Imaging and fluorescence signal analysis

SPAchip® kits are designed to dynamically shift fluorescence intensity values in response to intracellular changes in analyte concentration. Fluorescence intensity is measured by exciting the live cell sample at 488 nm and detecting fluorescence emission at 520 nm. Relative intensity units depend on the instrumental setup (excitation power, detector efficiency, etc). To obtain quantitative data, a calibration curve needs to be analyzed in the same run. Data acquisition can be adapted to fluorescence microscopy and flow cytometry. Additional detection channels, such as brightfield, phase contrast, FSC or SSC, are suggested to better identify individual SPAchips.

a) Emission spectra of Calcium--sensing probes when excited at the indicated wavelength. b) Brightfield image of SPAchips® once in solution (Image from Torras et al. 2015 DOI: 10.1002/adma.201504164), Scale bar = 10 µm. c) Calibration Curve of Fluorescence Intensity values from CytoCHECK SPAchip® Calcium Single Detection kit at different concentrations.

SPAchip® fluorescence signal quantification can be adapted to any analysis image software. Object segmentation should be performed to identify SPAchips inside cellular region to every sample. Fluorescence intensity units can be exported as a spreadsheet to plot graphical summaries of the results. Quantitative data need to be interpolated from the calibration curve values to each experiment. Normalization of results to experimental controls offer a semi-quantitative approach.

Image Analysis Workflow. From sample data acquisition to object identification, quantification and result presentation.