Literature
Exam: Monday the 7th of Febraury 2011
Prev week's lecture:
http://www.nanoimaging.de/Lectures/Biophotonics2010/Biophotonics_2010_01.ppt
Thursday lectures: Room 250 in the Hauptgebäude" (main building) of the University.
They will start this Thursday!
Literature:
S.G. Lipson, H. Lipson, and D.S. Tannhauser, "Optical Physics", 3rd 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, 3rd 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", 3rd 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 A0 –A1,2
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
fObj
back focal plane
sample plane
7
fObj
fTL
fTL
image plane
Tube Lens
M = fTL / fObj
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

fTL
RBF
8
Telecentric:
fTL
fTL
 f obj sin(  ) 
NA
M
fobj
immersion
medium
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
k
y
kz
Oil
14
IPC Friedrich-Schiller-Universität Jena
kx
Epifluorescent PSF
I(x) =
2
|A(x)|
= A(x)
*
· A(x)
Fourier Transform
~*
OTF
15
~
~
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
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 3D 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