
Aperture Planes IPC Friedrich-Schiller-Universitt Jena The modern
microscope: Infinity optics
fTL Tube Lens image plane Objective Lens fObj fObj back focal plane
sample plane M = fTL / fObj infinity path : Filters do not hurt BFP
Telecentric: fTL fTL fobj Meaning of the back focal plane
(BFP)
Object plane BFP Image plane coverslip Tube lense R a Telecentric:
fTL immersion medium fTL fobj 8 Meaning of the back focal plane
(BFP)
9 Optical Aberrations: Spherical Aberration
Perfect Lens Real Lens Optical Aberrations: Spherical
Aberration
The Concept of a Amplitude Spread Function
2. Contrast modes in light microscopy: Bright field Blackboard
exercises: Coherent vs. Incoherent imaging The Concept of a
Amplitude Spread Function Image Field as a Convolution of Object
with ASF The Concept of a Point Spread Function Imaging as a
Convolution of Object with PSF Fourier-space & Optics Intensity
in Focus (PSF)
Real Space (PSF) x z y Lens Focus Oil Cover Glass Reciprocal Space
(ATF) kx kz ky I(x) = |A(x)|2 = A(x) A(x)*
Epifluorescent PSF I(x) = |A(x)|2= A(x) A(x)* Fourier Transform
I(k) =A(k) A(-k) OTF ATF ~ ~* ? Convolution: Drawing with a
Brush
kx,y kz Region of Support Optical Transfer Function (OTF)
! kx,y kz Widefield OTF support Missing Cone Scattering /
Absorbtion
2. Contrast modes in light microscopy: Bright field Scattering /
Absorbtion Bright Field Transmission Back Focal Plane CCD Tube
Lense Objective Lense Dark object on bight background Relative
scattering angle and wavelength defines resolution Condensor AND
objective Numerical Aperture matter Contrast decreases when
resolution increases 2. Contrast modes in light microscopy: Bright
field
Interference of diffracted light with the undiffracted reference
(first Born approx.) Range of Detection Angles kout Kobj kin "Bragg
condition" Holgraphy with plane wave illumination:infinitely little
3D information is acquired! 1.? 2.? 3.? 6.? 4.? 5.?
http://biology.about.com
2. Contrast modes in light microscopy: Bright field 1.? 2.? 3.? 6.?
4.? 5.?