1. IPC Friedrich-Schiller-Universität Jena 1 From Wednesday, 9 November 2011 the weekly lectures will be every Wednesday, 8:30 Humboldtstr. 8, Seminar Room 3 (SR 3)

2. IPC Friedrich-Schiller-Universität Jena 2 Blackboard exercises: Coherent vs. Incoherent imaging The Concept of a Point Spread Function Imaging as a Convolution of Object with PSF 2. Contrast modes in light microscopy: Bright field The Concept of a Amplitude Spread Function Image Field as a Convolution of Object with ASF

3. IPC Friedrich-Schiller-Universität Jena 3 Fourier-space & Optics

4. IPC Friedrich-Schiller-Universität Jena 4 k [1/m] Real space: Intensity Amplitude Spatial Frequencies and Fourier Transforms Fourier space: x [m]

5. IPC Friedrich-Schiller-Universität Jena 5 : from: http://members.nbci.com/imehlmir/ Even better approximation: Fourier Analysis : from: http://www-groups.dcs.st-and.ac.uk/ ~history/PictDisplay/Fourier.html

6. IPC Friedrich-Schiller-Universität Jena 6 Cosine transform Fourier transform

7. IPC Friedrich-Schiller-Universität Jena 7 Constructing images from waves Sum of Waves Corresponding Sine-Wave kxkx kyky kxkx kyky Accumulated Frequencies Spatial Frequency

8. IPC Friedrich-Schiller-Universität Jena 8 Constructing images from waves Sum of Waves Corresponding Sine-Wave Accumulated Frequencies Spatial Frequency

9. IPC Friedrich-Schiller-Universität Jena 9 Intensity in Focus (PSF) Reciprocal Space (ATF) kxkx kzkz kyky Real Space (PSF) x z y Lens Focus Oil Cover Glass

10. IPC Friedrich-Schiller-Universität Jena 10 Ewald sphere McCutchen generalised aperture

11. IPC Friedrich-Schiller-Universität Jena 11 IFT A(k)a(r)

12. IPC Friedrich-Schiller-Universität Jena 12 |a| 2 (r)a(r)

13. IPC Friedrich-Schiller-Universität Jena 13 IFT FT |.| 2 ? ? |a| 2 (r) a(r)A(k)

14. IPC Friedrich-Schiller-Universität Jena 14 Epifluorescent PSF ? I(x) = |A(x)| 2 = A(x) · A(x) * I(k) = A(k)  A(-k) OTF ATF ~~ ~* Fourier Transform

15. IPC Friedrich-Schiller-Universität Jena 15 Convolution: Drawing with a Brush k x,y kzkz Region of Support

16. IPC Friedrich-Schiller-Universität Jena 16 Optical Transfer Function (OTF) ! k x,y kzkz

17. IPC Friedrich-Schiller-Universität Jena 17 Missing cone

18. IPC Friedrich-Schiller-Universität Jena 18 Scattering / Absorbtion Bright Field Transmission Objective Lense Tube Lense Back Focal Plane Dark object on bight background Relative scattering angle and wavelength defines resolution Condensor AND objective Numerical Aperture matter Contrast decreases when resolution increases CCD 2. Contrast modes in light microscopy: Bright field

19. IPC Friedrich-Schiller-Universität Jena 19 Interference of diffracted light with the undiffracted reference (first Born approx.) 2. Contrast modes in light microscopy: Bright field k in k out K obj Range of Detection Angles Holgraphy with plane wave illumination: infinitely little 3D information is acquired! "Bragg condition"

20. IPC Friedrich-Schiller-Universität Jena 20 http://biology.about.com 1.? 2.? 3.? 6.? 5.? 4.? 2. Contrast modes in light microscopy: Bright field