Transcript Lecture 4. Fluorescence microscopy II

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
Epi-illumination separates
light source,
Fluorescence signal
First barrier filter
Selects excitation
Arc
lamp
Second
barrier filter
Selects signal
From background
dichroic
mirror
objective lens
specimen
•Excitation filter typically interference bandpass
•Dichroic is longwave pass
•For 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 on
Wavelength, film thickness
material (index*length), incident
angle.
Fabry-Pérot interferometer
Use of bandpass interference filters in wavelength selection
Block 3-6 OD outside of band Transmit 10-50% (worse for UV)
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?
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Collection efficiency of microscopy: ~25%
Detector quantum yield: ~70-90%
Thermal noise
Shot noise (quantum noise):
Read noise (A/D conversion)
CCD Dark counts
Cooling methods:
Liquid Nitrogen
Thermal Electric
Thermal Electric
in ultrahigh vacuum
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
Single molecule fluorescence:
experimental considerations
• 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
• Emission
– Wavelength dependence
of detectors
– Spectral separation from
excitation
– Efficient detection optics
– Autofluorescence and
contaminant
fluorescence
– Photobleaching and ISC
– Scatter:
• elastic (Rayleigh)
• inelastic (Raman)
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
CCD
APD: Avalanche Photodiode
PMT
APD
Both can work under
Single-photon Counting
mode
Typical Dark Counts
CCD
Temperature
Sensitive Area
Dark Counts
-70 C
10-20 m
0.001 e/sec/pixel
APD
-20 C
100-500 m
10-100 e/sec/pixel
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.
Snell’s law
dp 

0

4n2 sin(1 ) / sin( g )  1
2
Application Example 1 – Cytoskeleton
TIRF
Epi
Setting up the TIRF microscope
Prism-TIRF
Objective-TIRF
A little History: EVDLS
1980s: start to apply TIR principle
to fluorescence and bio-imaging.
Daniel Axelrod
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
Key Points:
• NA requirement
• Oil immersion
• Size of the beam
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