Epi-illumination is form of Kohler Illumination: Objective is also condenser Lamp or laser detector Detect at 90 degrees Split with dichroic mirror Greatly.

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