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Spectroscopic applications in multilabel readers

3rd December 2013


In the early days of multilabel readers (MLR), the main focus lay on luminescence, absorbance and fluorescence intensity measurements. Now spectroscopic techniques have dramatically extended their capabilities. Dr Frank Schleifenbaum and Bernd Hutter report.

Starting from basic ELISA-readout devices, in the past decade multilabel readers (MLR) have developed to become a highly-versatile tool for optical investigations of small sample amounts, typically arranged in handy microtitre plates.

The variety of different read-out modes is large, all guided by the aim to obtain the deepest information content of a given sample while maintaining highest flexibility at the same time.

Fluorescence emission

In general, the spectroscopic techniques can be subdivided in frequency-resolved and time-resolved techniques. The latter exploit the intrinsic property of fluorescence emission being fast but not instantaneous. This means, that the fluorescence light emitted by a sample is slightly delayed with respect to the excitation light. Measuring this delay can offer valuable insights into the molecular properties of a sample or can be used to separate a target signal from unintended background-emission.

On the other hand, the possibility to record absorbance and fluorescence spectra in the frequency domain offers the opportunity to visualise biochemical reactions and biophysical properties by monitoring interchanging spectroscopic bands. This way, for example changes in the overall concentration, the pH, the redox potential or the presence of distinct functional groups can be identified.

Recent MLRs are not only limited to mere data acquisition but are also able to analyse the raw data to provide ready-to-use spectroscopic data. This way, for example, kinetic studies can be run in a one-click procedure and the individual reaction rate constants are directly displayed for each sample well.

Optical techniques

MLRs are not only limited to measure samples in homogeneous phase, but are also well-suited to analyse live cells non-invasively by optical techniques. Here, spectroscopic capabilities play a key role in offering live monitoring of cells. 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 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. This 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.

Another important field of spectroscopic applications in MLRs addresses the analysis of protein-protein interaction studies in a living cell context. On a molecular scale, cell functionality is understood to be controlled by the binding and dissociation of molecular species. Hence, the analysis of this interaction plays a key-role in cell biology, pharmacy and medicine and can be a highly-sensitive read-out of, eg, drug response in pharmaceutical screening studies. Whereas molecular interaction can hardly be visualised by spatially resolved co-localization 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.

A more sophisticated analysis utilises the time-domain domain approach to analyse the reduced fluorescence lifetime of a donor chromophore in presence of a FRET acceptor.

Integration

Future developments in MLR techniques might include an even deeper integration of spectroscopic techniques into these devices. With the availability of cost-efficient flexible laser sources, a complete new field of applications in the MLR techniques opens up, which comprise, eg, the combination of high throughput applications with spectroscopic read-outs or next-level spectroscopy techniques such as Raman scattering or energy up-conversion. Also, advances in the field of plasmonic structures might find its way into MLR applications.

Here at Berthold Technologies many of the described requirements and opportunities have been taken care of by developing the Mithras2 multilabel reader. Besides filter technology - which has been improved by the use of RFID coding of each individual filter in terms of application security - the instrument utilises dual monochromators for absorbance/excitation and emission respectively.

The monochromators, lamps and detectors cover a wide spectrum from 230 to 1000nm matching the needs of bio-analytical 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 tip located at reading positions enable the addition of subtrates and triggers right before or even during the measurements.

For more information at www.scientistlive.com/eurolab

Dr Frank Schleifenbaum and Bernd Hutter are with Berthold Technologies GmbH & Co KG, Bad Wildbad, Germany. www.Berthold.com/bio





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