1. Epi-illumination is form of Kohler Illumination:
Objective is also condenser
White light (regular Kohler)
Brightfield, phase, etc
Light is focused
At back aperture
Of the objective,
Conjugate to
condenser
aperture
Different illumination
And image paths
Lamp or
laser
lens
detector
Detect at 90 degrees
Split with dichroic mirror
Greatly increases S/N

2. dichroic mirror objective lens specimen Epi-illumination separates
light source,
Fluorescence signal
Second
barrier filter
Selects signal
From background
First barrier filter
Selects excitation
Arc
lamp
dichroic
mirror
objective lens
specimen

3. Excitation filter typically interference bandpass
Dichroic is longwave pass
For one dye-maybe no emission filter

4. Dielectric layers or Metallic layers used as filter coating
Reflect, transmit colors of choice by using multilayers

5. Coatings work by interference
Reflectance depends on
Wavelength, film thickness material (index*length), incident angle.
Fabry-Pérot interferometer

6. Use of bandpass interference filters in wavelength selection
Block 3-6 OD outside of band Transmit 10-50% (worse for UV)

7. Dichroic Mirrors: separate colors by using coatings
Beam separator:
Separate different colors (fluorescence)
At right angles: used in microscopes
Beam combiner:
Multiple lasers
Transition should be sharp

8. How CCD Camera Works
Serial readout limit speed. A partial solution is using Frame-Transfer.

9. Comparison: Detector Quantum Yield

10. Efficiency & Signal/Noise?
Collection efficiency of microscopy: ~25%
Detector quantum yield: ~70-90%
Thermal noise
Shot noise (quantum noise):
Read noise (A/D conversion)
V^2 = 4kTR

11. CCD Dark counts Cooling methods: Liquid Nitrogen Thermal Electric
in ultrahigh vacuum

12. EM-CCD Largely eliminate read noise Introduces amplification noise
Net effect is S/N improvement for extremely low light level situation

14. Detecting A Single Fluorescent Molecule?
Size: ~ 1nm
Absorption Cross-section: ~ cm2
Quantum Yield: ~1
Absorbance of 1 molecule = ?
How many fluorescence photons per excitation photons?

15. Single Molecule “Blinks”

16. How to Analyze Single Molecule Measurements (I) -- Histograms
Most Probable Value vs Average value

17. Single molecule fluorescence: experimental considerations
• Emission
– Wavelength dependence
of detectors
– Spectral separation from
excitation
– Efficient detection optics
– Autofluorescence and
contaminant
fluorescence
– Photobleaching and ISC
– Scatter:
• elastic (Rayleigh)
• inelastic (Raman)
• Excitation
– High NA objective lens
– “Bright” fluorophores
• High extinction
coefficient
• High quantum yield
– Exclude quenchers
• particularly molecular
oxygen!
• O2 scavengers include
β-mercaptoethanol
(BME), catalase

18. Back to Single Protein Detection
Myosin V has two heavy chains like myosin II, but myosin V has a longer neck region, and a shorter coiled coil region is followed by a  globular domain at the end of each heavy chain tail.
Light chains that associate with each heavy chain in the neck region are calmodulin or calmodulin-like proteins. Light chains may provide stiffening in neck domains and in some cases have a regulatory role. For example, each myosin II monomer binds two distinct light chains, designated essential and regulatory. The neck domain of each myosin V monomer has 6 binding sites for calmodulin, which serves as light chains. (To review the structure and role of calmodulin, see notes on calcium signals.)
Myosin V -- a motor protein.

20. De-convolution Microscopy
Thompson, RE; Larson, DR; Webb, WW, Biophys. J. 2002,

21. Paul Selvin

22. Photodiode
PMT: photomultiplier
CCD
APD: Avalanche Photodiode

23. PMT APD Both can work under Single-photon Counting mode
Both can work under
Single-photon Counting
mode

24. Typical Dark Counts CCD APD Temperature -70 C -20 C Sensitive Area
0.001 e/sec/pixel
e/sec/pixel

25. Total Internal Reflection Fluorescence Microscopy
TIRFM
Total internal reflection: the reflection
that occurs when light, in a higher
refractive-index medium, strikes an
interface with a medium that has a lower
refractive index, at an angle of incidence
(α1) greater than the critical angle.

26. Snell’s law

28. Minimal depth is about 1/7 of wavelength

29. Application Example 1 – Cytoskeleton
TIRF
Epi

30. Setting up the TIRF microscope
Prism-TIRF
Objective-TIRF

31. A little History: EVDLS

32. 1980s: start to apply TIR principle
to fluorescence and bio-imaging.
Daniel Axelrod

33. Prism Based TIRF Setup 1
Add the webpage demostration

34. Spherical Aberration from Aqueous Sample
Sample near glass coverslip
Sample in the bulk water

35. Water Immersion Objective
Fully water immersion
Water immersion with coverslip

36. Prism-TIRF
Objective-TIRF

37. Key Points: NA requirement Oil immersion Size of the beam 柳田敏雄
Toshio Yanagida

38. Through Objective TIR Design 1: direct coupling

39. Through Objective TIR Design 2: Fiber Optics
Optical fiber based light delivery
Easy conversion from non-TIR to TIR
Compatible with Arc lamp

41. Other Practical Concerns:
Upright or inverted microscope?
Light sources?
Polarization?

42. Arc Lamp TIRF

43. Fresnel equations

44. Polarization Control