1. Literature
Exam: Monday the 7th of Febraury Prev week's lecture: 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 X (hard back), I (paper back) Jerome Mertz, "Introduction to Optical Microscopy" Roberts & Company Publishers, 2010, ISBN     , Greenfield Sluder "Digital microscopy", vol 81 of "Methods in cell biology" eds: Greenfield Sluder, David E. Wolf, 3rd edition, Elsevier Academic Press, 2007 ISBN     , , 608 pages and for more advanced coverage of some topics: Pawley (ed), "Handbook of Biological Confocal Microscopy", 3rd edition, Springer (2006) ISBN-10: X, ISBN-13: 1

2. 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 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
An amplitude object absorbs light at certain wavelengths and therefore reduces the amplitude of the light passing through the object
Amplitude difference
Wavelength l
Uniform bright field image
Bright field image of Moss reeds 2

3. 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

4. 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 (blue- purple) 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.
hematoxylin and eosin staining of cancer cells 4

5. 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
Pseudomonas aeruginosa: Gram-negative 5

6. Geometric Optics of a Microscope
2. Contrast modes in light microscopy: Bright field
Blackboard exercise:
Geometric Optics of a Microscope
Image Planes and Aperture Planes
IPC Friedrich-Schiller-Universität Jena

7. The modern microscope: Infinity optics
fTL
Tube Lens
image plane
Objective Lens
fObj
fObj
back focal plane
sample plane
M = fTL / fObj
infinity path : Filters do not hurt

8. BFP Telecentric: fTL fTL fobj Meaning of the back focal plane (BFP)
Object plane
BFP
Image plane
coverslip
Tube lense R a
Telecentric: fTL
immersion medium
fTL
fobj 8

9. Meaning of the back focal plane (BFP)9

10. Optical Aberrations: Spherical Aberration
Perfect Lens
Real Lens

11. Optical Aberrations: Spherical Aberration

12. The Concept of a Amplitude Spread Function
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

13. Fourier-space & Optics

14. Intensity in Focus (PSF)
Real Space (PSF) x z y
Lens
Focus
Oil
Cover Glass
Reciprocal Space (ATF) kx kz ky

15. I(x) = |A(x)|2 = A(x) · A(x)*
Epifluorescent PSF
I(x) = |A(x)|2 = A(x) · A(x)*
Fourier Transform
I(k) = A(k)  A(-k)
OTF
ATF ~ ~* ?

16. Convolution: Drawing with a Brush
kx,y kz
Region of Support

17. Optical Transfer Function (OTF)!
kx,y kz

18. Widefield OTF support
Missing Cone

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

20. 2. Contrast modes in light microscopy: Bright field
Interference of diffracted light with the undiffracted reference (first Born approx.)
Range of Detection Angles
kout
Kobj
kin
"Bragg condition"
Holgraphy with plane wave illumination: infinitely little 3D information is acquired!

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2. Contrast modes in light microscopy: Bright field
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