Fluorescence Workshop UMN PhysicsJune 8-10, 2006

Steady-state FluorometerJoachim Mueller

Instrumental Setup, light sources, wavelength selection, detectors, polarizers, corrections, inner filter effect

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

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

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

Fluorometer Components

Fluorometer: The Basics

Excitation WavelengthSelection

EmissionWavelength

Selection

Sample

Light Source

Detector

Computer

Excitation Polarizer

Emission Polarizer

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

OPTICAL LAYOUT: PTI Model QM-4

1 Lamp housing2 Adjustable slits3 Excitation Monochromator4 Sample compartment5 Baffle6 Filter holders7 Excitation/emission optics8 Cuvette holder9 Emission port shutter10 Excitation Correction11 Emission Monochromator12 PMT detector

Light Sources

Lamp Light Sources

UV

Ozone Free

Visible

Xenon Arc Lamp Profiles

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)

Mercury-Xenon Arc Lamp Profile

4. Tungsten-Halogen Lamps

Detectors

Photomultiplier tube (PMT)http://micro.magnet.fsu.edu/primer/flash/photomultiplier

Principle of operation:

Pictures:

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

DiscriminatorSets Level

PMT

TTL Output(1 photon = 1 pulse)

PMT

Variable Voltage Supply

Computer

Anode Current=

Pulse averaging

Continuous Current Measurement

Photon Counting: Analog:

level

Wavelength Selection

Fixed Optical Filters

Tunable Optical Filters

Monochromators

Long Pass Optical Filters

Hoya O54

100

80

60

40

20

0800700600500400300

Wavelength (nm)

Tran

smis

sion

(%)

Spectral ShapeThicknessSizeFluorescence (!?)

More Optical Filter Types…

100

80

60

40

20

0700600500400300

Tran

smis

sion

(%)

Wavelength (nm)

Broad Bandpass Filter(Hoya U330)

Interference Filters(Chroma Technologies)

Neutral Density(Coherent Lasers)

Monochromator: Principle

Diagram of a Czerny-Turner monochromator.Light (A) is focused onto an entrance slit (B) and is collimated by a curved mirror (C). The collimated beam is diffracted from a rotatable grating (D) and the dispersed beam re-focused by a second mirror (E) at the exit slit (F). Each wavelength of light is focused to a different position at the slit, and the wavelength which is transmitted through the slit (G) depends on the rotation angle of the grating.

The Inside of a Monochromator

Mirrors

Grating

Nth Order(spectral distribution)

Zero Order(acts like a mirror)

0-th order (acts like a mirror)

2-nd order

1-st order

Order of diffraction

Monochromator SlitsThe slit size determines the bandpassand the throughput

Reducing the slit size leads to …

1. Drop in intensity2. Narrowing of the spectral selection

1.0

0.8

0.6

0.4

0.2

0.0580560540520

1.0

0.8

0.6

0.4

0.2

0.0

x106

580560540520

17 nm

2.125 nm

4.25 nm

8.5 nm

Fluo

resc

ence

(au)

Wavelength (nm)

Changing the Emission Bandpass

Wavelength (nm)

17 nm

8.5 nm

4.25 nm

2.125 nm

Collected on a SPEX Fluoromax - 2

Typical Background Emission Spectrum

Emission Scan:Excitation 300 nmGlycogen in PBS

350

300

250

200

150

100

50

0

x103

700600500400300200

Wavelength (nm)

Fluo

resc

ence

(au)

Excitation (Rayleigh) Scatter(300 nm)

2nd Order Scatter(600 nm)

Water RAMAN(334 nm)

2nd Order RAMAN(668 nm)

Fluorescent Contaminants

Raman scatter of water

Vibrational modes of water

Resonant Stokes Raman scattering

Energy for the OH stretch vibrational mode in water (expressed in inverse wavenumbers): 3400 cm-1

Simple formula to calculate the wavelength of the Raman peak:7

7

490

10 58710 3400

=−

(1) Take the excitation wavelength (say 490 nm) and insert in the following equation:

(2) The result specifies the position of the raman peak in nanometers (i.e. the raman peak is at 587nm for an excitation wavelength of 490nm.

Monochromator Polarization BiasTungsten Lamp Profile Collected on an SLM Fluorometer

Wood’s AnomalyParallel Emission

Perpendicular Emission

No Polarizer

Fluo

resc

ence

Fluo

resc

ence

800 250

250 800

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

Distortion of Excitation and Emission Spectra

Ideal Fluorometer:

Ligh

t in

tens

ity

wavelength wavelength wavelength

Light Source Monochromator Detector

Effi

cien

cy

Effi

cien

cy

Excitation Correction:

A beam splitter diverts a few percent of the excitation light impinging on the sample. This light is measured with a calibrated silicon photodiode (10). Thus the instrument monitors at all times the intensity of the excitation light.

Emission Correction:

Correcting the emission spectra can be quite complicated and requires specialized techniques.

Luckily, the wavelength and polarization dependent correction factors have been determined and are electronically available (see lab section).

Sample Issues

Signal Attenuation of the Excitation Light PMT Saturation

30

25

20

15

10

x106

700680660640620600580560540

3

2

1

x106

Wavelength (nm)

Excess Emission

Fluorescence vs Signal

LINEAR REGION

Inst

rum

ent S

igna

l

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

Polarizers

Common Types:

Glan Taylor (air gap)

Glan Thompson

Sheet Polarizers

The Glan Taylor prism polarizer

0

900

Two Calcite Prisms

Sheet polarizer

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.