epi-illumination is form of kohler illumination: objective is also condenser lamp or laser detector...
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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
First barrier filterSelects excitation
dichroicmirror
Secondbarrier filterSelects signalFrom background
objective lens
specimen
Epi-illumination separates light source,Fluorescence signal
Arclamp
•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
Dielectric layers or Metallic layers used as filter coating
Reflect, transmit colors of choice by using multilayers
Coatings work by interference
Reflectance depends onWavelength, film thickness material (index*length), incident angle.
Fabry-Pérot interferometer
Block 3-6 OD outside of band Transmit 10-50% (worse for UV)
Use of bandpass interference filters in wavelength selection
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
How CCD Camera Works
Serial readout limit speed. A partial solution is using Frame-Transfer.
Comparison: Detector Quantum Yield
Efficiency & Signal/Noise?
• Collection efficiency of microscopy: ~25%
• Detector quantum yield: ~70-90%
• Thermal noise
• Shot noise (quantum noise):
• Read noise (A/D conversion)
CCD Dark counts
Liquid Nitrogen
Thermal Electric
Thermal Electric in ultrahigh vacuum
Cooling methods:
EM-CCD
- Largely eliminate read noise
- Introduces amplification noise
- Net effect is S/N improvement for extremely low light level situation
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?
Single Molecule “Blinks”
How to Analyze Single Molecule Measurements (I)-- Histograms
Most Probable Value vs Average value
• 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
Back to Single Protein Detection
Myosin V -- a motor protein.
De-convolution Microscopy
Thompson, RE; Larson, DR; Webb, WW, Biophys. J. 2002,
Paul Selvin
Photodiode
PMT: photomultiplier
APD: Avalanche Photodiode
CCD
PMT
APD
Both can work under Single-photon Countingmode
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
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
Snell’s law
1)sin(/)sin(4 212
0
g
pn
d
Application Example 1 – Cytoskeleton
TIRF Epi
Prism-TIRF Objective-TIRF
Setting up the TIRF microscope
A little History: EVDLS
Daniel Axelrod
1980s: start to apply TIR principleto fluorescence and bio-imaging.
Prism Based TIRF Setup 1
Spherical Aberration from Aqueous Sample
Sample near glass coverslip Sample in the bulk water
Water Immersion Objective
Fully water immersion Water immersion with coverslip
Prism-TIRF Objective-TIRF
• NA requirement
• Oil immersion
• Size of the beam
Key Points:
柳田敏雄 Toshio Yanagida
Through Objective TIR Design 1: direct coupling
Through Objective TIR Design 2: Fiber Optics
Optical fiber based light delivery
Easy conversion from non-TIR to TIR
Compatible with Arc lamp
Other Practical Concerns:
• Upright or inverted microscope?
• Light sources?
• Polarization?
Arc Lamp TIRF
Fresnel equations
Polarization Control