Sensitivity measurement and compensation in spectral imaging.

Area of Science:

• Cellular imaging
• Flow cytometry
• Optical microscopy

Background:

• The ImageStream system integrates advanced CCD technology with a novel optical design for sensitive, multispectral cell imaging in flow.
• It enables high sensitivity and dynamic range, with a presented methodology for spectral compensation.

Purpose of the Study:

• To present the sensitivity, dynamic range, and spectral compensation methodology of the ImageStream system.
• To compare ImageStream performance with conventional flow cytometry.

Main Methods:

• Running multicolored fluorescent beads and single-color cell controls on ImageStream and a flow cytometer.
• Quantifying spectral overlap using single-color samples and compensating a multi-color sample across six spectral channels.

Main Results:

• Empirical data on sensitivity and dynamic range aligned with theoretical predictions.
• ImageStream demonstrated fluorescence sensitivity comparable to photomultiplier tube (PMT)-based flow cytometry.
• A methodology for spectral compensation, addressing spectral overlap and pixel anomalies, was successfully demonstrated on K562 cells.

Conclusions:

• Object size influences dynamic range and fluorescence sensitivity in pixilated detection systems, unlike conventional flow cytometers.
• Simultaneous imaging of alternate modalities can enhance fluorescence sensitivity.
• Complex, multi-band spectral compensation is achievable in a semi-automated fashion.