Literature Exam: Monday the 7th of Febraury 2011 Prev week's lecture: http://www.nanoimaging.de/Lectures/Biophotonics2010/Biophotonics_2010_01.ppt Thursday lectures: Room 250 in the Hauptgebäude" (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 0521 43047 X (hard back), 0521 43631 I (paper back) Jerome Mertz, "Introduction to Optical Microscopy" Roberts & Company Publishers, 2010, ISBN    0981519482, 9780981519487 Greenfield Sluder "Digital microscopy", vol 81 of "Methods in cell biology" eds: Greenfield Sluder, David E. Wolf, 3rd edition, Elsevier Academic Press, 2007 ISBN    0123740258, 9780123740250, 608 pages and for more advanced coverage of some topics: Pawley (ed), "Handbook of Biological Confocal Microscopy", 3rd edition, Springer (2006) ISBN-10: 0-381-25921-X, ISBN-13: 987-381-25921-5 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 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

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 to identify malignant changes The basic dye hematoxylin colors (blue- purple) basophilic structures which are usually the ones containing nucleic acids: ribosomes chromatin-rich cell nucleus RNA in cytoplasm Eosin colors (bright pink) eosinophilic structures which are generally composed of protein. hematoxylin and eosin staining 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 by fuchsine 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-Universität 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 http://en.wikipedia.org/wiki/Spherical_aberration Real Lens http://en.wikipedia.org/wiki/File:Spherical_aberration_2.svg

Optical Aberrations: Spherical Aberration http://en.wikipedia.org/wiki/File:Spherical-aberration-slice.jpg http://www.olympusmicro.com/primer/java/aberrations/pointspreadaberration/index.html

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.? http://biology.about.com