Workshop UMN Physics June 8-10, 2006 Steady-state Fluorometer Joachim 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

Excitation Polarizer Sample

Emission Excitation Wavelength Light Source Polarizer Selection Emission Wavelength Computer Selection

Fluorometer: DetectorThe Basics

Note: Both polarizers can be removed from the optical beam path OPTICAL LAYOUT: PTI Model QM-4

1 Lamp housing 2 Adjustable slits 3 Excitation Monochromator 4 Sample compartment 5 Baffle 6 Filter holders 7 Excitation/emission optics 8 Cuvette holder 9 Emission port shutter 10 Excitation Correction 11 Emission Monochromator 12 PMT detector Light Sources Lamp Light Sources Xenon Arc Lamp Profiles

1. Xenon Arc Lamp (wide Ozone Free range of wavelengths)

Visible UV 2. High Pressure Mercury Lamps (High Intensities but concentrated in specific lines) Mercury-Xenon Arc Lamp Profile 3. Mercury-Xenon Arc Lamp (greater intensities in the UV)

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

time

Continuous Current Measurement Signal

Photon Counting: Analog:

Constant Variable High Voltage Supply Voltage Supply PMT PMT

level Anode Current Discriminator = Sets Level TTL Output Pulse averaging (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 Long Pass Optical Filters

100

80 Spectral Shape 60 Thickness Size 40 Fluorescence (!?)

Transmission (%) Transmission 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

60

40

20 Transmission (%)

0 300 400 500 600 700 Wavelength (nm)

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) Order of diffraction

1-st order

0-th order (acts like a mirror)

2-nd order Monochromator Slits The slit size determines the bandpass and the throughput

Reducing the slit size leads to … 1. Drop in intensity 2. Narrowing of the spectral selection

Changing the Emission Bandpass 1.0 1.0

17 nm 0.8 17 nm 0.8 u) a 0.6 8.5 nm 0.6

6 8.5 nm x10 0.4 4.25 nm 0.4

2.125 nm 4.25 nm 0.2 0.2 Fluorescence ( 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 Typical Background

Emission Scan: Excitation 300 nm Glycogen in PBS

350 nd Excitation (Rayleigh) Scatter 2 Order Scatter 300 (300 nm) (600 nm) u) 250 a

3 200 Water RAMAN 2nd Order RAMAN x10 150 (334 nm) (668 nm)

100 Fluorescence ( 50

0 200 300 400 500 600 700

Wavelength (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: 107 (1) Take the excitation wavelength (say = 587 107 490 nm) and insert in the following − 3400 equation: 490 (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 Bias

Tungsten Lamp Profile Collected on an SLM Fluorometer

Wood’s Anomaly Parallel Emission

No Polarizer

Fluorescence Perpendicular Emission Fluorescence

250 800

250 800

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

Light Source Monochromator Detector

Ideal Fluorometer: Efficiency Efficiency Light intensity wavelength wavelength wavelength

Emission Correction: Excitation 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).

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. Sample Issues

Signal Attenuation of the Excitation Light PMT Saturation Excess Emission

30 Fluorescence vs Signal 3 25 x1

6 2 0 6 al

20

LINEAR REGION x10 n

15 1 10

540 560 580 600 620 640 660 680 700

Instrument Sig Wavelength (nm)

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

Common Types: The Glan Taylor prism polarizer Glan Taylor (air gap) Two Calcite Prisms Glan Thompson 0 Sheet Polarizers

90 0

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.