Literature Exam Monday the 30 th of January

Literature Exam: Monday the 30 th of January 2011, 12: 30 Prev week's lecture: http: //www. nanoimaging. de/Lectures/Biophotonics 2011/ Thursday lectures will start this Thursday! Literature: S. G. Lipson, H. Lipson, and D. S. Tannhauser, "Optical Physics", 3 rd 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, 3 rd 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", 3 rd edition, Springer (2006) ISBN-10: 0 -381 -25921 -X, ISBN-13: 987 -381 -25921 -5 1 IPC Friedrich-Schiller-Universität Jena

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 difference amplitude is called amplitude object. §Contrast is A 0 –A 1, 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 Wavelength l §An amplitude object absorbs light at certain wavelengths and therefore reduces the amplitude of the light passing through the object Uniform bright field image 2 Bright field image of Moss reeds IPC Friedrich-Schiller-Universität Jena

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 IPC Friedrich-Schiller-Universität Jena

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 (bluepurple) 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. 4 hematoxylin and eosin staining of cancer cells IPC Friedrich-Schiller-Universität Jena

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 5 Pseudomonas aeruginosa: Gram-negative IPC Friedrich-Schiller-Universität Jena

2. Contrast modes in light microscopy: Bright field Blackboard exercise: Geometric Optics of a Microscope Image Planes and Aperture Planes 6 IPC Friedrich-Schiller-Universität Jena

The modern microscope: Infinity optics Objective Lens f. Obj back focal plane sample plane 7 f. Obj f. TL image plane Tube Lens M = f. TL / f. Obj infinity path : Filters do not hurt IPC Friedrich-Schiller-Universität Jena

Meaning of the back focal plane (BFP) Object plane coverslip BFP Image plane Tube lense R a f. TL Telecentric: f. TL fobj immersion medium 8 IPC Friedrich-Schiller-Universität Jena

Perfect Lens Real Lens 10 http: //en. wikipedia. org/wiki/Spherical_aberration Optical Aberrations: Spherical Aberration http: //en. wikipedia. org/wiki/File: Spherical_aberration_2. svg IPC Friedrich-Schiller-Universität Jena

IPC Friedrich-Schiller-Universität Jena 11 http: //www. olympusmicro. com/primer/java/aberrations/pointspreadaberration/index. html http: //en. wikipedia. org/wiki/File: Spherical-aberration-slice. jpg Optical Aberrations: Spherical Aberration

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 12 IPC Friedrich-Schiller-Universität Jena

Fourier-space & Optics 13 IPC Friedrich-Schiller-Universität Jena

Intensity in Focus (PSF) Real Space (PSF) Lens Reciprocal Space (ATF) Cover Glass Focus y z x ky kz Oil 14 IPC Friedrich-Schiller-Universität Jena kx

Epifluorescent PSF I(x) = 2 |A(x)| = A(x) · Fourier Transform OTF 15 * A(x) ~* ~ ~ I(k) = A(k) A(-k) ? ATF IPC Friedrich-Schiller-Universität Jena

Convolution: Drawing with a Brush kx, y Region of Support 16 kz IPC Friedrich-Schiller-Universität Jena

Optical Transfer Function (OTF) ! kx, y kz 17 IPC Friedrich-Schiller-Universität Jena

Widefield OTF support Missing Cone 18 IPC Friedrich-Schiller-Universität Jena

2. Contrast modes in light microscopy: Bright field Bright Field Transmission Scattering / Absorbtion Objective Tube Back Focal Lense Plane Dark object on bight background Relative scattering angle and wavelength defines resolution Condensor AND objective Numerical Aperture matter Contrast decreases when resolution increases 19 CCD IPC Friedrich-Schiller-Universität Jena

2. Contrast modes in light microscopy: Bright field Interference of diffracted light with the undiffracted reference (first Born approx. ) kout Range of Detection Angles Kobj kin "Bragg condition" Holgraphy with plane wave illumination: infinitely little 3 D information is acquired! 20 IPC Friedrich-Schiller-Universität Jena

2. Contrast modes in light microscopy: Bright field 1. ? 2. ? 3. ? 4. ? 6. ? 5. ? 21 IPC Friedrich-Schiller-Universität Jena http: //biology. about. com