chromatography • spectroscopy 45
Spectroscopic applications in multilabel readers
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.
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tarting 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.
Te 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. Te latter exploit the intrinsic property of fluorescence emission being fast but not instantaneous. Tis 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. Tis 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. Tis 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. 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.
Fig. 1. Mithras2 multilabel reader with filter and monochromator technology for spectroscopic applications.
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.
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