By Dr Stefan Haberstock

As we have seen in the previous posts in this series, implementing fluorescence detection will be a quick and effective route to improving the quality and sensitivity of your assays. Achieving optimal fluorescence assays requires an optics system with both sensitivity and flexibility. A high-performance photomultiplier tube (PMT) will help you to develop multiplex assays with fluorescent dyes that emit light across the entire wavelength spectrum, giving clear signal separation and no compromise in detection sensitivity.

Fluorescence cell-based assays add other layers of complexity: the challenge of analyzing adherent cells with an inhomogeneous distribution on the well bottom, and minimizing autofluorescence from the culture medium. Tecan Spark multimode microplate reader, with fluorescence Fusion Optics™ meets these challenges and offers everything you need to develop and run advanced biochemical and cell-based fluorescence assays.

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The Spark multimode reader from Tecan performs accurate and sensitive measurements of fluorescent cell-based assays.

Developing highly sensitive fluorescence assays using flexible Fusion Optics

The unique Fusion Optics of Spark offers flexibility for your assay development without compromising on sensitivity. With Fusion Optics, you can freely combine the use of filters and monochromators in the same measurement. This gives a major leap in performance compared to hybrid readers.

Flexible filter choices can both maximize light input on the excitation side as well as signal detection on the emission side, while the monochromator allows scanning to identify the optimal wavelength settings. The user-selectable deep blocking dichroic mirrors increase sensitivity for popular dyes in the low end of the wavelength spectrum. The high-power xenon flash lamp reduces the number of flashes needed for reliable and sensitive results, so you don’t have to compromise between sensitivity and speed. Combined with SparkControl software, the system extends the dynamic range with automated gain regulation to avoid fluorescence measurements running into saturation.

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Scanning for excitation and emission maxima with monochromator/monochromator optics (light green) and monochromator/filter optics (dark green). The latter combination gives a much more distinct and sensitive identification of the maxima.

Minimize background autofluorescence in cell based assays with focused bottom reading

Using traditional bottom reading for cell based fluorescence assays reduces assay sensitivity because the light needs to pass through either plastic or glass before it reaches the sample, which reduces the amount of light available for excitation. Tecan Spark provides you with a high-performance bottom reading mode that overcomes this problem. A lens-based bottom reading system, combined with the Z-focusing guides the light to a focal point on the sample that delivers outstanding sensitivity. The Optimal Read function minimizes variation originating from inhomogeneous cell distribution by performing multiple measurements on spatially separated spots arrayed across each well.

Secure and robust results from cell-based assays

To get results from cell based assays that can be compared from experiment to experiment and well to well, you need to pay special attention to cell numbers, cell distribution and cell health. Tecan Spark allows you to check these parameters automatically with its bright field imaging and label free, laser based autofocus. Images and confluence can be measured automatically. With the live viewer in SparkControl, you can document the last view before starting the experiment with the Snapshot function.

Tecan Spark’s cell incubation features like heating, built-in gas control and humidity control allow cells to be incubated in the reader for days. Together with the automatic lid lifter, the injector and the possibility to program conditioned kinetics, Tecan Spark allows smart automation of complete assays. For example, cells can be grown in the reader with regular growth control; upon reaching a predefined confluence a substance can be injected that triggers GFP production. This is monitored as an additional fluorescence kinetic while simultaneously maintaining image based growth control.

Summary

Tecan Spark multimode reader offers excellent performance for fluorescence assays with its unique Fusion Optics. The combination of filter and monochromator in the same measurement give unprecedented flexibility without compromise on the sensitivity.

The environmental control features, together with imaging capability and conditioned kinetics, allows you to automate and standardize cell based assays with very high reproducibility.

In combination with the special read functions like lens-based bottom read, automatic z-focus and optimum read function, Tecan Spark is the ideal multimode reader when working with cells and fluorescence.

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About the author

Dr Stefan Haberstock

Dr Stefan Haberstock works as a sales development specialist for benchtop solutions at Tecan with over 5 years in experience in field marketing and supporting product launches. He studied Biology at Gutenberg University Mainz and Trinity College Dublin and received a PhD in Biochemistry from Goethe University Frankfurt am Main.

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