Literature Exam: Monday the 7th of Febraury 2011 Prev week's lecture: Thursday.

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.?