F980 Software

Excitation and Emission Scans

Excitation and emission spectra are standard measurements in fluorescence spectroscopy. The figure demonstrates a measurement of a well documented standard test solution of anthracene in degassed cyclohexane.

Sample: Anthracene in cyclohexane (10^-5M). Measurement conditions: λex = 358 nm for emission scan, λem = 400 nm for corrected excitation scan, Δλex = Δλem = 0.4 nm, step size = 1 nm, integration time = 1 s.

Synchronous Scans

In synchronous scans, both excitation and emission monochromators are scanned synchronously with a pre-set offset. The figure demonstrates a sample of five different aromatic hydrocarbons dissolved in cyclohexane, measured with a conventional emission scan (red) and a synchronous scan with zero offset (green). The five hydrocarbons are resolved by the synchronous scan.

Sample: Five aromatic hydrocarbons dissolved in cyclohexane. Measurement conditions: λex = 280 nm for emission scan, Δλex = Δλem = 0.5 nm, step size = 0.5 nm, integration time = 1 s, offset = 0 nm.

Kinetic Scans

Kinetic scans reveal temporal changes of the sample fluorescence at fixed excitation and emission wavelengths. Luminescence emission in the milliseconds to seconds range, such as long phosphorescence, chemical reactions or chemical migration in cells, can be studied. As an example, using the FLS980 in T-geometry for dual wavelength detection, simultaneous measurements of the Ca²⁺ active fluorophore Indo-1 can be made with both emission arms set to different wavelengths.

Sample: Human platelets cells loaded with Indo-1 in 1 mM Ca²⁺. Measurement conditions: λex = 340 nm, λem1 = 485 nm, λem2 = 410 nm, Δλex = Δλem = 1 nm, integration time = 0.5 s.

Excitation – Emission Maps (EEM)

The variety of measurement, display and analysis options allows easy and fast investigation of unknown luminescent samples or samples which contain different fluorophores. One method is to measure a series of emission scans within a selected range of excitation. The result is then demonstrated either in a 3D plot or in a contour plot.

Temperature Maps

The F980 software can communicate with Oxford Instruments Optistat DN (liquid nitrogen) and Optistat CF (liquid helium) cryostats (along with TE controlled sample holders). Temperature maps can be made by acquiring a series of emission, excitation or synchronous scans for a predefined temperature range. The individual measurements are automatically started when the target temperatures are reached.

f980-temp-map

Sample: CuInSe₂ (a material used for photovoltaic cells). Measurement conditions: F980 controlled Optistat, Xe1, NIR-PMT, λex = 694 nm, Δλex = 10 nm, Δλem = 5 nm, step size = 1 nm, integration time = 0.2 s. Temperature range: 6 K – 106 K, step 20 K.

Absolute Quantum Yield Measurements

The absolute method for fluorescence quantum yield measurements is becoming more widely used than the relative method, as it does not require a quantum yield standard. This is readily applicable to liquids, films and powders and can be extended into the near infrared spectral range.

The picture shows the independence of the fluorescence quantum yield from the wavelength of excitation for a standard organic dye. The graph shows the area of absorption for eight different excitation wavelengths on the left, while on the right it shows the corresponding emission spectra, scaled by a factor of 5. The inset shows the calculated quantum yields.

Sample: Quinine bisulphate in perchloric acid. Measurement conditions: integrating sphere, Δλex = 5.0 nm, Δλem = 0.5 nm, integration time = 0.3 s.

Singlet Oxygen Emission

The emission of singlet oxygen is known to be very weak and, historically, powerful laser excitation has been used to monitor this. However, both excitation and emission spectra of singlet oxygen can be measured using the FLS980 with a broadband xenon lamp. The figure demonstrates a measurement of singlet oxygen luminescence generated from hematoporphyrin in ethanol. In a mixture of photosensitizers, the excitation spectrum may be used to identify the singlet oxygen generator.

Sample: 10^-5 M hematoporphyrin in ethanol. Measurement conditions: λex = 380 nm for emission scan, λem = 1270 nm for excitation scan, Δλex = Δλem = 2.0 nm, step size = 1.0 nm, integration time = 3.0 s.

Fluorescence Upconversion

Fluorescence upconversion materials absorb light in the near infrared spectral range and produce emission of shorter wavelengths in the visible spectral range. These upconversion materials are currently the focus of research for their use in dye sensitized solar cells (DSSC). Efficient upconversion is important for the efficiency of solar panels in the near infrared part of the sun’s spectrum.

The picture below shows the upconversion emission of an erbium-ytterbium doped TiO₂ powder, for four different levels of excitation power at 980 nm. As shown in the insert, some of the upconversion lines scale close to linear with excitation power, whereas others scale approximately to the square of the excitation power.

Samples: Er³⁺/Yb³⁺ doped TiO2. Measurement conditions: 1 W diode laser with power adjustment @ 980 nm, R928P, Δλem = 0.25 nm, integration time = 0.5 s.

Other steady state measurement examples: Steady state fluorescence anisotropy, contour plots, water quality assessments, excimer equilibrium, reflection, absorption and quantum yield measurements of phosphor powders, chromaticity and much more.

Single and Multiple Exponential Decays

Fluorescence lifetime is an excellent parameter for analysing the interaction of fluorophores with their micro-environment, such as solvents, neighbouring fluorophores or non-fluorescing molecules. These “environmental” effects will reduce the natural decay process (characterised by the natural lifetime), to shorter and often more complex decay kinetics.

Most fluorescence decay kinetics are analysed by single or multiple exponential models. The user fits the raw data to a specific model. The quality of the fit will determine whether or not the selected model was appropriate, and – if it was – the result will provide the fit parameter such as lifetimes and pre-exponential factors.

The example shows two measurement results of the same homogeneous solution, taken at two different emission wavelengths. The decay at the shorter wavelength is clearly a single exponential, the decay at the longer wavelength is best characterised by three exponential components.

Sample: Hematoporphyrin IX in phosphate buffer (pH 7.2)

Measurement conditions: EPL 405, MCP-PMT, λex = 398 nm, Δλem = 1.0 nm, rep rate = 1 MHz, λem = 620 nm (left and right graph)

Data analysis: Multi-exponential reconvolution, confidence intervals verified by support plane analysis (FAST). τ₁ = 15.02 ± 0.03 ns (left). τ₁ = 14.80 ± 0.20 ns, τ₂ = 4.62 ± 0.55 ns, τ₃ = 0.81 ± 0.20 ns (right).

Time-Resolved Fluorescence Anisotropy

By exciting the sample with vertically polarised light and recording the emission in both the vertical and horizontal plane, one can calculate the fluorescence anisotropy of a homogeneous sample. The fluorescence anisotropy reveals the average rotational diffusion time of the molecules.

The measurement example shows that rotational diffusion in the picosecond time scale can be accurately measured. Most samples show rotational diffusion. To avoid this effect when precise fluorescence lifetime measurements are required, the emission polariser must be set to magic angle conditions (and vertically polarised excitation used).

Sample: POPOP in cyclohexane (IRF, decays with parallel and crossed polariser – left plot), fluorescence anisotropy (raw data and fit – right plot). Measurement conditions: EPL 375, MCP-PMT, λex = 375 nm,  Δλex = 2.0 nm, λem = 390 nm,  Δλem = 2.0 nm.

Data analysis: Full anisotropy reconvolution (FAST) with ellipsoidal rotor model. The rotation diffusion times are 110 ps, 150 ps and 620 ps respectively. A spherical rotor model results in a fit with significantly increased chi-square. POPOP is a rod like molecule.

Other TCSPC measurement examples: Time-resolved emission spectroscopy (TRES), monomer-excimer kinetics, solvent relaxation dynamics and much more.

Time-Resolved Measurements of Lanthanides

The photoluminescence emission lifetime of lanthanides extends over a large time range from nanoseconds to seconds where the method of choice for time-resolved measurements is the MCS technique. Due to the high dynamic range and the accuracy resulting from counting statistics, complex decay analysis can be performed.

The pictures show time resolved measurements from a lanthanide doped glass sample at two different emission wavelengths. At the shorter wavelength the decay is best fitted with a three exponential terms, while at the longer emission wavelength the initial rise is followed by a millisecond decay.

Sample: Rare-earth doped glass

Measurement conditions: μF2, λex = 370 nm,  Δλex = Δλem = 2.5 nm, rep rate 100 Hz, step size = 10 nm, spectra produced for every 50 μs (Top Left). μF2, λex = 370 nm,  λem = 430 nm, Δλex = Δλem = 2.5 nm, rep rate 100 Hz, step size = 10 nm, measurement time = 2 min (Top Right). μF2, λex = 370 nm,  λem = 612 nm, Δλex = Δλem = 1.7 nm, rep rate 20 Hz, measurement time = 8 min (Bottom Left).

Data Analysis: Multi-exponential reconvoltution. Good fit results were achieved with four exponential decay model (Top Right) and model comprising two exponential rise and one decay function (Bottom Left).

Other MCS measurement examples: Time-resolved singlet oxygen measurements, time-resolved FRET measurements and much more.