Bio 321 Lightmicroscopy Electronmicrosopy Image Processing

Transcription

1 Bio 321 Lightmicroscopy Electronmicrosopy Image Processing Urs Ziegler Center for Microscopy and Image Analysis

2 Light microscopy (Confocal Laser Scanning Microscopy)

3 Light microscopy (Confocal Laser Scanning Microscopy)

4 Light microscopy (Confocal Laser Scanning Microscopy)

5 Light microscopy (Confocal Laser Scanning Microscopy)

6 Live cell microscopy

7 Electron microscopy 40 m

8 Electron microscopy 1 m

9 Electron microscopy 1 m

10 Electron microscopy 100 nm

11 Literatur Fundamentals of light microscopy and electronic imaging, Douglas B. Murphy; Wiley-Liss, 2001 ISBN X (Sehr verständliches Buch mit allem nötigen Grundlagenwissen zu Lichtmikroskopie) Light Microscopy in Biology A practical approach, A. J. Lacey; Oxford University Press, 2004 (Einfache Beschreibung der Lichtmikroskopie mit praktischen Übungen und Anleitungen) Light and Electron Microscopy, E. M. Slayter, H. S. Slayter; Cambridge University Press, 1992 (Detailierte und oft mathematische Beschreibung der Licht und Elektronenmikroskopie. Gutes Referenzwerk) (Ausführliche und vorzügliche Beschreibung der Lichtmikroskopie mit Demonstrationen, sehr empfehlenswert)

12 Magnification Resolution Resolving power

13 Resolution - Resolving power Minimum resolvable distance (e. g. periodic spacings) Resolving power: specified minimal resolvable distance that can be obtained by the instrument Resolution: minimal resolvable distance that can be obtained with a real sample Light microscopy: 200nm; can be achieved with actual biological samples Electron microscopy: theoretically 10-3 nm, currently 0.1nm; actual resolution depends very strongly on preparation of biological samples (1nm 5nm)

14 Resolution in biomedical imaging

15 Resolution in biomedical imaging Resolution Limit Wavelength Object MRI, CT 1 mm Radio Human eye 100 m Cells Infrared 10 m Red blood cells Visible 1 m Bacteria Light microscope Ultraviolet 100 nm Mycoplasma Viruses 10 nm Proteins x, -rays 1 nm Amino acids Electron microscope 0.1 nm Atoms

16 Resolution limit in biomedical imaging Concept Interaction of a probe with sample Example: Atomic force microscopy: Resolving power: Physical nature of probe and sample Properties of microscope

17 Fundamental Setup of Light Microscopes

18 Fundamental Setup of Light Microscopes Bright Field Microscopy (including DIC / Phase Contrast) Ocular Polarizer Wollaston Prism Objectives Sample Plane Condenser Wollaston Prism Phase Ring Polarizer Z Focus Light Source

19 Geometrical Optics of a Simple Lens a a' f f' y F F' 1. A light ray passing through the center of a lens is not deviated y' 2. A light ray parallel with the optic axis will, after refraction, pass through the rear focal point Equations: 3. A ray passing through the front focal point will be refracted in a direction parallel to the axis. Magnification: M= y' y = n'a' n'a f' f a ' a 1 (diffractive index identical on both sides of lens)

20 Geometrical Optics of a Simple Microscope F objective F eyepiece Object Objective Primary image Eyepiece Virtual image seen by eye Remark: Camera will record the primary image!

21 Properties of Light monochromatic polychromatic Linear polarized Non polarized Coherent Non coherent Collimated Divergent

22 Ernst Abbe ( )

23 Grating Lens Object plane

24 Grating Lens Planar wave Object plane Back focal plane Image plane

25 Grating Lens Planar wave Object plane Back focal plane Image plane

26 Grating Lens 0 th order Planar wave Object plane Back focal plane Image plane

27 Grating Lens 0 th order Planar wave Object plane Back focal plane Image plane

28 Grating Lens n th order Planar wave Object plane Back focal plane Image plane

29 Grating Lens n th order Planar wave Object plane Back focal plane Image plane

30 Grating Lens n th order 0 th order Planar wave Object plane Back focal plane Image plane

31 Diffraction of a grating in the back focal plane of the objective Grating Back focal plane

32 Diffraction of grids in the back focal plane of the objective Grating Back focal plane

33 Diffraction patterns in the back focal Generation of an image by interference requires collection of two adjacent orders of diffracted light! a,b: no image is formed c: image is formed d: image with high definition due to multiple diffracted orders collected

34 Modification of the diffraction in the back focal plane of the objective Object Back focal plane

35 Back focal plane Image Diffraction image relation

36 Numerical aperture NA=n sin

37 Resolution Microscope Light microscopy: Aperture of objective determines the resolution, not the magnification! Objective with high aperture (NA 1.25) Objective with low aperture (NA 0.3)