+ All Categories
Home > Documents > Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June...

Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June...

Date post: 31-Mar-2015
Category:
Upload: abby-woodham
View: 216 times
Download: 3 times
Share this document with a friend
Popular Tags:
45
Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore Hazlett
Transcript
Page 1: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Basic Instrumentation

Joachim Mueller

Principles of Fluorescence Spectroscopy Genova, ItalyJune 19-22, 2006

Figure and slide acknowledgements: Theodore Hazlett

Page 2: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

ISS PC1 (ISS Inc., Champaign, IL, USA)

Fluorolog-3 (Jobin Yvon Inc, Edison, NJ, USA )

QuantaMaster (OBB Sales, London, Ontario N6E 2S8)

Page 3: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Fluorometer: The Basics

Note: Both polarizers can be removed from the optical beam path

Excitation WavelengthSelection

EmissionWavelength

Selection

Sample

Light Source

Detector

Computer

Excitation Polarizer

Emission Polarizer

Fluorometer Components

Page 4: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Fluorometer Components

Light Source

Detectors

Wavelength Selection

Polarizers

Page 5: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

The Laboratory Fluorometer

Pem

Pex

Pem

ISS (Champaign, IL, USA) PC1 Fluorometer

Standard Light Source: Xenon Arc Lamp

Exit Slit

Page 6: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Light Sources

Page 7: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Lamp Light Sources

1. Xenon Arc Lamp (wide range of wavelengths)

2. High Pressure Mercury Lamps (High Intensities but concentrated in specific lines)

3. Mercury-Xenon Arc Lamp (greater intensities in the UV)

4. Tungsten-Halogen Lamps

5. Light emitting diodes (LEDs) Multiple color LEDs can be bunched to provide a broad emission range)

Xenon Arc Lamp Profiles

Mercury-Xenon Arc Lamp Profile

UV

Ozone Free

Visible

Page 8: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Light Emitting Diodes (LED)

Wavelengths from350 nm to 1300 nm

Near UV LED

Page 9: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Lasers Light Sources

Argon-ion100 mW

Helium-cadmium Nd-YAG GreenHe-Ne10 mW

OrangeHe-Ne10 mW

He-Ne >10 mW

200 300 400 500 600 700 Wavelength (nm)

295nm

325nm

351 nm 364 nm

442nm

488nm

514nm528nm 532nm 543nm

633nm

576nm

Titanium:Sapphire690 nm – 990 nm

Page 10: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Laser Diodes

700600500400300

Wavelength (nm)

Page 11: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Detectors

Scallop

Image courtesy of BioMEDIA ASSOCIATES http://www.ebiomedia.com

Scallop Eyes

From http://www.eyedesignbook.com/index.html

Page 12: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

MCP & Electronics (ISS Inc. Champaign, IL USA)

APD

The silicon avalanche photodiode (Si APD) has a fast time response and high sensitivity in the near infrared region. APDs can be purchased from Hamamatsu with active areas from 0.2 mm to 5.0 mm in diameter and low dark currents (selectable). Photo courtesy of Hamamatsu

Page 13: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

DynodesPhotocathode

High Voltage Supply(-1000 to -2000 V)

Ground

e-Anode

Current Output

e-

e-e-

Constant Voltage (use of a Zenor Diode)

resister series

(voltage divider) capacitor series(current source)

e-

e-e-e-

e-e-

e-

e-e-

Vacuum

The Classic PMT Design

Window

Page 14: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Hamamatsu R928 PMT Family

Window with Photocathode Beneath

R2949

Page 15: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Cathode Material

Window Material

PMT Quantum Efficiencies

Page 16: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Photon Counting (Digital) and Analog Detection

Primary Advantages:1. Sensitivity (high signal/noise)2. Increased measurement stability

Primary Advantage:1. Broad dynamic range2. Adjustable range

Sig

nal

time

Constant High Voltage Supply

Discriminator Sets Level

PMT

TTL Output(1 photon = 1 pulse)

PMT

Variable Voltage Supply

Computer

Anode Current=

Pulse averaging

Continuous Current Measurement

Photon Counting: Analog:

level

Page 17: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Wavelength Selection

Fixed Optical Filters

Tunable Optical Filters

Monochromators

                                                                                                 

                                                                                                                                  

Page 18: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Optical Filter Channel

Pem

Pex

Pem

Page 19: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Hoya O54

100

80

60

40

20

0800700600500400300

Wavelength (nm)

Tra

nsm

issi

on

(%

)

Spectral ShapeThicknessPhysical ShapeFluorescence (!?)

Long Pass Optical Filters

Page 20: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

100

80

60

40

20

0700600500400300

More Optical Filter Types…

Tra

nsm

issi

on

(%

)

Wavelength (nm)

Interference Filters(Chroma Technologies)

Broad Bandpass Filter(Hoya U330)

Neutral Density(Coherent Lasers)

Page 21: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Tunable Optical Filters

An electrically controlled liquid crystal elements to select a specific visible wavelength of light for transmission through the filter at the exclusion of all others.

Liquid Crystal Filters:

AO Tunable Filters:

The AOTF range of acousto-optic devices are solid state optical filters. The wavelength of the diffracted light is selected according to the frequency of the RF drive signal.

Isomet (http://www.isomet.com/index.html)

Page 22: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Monochromators

Czerny-Turner design

Mirrors

Rotating Diffraction Grating(Planar or Concaved)

Entrance slit

Exit Slit

1. Slit Width (mm) is the dimension of the slits.

2. Bandpass is the FWHM of the selected wavelength.

3. The dispersion is the factor to convert slit width to bandpass.

Page 23: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Zero Order(acts like a mirror)

Nth Order(spectral distribution)

Mirrors

Grating

The Inside of a Monochromator

Page 24: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

1.0

0.8

0.6

0.4

0.2

0.0580560540520

Changing the Bandpass

1.0

0.8

0.6

0.4

0.2

0.0x1

06

580560540520

Fixed Excitation Bandpass = 4.25 nm

17 nm

2.125 nm

4.25 nm

8.5 nm

1. Drop in intensity2. Narrowing of the spectral selection

Flu

ore

scen

ce

(au

)

Wavelength (nm)

Changing the Emission Bandpass

Collected on a SPEX Fluoromax - 2

Wavelength (nm)

17 nm

8.5 nm

4.25 nm

2.125 nm

Page 25: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Higher Order Light Diffraction

350

300

250

200

150

100

50

0

x1

03

700600500400300200

Wavelength (nm)

Flu

ore

scen

ce

(au

)

Emission Scan:Excitation 300 nmGlycogen in PBS

Excitation (Rayleigh) Scatter(300 nm)

2nd Order Scatter(600 nm)

Water RAMAN(334 nm)

2nd Order RAMAN(668 nm)

Fluorescent Contaminants

Page 26: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Monochromator Polarization Bias

No Polarizer

Parallel Emission

Perpendicular Emission

Wood’s Anomaly

Adapted from Jameson, D.M., Instrumental Refinements in Fluorescence Spectroscopy: Applications to Protein Systems., in Biochemistry, Champaign-Urbana, University of Illinois, 1978.

250

250

800

800

Flu

ore

scen

ce

Flu

ore

scen

ce

Tungsten Lamp Profile Collected on an SLM Fluorometer

Page 27: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

300 350 400 450 500 550 600

vertical horizontal

Wavelength (nm)

ISSPC1Correction Factors

400 450 500 550 600

Inte

nsity

(a.

u.)

Wavelength (nm)

B

400 450 500 550 600

Inte

nsity

(a.

u.)

Wavelength (nm)

C

Correction of Emission Spectra

from Jameson et. Al., Methods in Enzymology, 360:1

Wavelength Wavelength

Flu

ore

scen

ce

Flu

ore

scen

ce

ANS Emission Spectrum, no polarizer ANS Emission Spectrum, parallel polarizer

uncorrected

corrected

Wavelength

Page 28: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Pem

Pex

Pem

Exit Slit

Quantum Counter

Excitation Correction

Page 29: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

The Instrument Quantum Counter

FluorescenceHere we want the inner filter effect!

Optical Filter

ReferenceDetector

Quantum Counter

Common Quantum Counters (optimal range)*

Rhodamine B (220 - 600 nm)

Fluorescein (240 - 400 nm)

Quinine Sulfate (220 - 340 nm)

* Melhuish (1962) J. Opt. Soc. Amer. 52:1256

Wavelength (nm)

200 600400

1.2

0.8

0.4

0.0

Ep

ple

y T

he

rmo

pil

e/

QC

Linearity of Rhodamine as a quantum counter

Page 30: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

A

Wavelength (nm)250 300 350 400 450

0.0

0.2

0.4

0.6

0.8

1.0

B

Wavelength (nm)

250 300 350 400 4500.0

0.2

0.4

0.6

0.8

1.0

C

Wavelength (nm)

Excitation Correction

from Jameson et. Al., Methods in Enzymology, 360:1

Wavelength Wavelength

Wavelength

Flu

ore

scen

ce

Flu

ore

scen

ce

Flu

ore

scen

ce

Absorption (dotted line) and Excitation Spectra (solid line) of ANS in Ethanol

UncorrectedRatio Corrected

Lamp Corrected

Page 31: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Two UV selected calcite prisms are assembled with an intervening air space. The calcite prism is birefringent and cut so that only one polarization component continues straight through the prisms. The spectral range of this polarizer is from 250 to 2300 nm. At 250 nm there is approximately 50% transmittance.

The Glan Taylor prism polarizer

Polarizers

Common Types:

Glan Taylor (air gap)

Glan Thompson

Sheet Polarizers

Two Calcite Prisms

0

90

90

0

Page 32: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

30

25

20

15

10

x10

6 700680660640620600580560540

3

2

1

x106

Wavelength (nm)

Sample Issues

Signal Attenuation of the Excitation Light PMT Saturation

Fluorescence vs Signal

Excess Emission

[Fluorophore]

Inst

rum

ent

Sig

nal

LINEAR REGION

Reduced emission intensity1. ND Filters 2. Narrow slit widths3. Move off absorbance peak

Page 33: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Attenuation of the Excitation Light through Absorbance

Sample concentration& the inner filter effect

Rhodamine B

from Jameson et. al., Methods in Enzymology (2002), 360:1

Page 34: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

3

2

1

x1

06

660640620600580560540

4

3

2

1

x1

06

The second half of the inner filter effect:attenuation of the emission signal.

Diluted Sample

(1) Spectral Shift(2) Change in Spectral Shape

1.0

0.8

0.6

0.4

0.2

700650600550500450

3

2

1

x1

06

Absorbance Spectrum

Wavelength (nm) Wavelength (nm)

Page 35: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

How do we handle highly absorbing solutions?

Quartz/Optical Glass/Plastic Cells

Emission Path Length

Detector

Excitation

Em

iss

ion

Excitation Path Length

4 Position Turret SPEX Fluoromax-2, Jobin-Yvon

Page 36: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Front Face Detection

Triangular Cells

Detector

Excitation

Reflected Excitation & Emission

Thin Cells & Special Compartments

Sample

Absorbance Measurements

ExcitationEmission

[1] Adapted from Gryczynski, Lubkowski, & Bucci Methods of Enz. 278: 538

[1]

Mirror

IBH, Glasgow  G3 8JUUnited Kingdom

Page 37: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Lifetime Instrumentation

Page 38: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Light Sources for Decay Acquisition:Frequency and Time Domain Measurements

Pulsed Light Sources (frequency & pulse widths)

Mode-Locked LasersND:YAG (76 MHz) (150 ps)Pumped Dye Lasers (4 MHz Cavity Dumped, 10-15 ps)Ti:Sapphire lasers (80 MHz, 150 fs)Mode-locked Argon Ion lasers

Directly Modulated Light SourcesDiode Lasers (short pulses in ps range, & can be modulated by synthesizer)LEDs (directly modulated via synthesizer, 1 ns, 20 MHz)

Flash Lamps Thyratron-gated nanosecond flash lamp (PTI), 25 KHz, 1.6 nsCoaxial nanosecond flashlamp (IBH), 10Hz-100kHz, 0.6 ns

Page 39: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Modulation of CW LightUse of a Pockel’s Cell

Mir

ror

Radio FrequencyInput

Pockel’s Cell

Polarizer

Polished on a side exit plane

CW Light Source

Double Pass Pockel’s Cell

Pulsed Emission

The Pockel’s Cell is an electro-optic device that uses the birefringment properties of calcite crystals to alter the beam path of polarized light. In applying power, the index of refraction is changed and the beam exiting the side emission port (0 polarized) is enhanced or attenuated. In applying RF the output becomes modulated.

90

0

Polarizer

Page 40: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Time Correlated Single Photon Counting

Pulsed Light Source

PM

T

TAC

Multichannel Analyzer

Constant FractionDiscriminator

Time

Co

un

tsSample Compartment

Filter or Monochromator

Time-to-Amplitude Converter (TAC)

Instrument Considerations

Excitation pulse width

Excitation pulse frequency

Timing accuracy

Detector response time (PMTs 0.2-0.9 ns; MCP 0.15 to 0.03 ns)

Photon Counting PMT

Timing Electronics or 2nd PMT Neutral density (reduce to one photon/pulse)

Page 41: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

4

6

80.01

2

4

6

80.1

2

4

6

81

300250200150100500

Channels (50 ps)

Flu

ore

scen

ce

Fluorescence Decay

Instrument Response Function

Histograms built one photon count at a time …

(1) The pulse width and instrument response times determine the time resolution.

(2) The pulse frequency also influences the time window. An 80 MHz pulse frequency (Ti:Sapphire laser) would deliver a pulse every 12.5 ns and the pulses would interfere with photons arriving later than the 12.5 ns time.

Page 42: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Polarization CorrectionThere is still a polarization problem in the geometry of our excitation and collection (even without a monochromator)!!

Will the corrections never end ???

[1]

[2]

[3]

[4]

[5]

[6]

Polarized Excitation

0

0

90

An intuitive argument:

[1] = I0 + I90

[2] = I0 + I90

[3] = I0 + I90

[4] = I0 + I90

[5] = 2 x I90

[6] = 2 x I90

Total = 4 x I0 + 8 x I90

The total Intensity is proportional to: I0 + 2 x I90

Setting the excitation angle to 0 and the emission polarizer to 54.7 the proper weighting of the vectors is achieved.*

*Spencer & Weber (1970) J. Chem Phys. 52:1654

Page 43: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Frequency Domain Fluorometry

CW Light SourceSample Compartment

Filter or Monochromator

PM

T

Analog PMTs (can also be done with photon counting)

PM

T

S1 = n MHz

S2 = n MHz + 800 Hz

R F

Digital Acquisition Electronics

Signal

Sig

nal

RF

Locking Signal

S1 S2Synthesizers

S1 and S2

Computer Driven Controls

Similar instrument considerations as With TCSPC

Reference Turret

Pockel’s Cell

Page 44: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

Lifetime Station #3, LDF, Champaign IL, USA

Page 45: Basic Instrumentation Joachim Mueller Principles of Fluorescence Spectroscopy Genova, Italy June 19-22, 2006 Figure and slide acknowledgements: Theodore.

& hiding under the table:

RF Amplifiers Frequency Synthesizers


Recommended