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Epi-illumination is form of Kohler Illumination:Objective is also condenser
Lamp orlaser
detector
Detect at 90 degreesSplit with dichroic mirrorGreatly increases S/N
Light is focusedAt back aperture Of the objective,Conjugate tocondenseraperture
Different illuminationAnd image paths
White light (regular Kohler)White light (regular Kohler)Brightfield, phase, etcBrightfield, phase, etc
lens
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First barrier filterSelects excitation
dichroicmirror
Secondbarrier filterSelects signalFrom background
objective lens
specimen
Epi-illumination separates light source,Fluorescence signal
Arclamp
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•Excitation filter typically interference bandpassExcitation filter typically interference bandpass•Dichroic is longwave passDichroic is longwave pass•For one dye-maybe no emission filterFor one dye-maybe no emission filter
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Dielectric layers or Metallic layers used as filter coating
Reflect, transmit colors of choice by using multilayers
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Coatings work by interference
Reflectance depends onWavelength, film thickness material (index*length), incident angle.
Fabry-Pérot interferometer
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Block 3-6 OD outside of band Transmit 10-50% (worse for UV)
Use of bandpass interference filters in wavelength selection
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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
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How CCD Camera Works
Serial readout limit speed. A partial solution is using Frame-Transfer.
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Comparison: Detector Quantum Yield
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Efficiency & Signal/Noise?
• Collection efficiency of microscopy: ~25%
• Detector quantum yield: ~70-90%
• Thermal noise
• Shot noise (quantum noise):
• Read noise (A/D conversion)
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CCD Dark counts
Liquid Nitrogen
Thermal Electric
Thermal Electric in ultrahigh vacuum
Cooling methods:
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EM-CCD
- Largely eliminate read noise
- Introduces amplification noise
- Net effect is S/N improvement for extremely low light level situation
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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?
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Single Molecule “Blinks”
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How to Analyze Single Molecule Measurements (I)-- Histograms
Most Probable Value vs Average value
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• Emission– Wavelength dependenceof detectors– Spectral separation fromexcitation– Efficient detection optics– Autofluorescence andcontaminantfluorescence– Photobleaching and ISC– Scatter:• elastic (Rayleigh)• inelastic (Raman)
Single molecule fluorescence:experimental considerations
• Excitation– High NA objective lens– “Bright” fluorophores• High extinctioncoefficient• High quantum yield– Exclude quenchers• particularly molecularoxygen!• O2 scavengers includeβ-mercaptoethanol(BME), catalase
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Back to Single Protein Detection
Myosin V -- a motor protein.
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De-convolution Microscopy
Thompson, RE; Larson, DR; Webb, WW, Biophys. J. 2002,
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Paul Selvin
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Photodiode
PMT: photomultiplier
APD: Avalanche Photodiode
CCD
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PMT
APD
Both can work under Single-photon Countingmode
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Typical Dark Counts
CCD APD
0.001 e/sec/pixel 10-100 e/sec/pixelDark Counts
Temperature -70 C -20 C
Sensitive Area 10-20 m 100-500 m
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Total internal reflection: the reflectionthat occurs when light, in a higherrefractive-index medium, strikes aninterface with a medium that has a lowerrefractive index, at an angle of incidence(α1) greater than the critical angle.
Total Internal Reflection Fluorescence Microscopy
TIRFM
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Snell’s law
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1)sin(/)sin(4 212
0
g
pn
d
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Application Example 1 – Cytoskeleton
TIRF Epi
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Prism-TIRF Objective-TIRF
Setting up the TIRF microscope
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A little History: EVDLS
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Daniel Axelrod
1980s: start to apply TIR principleto fluorescence and bio-imaging.
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Prism Based TIRF Setup 1
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Spherical Aberration from Aqueous Sample
Sample near glass coverslip Sample in the bulk water
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Water Immersion Objective
Fully water immersion Water immersion with coverslip
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Prism-TIRF Objective-TIRF
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• NA requirement
• Oil immersion
• Size of the beam
Key Points:
柳田敏雄 Toshio Yanagida
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Through Objective TIR Design 1: direct coupling
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Through Objective TIR Design 2: Fiber Optics
Optical fiber based light delivery
Easy conversion from non-TIR to TIR
Compatible with Arc lamp
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Other Practical Concerns:
• Upright or inverted microscope?
• Light sources?
• Polarization?
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Arc Lamp TIRF
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Fresnel equations
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Polarization Control