A single-photon imaging system for the simultaneous quantitation of luminescent emissions from multiple samples

Area of Science:

• Biophotonics
• Analytical Chemistry
• Biochemistry

Background:

• Accurate spatial quantitation of luminescence is crucial for biological and chemical assays.
• Existing methods may lack the resolution or sensitivity for certain applications.
• Developing cost-effective, high-resolution luminescence detection systems is an ongoing need.

Purpose of the Study:

• To describe and validate a novel system for space-resolved luminescence quantitation.
• To assess the sensitivity and spatial resolution of the system for different luminescence types.
• To demonstrate the system's applicability to microtiter plate-based assays.

Main Methods:

• Utilized a two-dimensional photon detector (double-microchannel plate) with single-photon sensitivity.
• Integrated an optical projection system for focusing luminescent emissions onto the detector.
• Employed a microtiter plate with luminescent samples (ATP bioluminescence and cell-derived luminescence) as the test object.
• Accumulated luminescent images in digital frame memory for photon count analysis.

Main Results:

• Achieved high spatial resolution and linearity of response for both chemically generated and cell-derived luminescence.
• Demonstrated sensitivity comparable to dedicated photon-counting luminometers, with only a 10-fold reduction in photon collection.
• Identified and proposed an arithmetic correction for diminished photon collection from peripheral wells.
• Successfully detected ATP bioluminescence and superoxide-dependent luminescence with lucigenin.

Conclusions:

• The described system offers a sensitive and spatially resolved method for luminescence quantitation.
• It provides a viable alternative to dedicated luminometers, particularly for microtiter plate-based assays.
• The system's ability to correct for optical artifacts enhances its reliability for diverse luminescence detection applications.