Reliability of confocal microscopy spectral imaging systems: Use of multispectral beads

Abstract

Background: There is a need for a standardized, impartial calibration, and validation protocol on confocal spectral imaging (CSI) microscope systems. To achieve this goal, it is necessary to have testing tools to provide a reproducible way to evaluate instrument performance. Methods: We evaluated fluorescent spectral beads (Focal-Check™) from Molecular Probes/Invitrogen that consist of four pairs with emissions between 500 and 725 nm and a europium macrocycle quantum dye bead. These bead tools compliment our previously published protocol for testing spectral imaging systems that used an inexpensive multi-ion discharge lamp (MIDL) that contains Hg+, Ar+, and inorganic fluorophores that emits distinct, stable spectral features. Results: We acquired the spectra of the FocalCheck™ beads on a Zeiss 510 Meta, a Leica TCS-SP1, a Leica SP2 AOBS, an Olympus FV 1000, and a Nikon C1Si confocal systems and a PARISS microscopic spectral system and of the europium beads on the Leica TCS-SP1 and PARISS spectral imaging systems. A lack of performance with some equipment between 650 and 750 nm was identified using the far red pair of the FocalCheck™ beads. The position of the slider in front of PMT 2 that reflects light into PMT 1 and PMT 3 affected the measurement of all bead intensities. Unmixing algorithms were used to separate beads with different fluorochromes and separate two fluorochromes on the same bead. Conclusions: The FocalCheck™ multi-spectral beads yielded similar profiles on four CSI systems and a PARISS spectral system. The utilization of the spectral FocalCheck™ beads is helpful to evaluate proper spectral performance, especially in the far red region. Europium beads provide a very narrow spectrum that can help to identify machines that have spectral problems. The dyes located on individual beads or mixed together in ring-core configuration can be used as test particles to demonstrate spectral unmixing with various algorithms.