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!