76 SPECTROSCOPY
properties by monitoring interchanging spectroscopic bands. Tis way, for example changes in the overall concentration, the pH, the redox potential, the presence of distinct functional groups but also the purity of sample can be identified.
Vast benefits Recording the entire excitation/ absorbance and/or emission spectrum or at least determining the intensity within multiple small spectral windows has immense benefits compared to single wavelength measurements. For example, the expression level of distinct fluorescent proteins can be investigated while the absolute cell number is monitored simultaneously. Moreover, the frequency domain spectroscopy allows users to record cellular fluorescence in a number of small spectral windows as a basis for spectral un-mixing.
Here, the spectral emission is subdivided into a number of spectral intercepts and the integrated intensity of each intercept is compared to the equivalent spectral
region of the spectrum of the pure dye. Tis technique has been successfully implemented to suppress cellular auto- fluorescence when detecting low-abundant proteins. Assuming that auto-fluorescence background has a different spectral fingerprint than the marker dye, any untargeted contribution can be suppressed.
Interaction studies Another important field of spectroscopic applications in MLR addresses the analysis of protein-protein interaction studies in a living cell context. Whereas molecular interaction can hardly be visualised by spatially resolved co-localisation studies, fluorescence resonance energy transfer (FRET) is a prominent technique to identify these interactions on a nanometer length-scale.
Based on the dipole-dipole coupling of two adjacent chromophores with overlapping emission and absorbance spectra, this technique intrinsically requires spectroscopic techniques. A quantitative approach records both the (partially quenched) emission
of the donor chromophore and the emission of the acceptor dye and ratiometrically calculates the absolute molecular interaction strength via an average chromophoric distance in a dynamic system.
Te concentration of nucleic acids and proteins can be determined label-free based on their specific absorption coefficient by measuring the absorbance at their respective absorption maxima at 260 and 280 nm.
In addition the respective ratios and in addition the ratios to 230 or 240 nm indicate the purity of the samples. Measuring the absorption at 977 nm allows for the determination of the path length and thus within a known sample geometry the calculation of the amount of analytes.
Te software of current MLRs is not only limited to mere data acquisition but is also feasible to analyse the raw data to provide ready-to- use spectroscopic data. In addition data deduced from the spectroscopic subset can
be applied for kinetic analysis as well as the calculations of interaction properties, purities and concentrations. An outlook on future developments in MLR techniques might include an even deeper integration of spectroscopic techniques into these devices. With the availability of cost-efficient tunable laser sources new possibilities for MLRs open up, e.g. highly sensitive high throughput spectroscopy, Raman scattering or up-conversion as well as label-free plasmonic methods.
Berthold Technologies has already adopted many of the described technologies and methods and implemented in its TriStar² S and Mithras² multilabel readers. Besides filter technology – which has been improved by the use of RFID coding of each individual filter in terms of application – the instruments utilise dual monochromators for absorbance/excitation and emission respectively.
A flexible approach Te monochromators, lamps and detectors cover a wide spectrum from 215 to 1000 nm, matching the needs of bioanalytical applications. Especially for cellular applications the Mithras² is equipped with bottom reading capabilities and temperature control for the samples within the microplate.
Reagent injectors with tips located at reading positions enable the addition of substrates and triggers right before or even during the measurements, thus enabling the user to monitor biochemical reactions in real-time.
For more information ✔ at
www.scientistlive.com/eurolab
Fig. 2. Reagent injectors enable the addition of substrates and triggers before or even during measurements.
www.scientistlive.com
Bernd Hutter & Dr Frank Schleifenbaum are with Berthold Technologies .
www.berthold.com/bio
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