Technical and Financial Feasibility Analysis of Bio Processing
Bio 321 Lightmicroscopy Electronmicrosopy Image Processing · Bio 321 Lightmicroscopy...
Transcript of Bio 321 Lightmicroscopy Electronmicrosopy Image Processing · Bio 321 Lightmicroscopy...
Bio 321Lightmicroscopy
ElectronmicrosopyImage Processing
Urs Ziegler
Center for Microscopy and Image Analysis
Light microscopy (Confocal Laser Scanning Microscopy)
Light microscopy (Confocal Laser Scanning Microscopy)
Light microscopy (Confocal Laser Scanning Microscopy)
Light microscopy (Confocal Laser Scanning Microscopy)
Live cell microscopy
Electron microscopy
40 m
Electron microscopy
1 m
Electron microscopy
1 m
Electron microscopy
100 nm
Literatur
Fundamentals of light microscopy and electronic imaging, Douglas B. Murphy; Wiley-Liss, 2001ISBN 0-471-25391-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)
http://microscopy.fsu.edu/primer/index.html (Ausführliche und vorzügliche Beschreibung der Lichtmikroskopie mit Demonstrationen, sehr empfehlenswert)
Magnification – Resolution – Resolving power
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 10-3nm, currently 0.1nm; actual resolution depends very strongly on preparation of biological samples (1nm – 5nm)
Resolution in biomedical imaging
Resolution in biomedical imaging
Atoms
1 mm
100 m
10 m
1 m
100 nm
10 nm
1 nm
0.1 nm
Cells
Red blood cells
Bacteria
Mycoplasma
Viruses
Proteins
Amino acids
Radio
Infrared
Visible
Ultraviolet
x,-rays
Human eye
Electron microscope
Light microscope
Resolution Limit Wavelength ObjectMRI, CT
Resolution limit in biomedical imaging
ConceptInteraction of a probe with sample
Example: Atomic force microscopy:
Resolving power:Physical nature of probe and sampleProperties of microscope
Fundamental Setup of Light Microscopes
Ocular
Sample PlaneObjectives
Condenser
Z Focus
Light Source
Phase RingWollaston Prism
Wollaston Prism
Bright Field Microscopy(including DIC / Phase Contrast)
Fundamental Setup of Light Microscopes
Polarizer
Polarizer
F F'
f f'
a a'
Geometrical Optics of a Simple Lens
1. A light ray passing through the center of a lens is not deviated
2. A light ray parallel with the optic axis
will, after refraction, pass through
the rear focal point
3. A ray passing through the front focal
point will be refracted in a direction
parallel to the axis.
M= y 'y
= n ' a 'n ' a
a
y
y'
(diffractive index identical on both sides of lens)
Equations:
Magnification:
f 'a '
fa
1
Geometrical Optics of a Simple Microscope
Virtual image seen by eye
Remark: Camera will record the primary image!
F eyepiece
Primary image Eyepiece
F objective
Object Objective
Properties of Light
monochromatic
Linear polarized
Coherent
Collimated
polychromatic
Non polarized
Non coherent
Divergent
Ernst Abbe (1840 – 1905)
LensGrating
Object plane
LensGrating
Object plane Image planeBack focal plane
Planar wave
LensGrating
Object plane
LensGrating
Object plane Image planeBack focal plane
Planar wave
LensGrating
Object plane Image planeBack focal plane
Planar wave
0th
order
LensGrating
Object plane Image planeBack focal plane
Planar wave
0th
order
LensGrating
Object plane Image planeBack focal plane
Planar wave
nth
order
LensGrating
Object plane Image planeBack focal plane
Planar wave
nth
order
LensGrating
Object plane Image planeBack focal plane
Planar wave
0th
ordern
thorder
Diffraction of a grating in the back focal plane of the objective
Grating Back focal plane
Diffraction of grids in the back focal plane of the objective
Grating
Back focal plane
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 formedc: image is formedd: image with high definition due to multiple diffracted orders collected
Modification of the diffraction in the back focal plane of the objective
Object
Back focal plane
Diffraction image relationIm
ageB
ack focal plane
Numerical aperture
NA=n sin
Resolution
Microscope
Objective with high aperture(NA 1.25)
Objective with low aperture(NA 0.3)
Light microscopy:
Aperture of objective determines the resolution, not the magnification!