Spectrofluorometers: Tools for Measuring Fluorescence Signature

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Spectrofluorometers (or fluorescence spectrophotometers) measure the fluorescence signature of an analyte in a sample based on its specific excitation and emission wavelengths. The fluorescence signature can be correlated to the concentration level of the analyte in the sample.

A spectrofluorometer can be used in basic and applied research, biofuels analysis, biotechnology applications, quality control, medical diagnostics, plasma monitoring and polymer analysis and as a tool in teaching laboratories. 

How a spectrofluorometer works

The essential components of a spectrofluorometer are a light source, an excitation monochromator, a sample cell/cuvette, an emission monochromator and a detector. 

The light source sends out light at the excitation wavelength of an analyte in a sample. Before it reaches the sample, the light passes through the excitation monochromator, which transmits a wavelength specific to the excitation spectrum of the analyte while blocking other wavelengths. The light from the excitation monochromator passes through the sample contained in the sample cell/cuvette holder and excites the analyte. Following excitation, the analyte relaxes and emits light at an emission wavelength longer than the excitation wavelength. The emitted light passes through the emission monochromator positioned at a right angle to the excitation light. The emission monochromator minimizes light scatter and screens the emission light before it reaches the detector. The detector measures the emitted light, displays the fluorescence value and produces the fluorescence signature of the analyte. The fluorescence value is proportional to the concentration level of the analyte in the sample.

Steady-state fluorescence method

Spectrofluorometers can utilize the steady-state (SS) fluorescence method. This method is based on the measurement of the long-term average fluorescence of a sample when irradiated with UV, visible or near-infrared (NIR) light. Examples of SS spectrofluorometers are the QuantaMaster 300, QuantaMaster 300 Plus and QuantaMaster 400 from Photon Technology International, Inc.; the FLS980 and FS5 from Edinburgh Instruments Ltd. and FluoroMax, FluoroLog-3, Fluorolog Extreme and Aqualog from HORIBA Scientific.

Fluorescence lifetime method

Spectrofluorometers can also employ the fluorescence lifetime (FLT) method, which is based on the measurement of the change in fluorescence over time of an analyte when irradiated with UV, VIS or NIR light.

Photon Technology International, Inc. offers the TimeMaster 3000, TimeMaster 50, TimeMaster 2000 and PicoMaster 1000 FLT spectrofluorometers. Time-correlated single-photon counting method The time-correlated single-photon counting (TCSPC) method is applicable to FLT measurements. The two terms, TCSPC and fluorescence lifetime, are commonly used in practice to mean the same thing. TCSPC is based on the detection times of the individual photons and the reconstruction of the waveform from the individual time measurements.

TCSPC spectrofluorometers include the DeltaFlex and DeltaPro from HORIBA Scientific and the LifeSpec II from Edinburgh Instruments Ltd. 

Purchasing considerations for spectrofluorometers 

Application

There is a wide range of spectrofluorometers to choose from, depending on the area of application.

The USB4000-FL spectrofluorometer from Ocean Optics is well-suited for general fluorescence measurements.

Photon Technology International, Inc. offers the QuantaMaster 800 high-speed multiwavelength spectrofluorometer.

RatioMaster and RatioMaster SL200 microscope-based ratio spectrofluorometers from Photon Technology International, Inc. are intended for imaging applications.

The NanoLog from HORIBA Scientific is applicable to nanotechnology and nanomaterials, and the Aqualog is for water quality analysis.

Edinburgh Instruments Ltd. offers the mini-τ, dedicated to fluorescence lifetimes for student teaching. The company also provides a modular spectrofluorometer, the FLS980, and a compact benchtop spectrofluorometer, the FS5.

The Z600 standard and Z620 fast nonimaging chlorophyll spectrofluorometers are from Qubit Systems Inc., as are the Z980 Probe AquaPen and Z990 FluorPen handheld chlorophyll spectrofluorometers.

HORIBA Scientific provides a modular spectrofluorometer, the FluoroLog-3, and a compact benchtop spectrofluorometer, the FluoroMax-4 and FluoroCube.

Light source

An important consideration in selecting a spectrofluorometer is the light excitation source. One type of light excitation source is a xenon arc lamp.

Olis, Inc. offers the DM 45 with a 75-W xenon arc lamp light source, the DM 245 with a 150-W xenon arc lamp light source, and the RSM 1000F1 and RSM 1000F4 with a 450-W xenon arc lamp light source.

The FP-8200, FP-8300, FP-8500 and FP-8600 from JASCO Analytical Instruments have a 150-W xenon lamp and shielded lamp housing. 

The Jenway 6270, Jenway 6280 and Jenway 6285 from Bibby Scientific Ltd. have a pulsed xenon light source.

Lasers provide light excitation when a greater amount of energy is needed. Light-emitting diodes (LEDs) provide light excitation when improved efficiencies are needed.

Photon Technology International, Inc. supplies the QuantaMaster 310 with a pulsed nitrogen/dye laser combination. 

The FLS980 and FS5 from Edinburgh Instruments Ltd. can be supplied with continuous and pulsed xenon light sources and proprietary picosecond pulsed LEDs and laser diodes.

The DeltaPro-DD and DeltaPro-NL from HORIBA Scientific feature a laser-diode light source.

The Z600 from Qubit Systems Inc. comes with three sets of LEDs and a PIN iode detector.

Wavelength range

Photon Technology International’s QuantaMaster 400 features a wavelength range of 185–900 nm. The QuantaMaster 500 has a wavelength range of 500–1700 nm (optional up to 3200 nm) and the QuantaMaster 600 has wavelength ranges of 950–1400 nm, 300–400 nm, 950–1700 nm and 300–1700 nm.

The FP-8500 from JASCO Analytical Instruments has a wavelength range of 200–850 nm and the FP-8600 a wavelength range of 200–850 nm (excitation) and 200–1010 nm for (emission).

A wavelength range of 220–900 nm can be found in the RF-5301PC from Shimadzu.

Bibby Scientific’s Jenway 6270, 6280 and 6285 have wavelength ranges of 190–1100 nm, 190–650 nm and 190–850 nm, respectively.

Wavelength accuracy

HORIBA Scientific’s FluoroMax has a wavelength accuracy of ±0.5 nm.

The FS5 from Edinburgh Instruments Ltd. has a wavelength accuracy of ±0.5 nm and the FLS980 has a wavelength accuracy of ±0.2 nm.

The FP-8200 from JASCO Analytical Instruments has a ±2.0-nm wavelength accuracy; the FP-8300 and FP-8500 have a wavelength accuracy of ±1.5 nm, and the FP-8600 has ±1.0-nm wavelength accuracy (excitation) and ±2.0-nm wavelength accuracy (emission).

Scanning speed

HORIBA Scientific’s FluoroLog-3 and NanoLog have a scanning speed of 150 nm/sec, and the FluoroMax has a scanning speed of up to 80 nm/sec.

The FLS980 from Edinburgh Instruments Ltd. has a scanning speed of 160 nm/sec, and the FS5 has a scanning speed of 100 nm/sec.

The FP-8600 from JASCO Analytical Instruments has a scanning speed of 20–60,000 nm/min (excitation) and 20–120,000 (emission).

Customization

Most providers offer customization options. The Q-5H system enhancement from Photon Technology International, Inc. converts the QuantaMaster 300 to the QuantaMaster 400.

Olis, Inc. offers a modernization procedure for the SLM SPF-500, 4800, 8000 and 8100 spectrofluorometers.

Representative spectrofluorometers

• Agilent Technologies: Cary Eclipse
• B&W Tek, Inc.: Glacier X
• BERTHOLD TECHNOLOGIES: Twinkle LB 970
• Bibby Scientific Ltd.: Jenway 6270, 6280, 6285
• Edinburgh Instruments Ltd.: FS5, FLS980, FLS920, LifeSpec II, mini-τ
• Hitachi High Technologies America, Inc.: F-2700, 2710
• Hoefer, Inc.: DQ300
• HORIBA Scientific: FluoroMax, FluoroLog-3, Fluorolog Extreme, Aqualog, NanoLog, Dual-FL, DeltaFlex, DeltaPro-DD, DeltaPro-NL, FluoroCube, FluoroCube Extreme
• ISS, Inc.: ChronosBH, ChronosFD, K2, PC1
• JASCO Analytical Instruments: FP-8200, FP-8300, FP-8500, FP-8600
• LEUKOS: HALCYONE
• Ocean Optics: USB4000-FL, USB4000-FL-395, USB4000-FL-450, QE Pro-FL
• Olis, Inc.: DM 45, DM 245, RSM 1000F1, RSM 1000F4, Cary 14F (modernized SLM SPF-500, 4800, 8000, 8100)
• PerkinElmer, Inc.: LS 45, 55
• Photon Technology International, Inc.: QuantaMaster 300, 300 Plus, 310, 400, 500, 510, 520, 600, 800; TimeMaster 3000, 50, 2000; PicoMaster 1000, RatioMaster, RatioMaster SL200
• PicoQuant: FluoTime 300, 200, 100
• Qubit Systems Inc.: Z600, Z620, Z985, Z980, Z995, Z990
• SAFAS Monaco: Xeniux SC, XM, XOF
• Shimadzu: RF-5301PC
• Standa Ltd.: S-FL
• StellarNet Inc.: BLACK-Comet-TEC
• Thermo Fisher Scientific: NanoDrop 3300

 Table 1 – Providers of spectrofluorometers

Providers

A list of providers of spectrofluorometers is given in Table 1.

Looking ahead

Future developments in nanotechnology and cell biology are expected to have an impact on the configuration of spectrofluorometers.

Lina Genovesi, Ph.D., JD, is a Technical, Regulatory, and Business Writer based in Princeton, NJ; e-mail: [email protected];  www.linagenovesi.com