Combination systems aid easy detection

Absorption, fluorescence and luminescence spectra in microplate format

We cannot imagine laboratory analysis today without microplates. Particularly with medium to high sample throughputs, they display their strengths and allow the parallel analysis of up to 1536 samples. Both the handling of liquid samples, and the reading of the microplates at the end of the analytical cycle are carried out by fast and parallel working pipettors and microplate readers. In most assays, reporter systems are used, which are based on the generation of a colourant, the release of a fluorescent dye or chemiluminescence. For the optical detection of these reporters, Analytik Jena offers a family of microplate readers, including both simple photometers and spectrally resolving instruments. The latest microplate readers of the FLASHScan Series from Analytik Jena are so-called combination systems allowing absorption, fluorescence and luminescence to be detected on a single instrument.

Technical design
The exceptional feature of the FLASHScan microplate readers is that they allow the analysis of both absorption and fluorescence spectra in the range from 200-900nm. Thus, the spectral information can be used as additional source of information without an increase of the measuring time per microplate, as this would be the case with scanning instruments. This is made possible by the use of a polychromator with CCD matrix detector (Figure 1).

Figure 1: Optical path of FLASHScan 550

The light source used is a xenon flash lamp the light of which is transmitted through six optical fibres to the sample, which results in a further reduction of measuring time. Because of the parallel measurement in six channels, a 96-well microplate can be completely read in less than 15 seconds. The transmitted light or the fluorescence or luminescence light is directed from the sample to the polychromator through six further optical fibres. There, the optical fibres are arranged as slit and imaged onto the CCD matrix via a holographic grating. On the FLASHScan 550, the CCD matrix is cooled thermoelectrically, allowing longer integration times in luminescence measurements. All functions of the microplate readers are controlled by a computer with WinFLASH software via the USB port.

Absorption measurements
In the absorption and transmission modes, the sample is illuminated with the complete spectrum of the flash lamp (white light). The technical design of the FLASHScan Series readers provides fast spectrally resolved absorption measurements to be taken on 96-well and 384-well microplates. That way, it is possible to record UV VIS spectra of 96 samples in less than 15 seconds. These readers are also suitable for end-point determinations and multiwavelength measurements. The optimised optical configuration does not only permit measurements to be taken at one position in the well. In the four-point measuring mode, the instrument measures at four different points in the well. The result is an averaged spectrum based on the spectra measured at these points. Moreover, an optimum signal-to-noise ratio is achieved in the measurement by varying the number of light flashes of the xenon lamp. Using the WinFLASH control and data analysis software, spectra can be automatically evaluated by different criteria (Figure 2).

Figure 2a: Survey presentation of absorbsion spectra

Figure 2b: Visualisation of absorbtion with colour scale
(from blue via green to red with increasing absorbtion at the maximum)

Figure 2c: Analysis of individual spectrum

Therefore the microplate readers do not only ensure a high scanning speed, but also provide comfortable result analysis options, which makes them universally applicable, either as single system or integrated in screening and robotic systems.

Fluorescence measurements
For fluorescence excitation, the user can adjust optional wavelengths in the range from 390-720nm. The wavelengths are selected from the spectrum of the flash lamp by means of a variable interference filter. For applications requiring fluorescence excitation in ultraviolet light, three filter holders are available, which can be fitted with user-specific filters and moved into the optical path via software control. The reader can be calibrated to the great variety of plate geometries commercially available to ensure optimum measurement results. Particularly the optimum adjustment of the position of the beam focus in the sample (Z focusing) is crucial for achieving high detection sensitivity. The optimum Z focus is automatically determined by the reader by means of a calibration standard. The systems also permit the Z focus to be adapted to user-specific samples, e.g. adherent or sedimented cells. In fluorescence measurements, there is always a certain percentage of the excitation light also entering the detection channel. On traditional, filter-based microplate readers, this percentage of excitation light may distort the result of the measurement. As the FLASHScan Series readers disperse the light spectrally in the polychromator, the excitation light can be clearly separated from fluorescence light. If desired, it appears in the spectrum as excitation peak.

Figure 3a: Fluorescence specrum of different NADH
concentraions with excitation peak

In the example shown in Figure 3, NADH (nicotinamide adenine dinucleotide) was measured once with (Figure 3a) and once without (Figure 3b) excitation peak. For this method, the fluorescence excitation is at 340nm, while the emission is to be expected at 465nm. Filtering out the excitation peak does not only improve the selectivity of the method, but also the detection limit.

Figure 3b: Fluorescence specrum of different NADH
concentraions without excitation peak

Kinetic measurements
Kinetic measurements require constant temperatures during and between the measurements. For this purpose, a sample temperature-control system for temperatures up to 45°C has been integrated in the readers. For the first time, three-dimensional fluorescence spectra (fluorescence emission as a function of wavelength and excitation wavelength) can be recorded at acceptable measuring times by the simultaneous measurement of complete fluorescence spectra. The fast microplate reading capability is also advantageous for kinetic measurements. Because of the spectral resolution of absorption or fluorescence, three-dimensional kinetics measurements are possible. In these measurements, the wavelength-dependent change of absorption can be plotted versus time (Figure 4).

Figure 4: Three-dimensional reaction kinetics with an example of the
enzymatic reaction of pyruvate with LDH (L-lactate dehydrogenase) and NADH (nicotinamide adenine dinucleotide). The concentration of NADH as fluorescent componet decreases in the course of the reation.

Conclusion
So far, microplate readers could measure absorption and fluorescence only at fixed wavelengths. With the newly developed reader technology of Analytik Jena, it has become possible to run spectrally resolved absorption and fluorescence measurements in microplate format. Many additional features provided by the microplate reader system, such as luminescence measurement, temperature control and shaking of microplates, allow users to easily cope with the requirements of standard applications. In addition, they open up new applications in research and development in the field of screenings and assays.

By Dr. Stefan Winter, Anja Jungnickel, Andreas Möbius, Dr. Alf Liebmann, Analytik Jena