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Epi-illumination is form of Kohler Illumination: Objective is also condenser

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.

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Epi-illumination is form of Kohler Illumination: Objective is also condenser

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  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. 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)

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

  13. Detecting A Single Fluorescent Molecule? • Size: ~ 1nm • Absorption Cross-section: ~ 10-16 cm2 • Quantum Yield: ~1 Absorbance of 1 molecule = ? How many fluorescence photons per excitation photons?

  14. Single Molecule “Blinks”

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

  16. 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

  17. Back to Single Protein Detection Myosin V -- a motor protein.

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

  19. Paul Selvin

  20. Photodiode PMT: photomultiplier CCD APD: Avalanche Photodiode

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

  22. Typical Dark Counts APD CCD -20 C Temperature -70 C Sensitive Area 10-20 m 100-500 m Dark Counts 0.001 e/sec/pixel 10-100 e/sec/pixel

  23. 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.

  24. Snell’s law

  25. Application Example 1 – Cytoskeleton Epi TIRF

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

  27. A little History: EVDLS

  28. 1980s: start to apply TIR principle to fluorescence and bio-imaging. Daniel Axelrod

  29. Prism Based TIRF Setup 1

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

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

  32. Objective-TIRF Prism-TIRF

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

  34. Through Objective TIR Design 1: direct coupling

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

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

  37. Arc Lamp TIRF

  38. Fresnelequations

  39. Polarization Control

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