Basic Instrumentation

Joachim Mueller

Principles of Fluorescence Spectroscopy

Genova, Italy Figure and slide acknowledgements:
June 19-22, 2006 Theodore Hazlett
 ISS PC1 (ISS Inc., Champaign,
IL, USA)

Fluorolog-3 (Jobin Yvon Inc, Edison,

NJ, USA )

QuantaMaster (OBB Sales, London,

Ontario N6E 2S8)
 Fluorometer Components

Excitation
Polarizer
Sample

Emission
Light Source Excitation Wavelength
Polarizer
Selection
Emission
Wavelength
Computer Selection

Detector
Fluorometer: The Basics

Note: Both polarizers can be removed from the

optical beam path
Fluorometer Components

Light Source
Detectors
Wavelength Selection
Polarizers
The Laboratory Fluorometer
Standard Light Source:
Xenon Arc Lamp

Exit Slit

Pex

Pem

Pem

ISS (Champaign, IL, USA) PC1 Fluorometer

Light Sources
 Xenon Arc Lamp Profiles
Lamp Light Sources

1. Xenon Arc Lamp (wide Ozone Free

range of wavelengths)
2. High Pressure Mercury Lamps
(High Intensities but
Visible
concentrated in specific lines) UV

3. Mercury-Xenon Arc Lamp

(greater intensities in the UV)
4. Tungsten-Halogen Lamps
Mercury-Xenon Arc Lamp Profile
5. Light emitting diodes (LEDs)
Multiple color LEDs can be
bunched to provide a broad
emission range)
Light Emitting Diodes (LED)

Wavelengths from
350 nm to 1300 nm

Near UV
LED
 Lasers Light Sources

Titanium:Sapphire
528nm
514nm 532nm 633nm 690 nm – 990 nm
543nm

442nm
325nm
488nm

295nm 351 nm
364 nm
576nm

200 300 400 500 600 700 Wavelength (nm)

Green Orange
Argon-ion Helium-cadmium Nd-YAG He-Ne He-Ne He-Ne
100 mW 10 mW 10 mW >10 mW
Laser Diodes

300 400 500 600 700

Wavelength (nm)
 Detectors

Scallop
Scallop Eyes

From http://www.eyedesignbook.com/index.html Image courtesy of BioMEDIA ASSOCIATES http://www.ebiomedia.com

 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

MCP & Electronics

(ISS Inc. Champaign, IL USA)
 The Classic PMT Design

Photocathode Vacuum Dynodes

e--
 e- e--
e-
e--e -
e -ee- Anode
e ee-
e-
Window

Constant Voltage
(use of a Zenor Diode) Current Output

resister series Ground

(voltage divider) capacitor series
High Voltage Supply
(-1000 to -2000 V) (current source)
 Hamamatsu R928 PMT Family

R2949

Window with
Photocathode Beneath
 PMT Quantum Efficiencies

Cathode Material

Window Material
 Photon Counting (Digital) and Analog Detection
time

Signal
Continuous
Current Measurement

Photon Counting: Analog:

Constant Variable
High Voltage Supply Voltage Supply

PMT PMT

level Anode Current

Discriminator =
Sets Level Pulse averaging
TTL Output
(1 photon = 1 pulse)

Computer

Primary Advantages: Primary Advantage:

1. Sensitivity (high signal/noise) 1. Broad dynamic range
2. Increased measurement stability 2. Adjustable range
 Wavelength Selection

Fixed Optical Filters

  Tunable Optical Filters

                                                                                                
Monochromators
                                                                                                         
                         
Optical Filter Channel

Pex

Pem

Pem
 Long Pass Optical Filters

100
Transmission (%)

80

Spectral Shape
60
Thickness
Physical Shape
40
Fluorescence (!?)

20

0
300 400 500 600 700 800
Wavelength (nm)

Hoya O54
 More Optical Filter Types…

Interference Filters
(Chroma Technologies)
Broad Bandpass Filter
(Hoya U330)
100

80
Transmission (%)

60

40

20

0
300 400 500 600 700
Wavelength (nm)

Neutral Density
(Coherent Lasers)
 Tunable Optical Filters
Liquid Crystal 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.

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)
 Monochromators
Mirrors

Czerny-Turner design
1. Slit Width (mm) is the
dimension of the slits.

2. Bandpass is the FWHM of

Exit Slit
the selected wavelength.

3. The dispersion is the factor

to convert slit width to
bandpass.

Entrance slit
Rotating Diffraction Grating
(Planar or Concaved)
The Inside of a Monochromator

Mirrors

Grating

Nth Order
(spectral distribution)
Zero Order
(acts like a mirror)
 Changing the Bandpass

1. Drop in intensity Fixed Excitation Bandpass =

2. Narrowing of the spectral selection 4.25 nm

Changing the Emission Bandpass

1.0 1.0

17 nm
0.8 17 nm 0.8

(au)
0.6 8.5 nm 0.6

Fluorescence
8.5 nm

6
x10
0.4 4.25 nm 0.4

2.125 nm 0.2 4.25 nm

0.2

2.125 nm
0.0 0.0
520 540 560 580 520 540 560 580

Wavelength (nm) Wavelength (nm)

Collected on a SPEX Fluoromax - 2

 Higher Order Light Diffraction

Emission Scan:
Excitation 300 nm
Glycogen in PBS

350
Excitation (Rayleigh) Scatter 2nd Order Scatter
300 (600 nm)
(300 nm)
(au)

250
Fluorescence

200
3

2nd Order RAMAN

x10

Water RAMAN
150 (334 nm) (668 nm)

100

50

0
200 300 400 500 600 700

Wavelength (nm) Fluorescent Contaminants

 Monochromator Polarization Bias
Tungsten Lamp Profile Collected on an SLM Fluorometer

Wood’s Anomaly
Parallel Emission

No Polarizer
Fluorescence

Fluorescence
Perpendicular Emission

250 800

250 800

Adapted from Jameson, D.M., Instrumental Refinements in Fluorescence

Spectroscopy: Applications to Protein Systems., in Biochemistry,
Champaign-Urbana, University of Illinois, 1978.
 Correction of Emission Spectra

ISSPC1 vertical
Correction Factors horizontal

300 350 400 450 500 550 600

Wavelength (nm)
Wavelength

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

B C
corrected
Fluorescence

Fluorescence
Intensity (a.u.)

Intensity (a.u.)

uncorrected

400 450 500 550 600 400 450 500 550 600

Wavelength (nm) Wavelength (nm)

Wavelength Wavelength

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

Excitation Correction

Quantum Counter

Exit Slit

Pex

Pem

Pem
 The Instrument Quantum Counter

Common Quantum Counters

(optimal range)*

Optical Filter
Rhodamine B (220 - 600 nm)
Fluorescein (240 - 400 nm) Quantum Counter

Quinine Sulfate (220 - 340 nm)

Reference
Detector
1.2
Eppley Thermopile/ QC

0.8
Linearity of Rhodamine
as a quantum counter
0.4
Fluorescence
Here we want the inner filter effect!
0.0
200 400 600
Wavelength (nm)

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

 Excitation Correction
Absorption (dotted line) and Excitation Spectra (solid line) of ANS in Ethanol
1.0 1.0
A
Uncorrected B
0.8
Ratio Corrected
0.8
Fluorescence

Fluorescence
0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0
250 300 350 400 450 250 300 350 400 450
Wavelength (nm)
Wavelength Wavelength (nm)
Wavelength
1.0
C
0.8 Lamp
Fluorescence

Corrected
0.6

0.4

0.2

0.0
250 300 350 400 450

Wavelength (nm)
Wavelength

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

 Polarizers

The Glan Taylor prism polarizer

Two Calcite Prisms

0

90
0
Common Types:
Glan Taylor (air gap)
Glan Thompson
Sheet Polarizers 90

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.
 Sample Issues

Signal Attenuation of the Excitation Light

PMT Saturation
Excess Emission

30
Fluorescence vs Signal 3
25

x10
6
2
Instrument Signal

x10
LINEAR REGION 20

6
15
1
10

540 560 580 600 620 640 660 680 700

Wavelength (nm)

[Fluorophore]
Reduced emission intensity
1. ND Filters
2. Narrow slit widths
3. Move off absorbance peak
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

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

1.0
4
3 3 Diluted Sample
0.8
3

x10
0.6

6
6

2 2

x10
x10

6
2
0.4
1 1
1
0.2

450 500 550 600 650 700 540 560 580 600 620 640 660

Wavelength (nm) Wavelength (nm)

Absorbance Spectrum (1) Spectral Shift

(2) Change in Spectral Shape
 How do we handle highly absorbing solutions?

Quartz/Optical Glass/Plastic Cells

Excitation
Emission
Path Length

4 Position Turret
Emission

SPEX Fluoromax-2, Jobin-Yvon

Detector

Excitation
Path Length
 Front Face Detection
Triangular Cells Thin Cells & Special Compartments

Excitation IBH, Glasgow  G3

8JU
United Kingdom
Mi Excitation
rr o
Emission r
Detector

Sample
[1]

Absorbance
Measurements
Reflected Excitation & Emission

[1] Adapted from Gryczynski, Lubkowski, & Bucci Methods of Enz. 278: 538
Lifetime Instrumentation
 Light Sources for Decay Acquisition:
Frequency and Time Domain Measurements

Pulsed Light Sources (frequency & pulse widths)

Mode-Locked Lasers
ND: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 Sources

Diode 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 ns
Coaxial nanosecond flashlamp (IBH), 10Hz-100kHz, 0.6 ns
 Modulation of CW Light
Use of a Pockel’s Cell

Pulsed Emission

Polished on a side
0 exit plane

Pockel’s Cell Polarizer

Mirror

90
Polarizer
CW Light Source
Radio Frequency
Input
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
Double Pass Pockel’s Cell 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.
 Time Correlated Single Photon Counting

Sample Compartment
Pulsed Light Source

Timing Electronics Filter or Monochromator

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

Photon Counting PMT

PMT
Constant Fraction
Discriminator
TAC
Time-to-Amplitude
Converter (TAC)
Multichannel
Analyzer Instrument Considerations
Excitation pulse width
Excitation pulse frequency
Counts

Timing accuracy
Time Detector response time (PMTs 0.2-
0.9 ns; MCP 0.15 to 0.03 ns)
 Histograms built one photon count at a time …
1
8
6

4
Fluorescence Decay
2
Fluorescence

0.1
8
6

4
Instrument Response Function

0.01
8
6

0 50 100 150 200 250 300

Channels (50 ps)

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

Will the corrections never end ???

[1] = I0 + I90
[2] = I0 + I90
An intuitive argument: [3] = I0 + I90
[6] [4]
[4] = I0 + I90
0
[1] [5] = 2 x I90
Polarized Excitation [3] [6] = 2 x I90
Total = 4 x I0 + 8 x I90

[5] The total Intensity is proportional to:

0
[2] I0 + 2 x I90
90
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
 Frequency Domain Fluorometry

Pockel’s Cell

Sample Compartment
CW Light Source

Filter or Monochromator

PMT

PMT
Analog PMTs (can also be done with photon counting)
RF

Reference Turret
RF

Signal
Synthesizers Signal
S1 and S2 S1 S2 Digital Acquisition
Electronics
Locking Signal

S1 = n MHz

S2 = n MHz + 800 Hz
Similar instrument
Computer Driven considerations as
Controls With TCSPC
Lifetime Station #3, LDF, Champaign IL, USA
& hiding under the table:

RF Amplifiers Frequency Synthesizers