High-Throughput Imaging of PPIX Using Confocal Microscopy.

ABSTRACT

Increases in levels of protoporphyrin IX (PPIX; a heme precursor) may be driven by xenobiotic induction of aminolevulinic acid synthase 1 (ALAS1) expression. ALAS1 is the rate-limiting enzyme of heme biosynthesis and may be upregulated to satisfy the increased need for heme in CYP450 enzymes. Therefore, a high-throughput fluorescence spectroscopy method that detects PPIX would enable the screening of drugs that increase ALAS1 through nuclear hormone receptor-mediated induction of transcription that may cause toxicity or even provide utility in the diagnosis or treatment of cancers that have elevated cellular PPIX levels. This chapter describes a high-throughput plate-based imaging technique for determining cellular protoporphyrin levels by using the GE Healthcare InCell 6000 confocal imaging system to detect the presence and location of PPIX in each cell and may be adapted for use with other imaging systems. Laser excitation and a scientific-grade complementary metal oxide semiconductor (CMOS) camera generate short exposure times, decreasing photobleaching in the target cells that may result in inaccurate measurements of PPIX and increasing screening throughput. Nuclear staining was detected by using a laser with 405-nm excitation and 455-nm emission wavelengths, and the presence of PPIX was measured using 405-nm excitation and 706-nm emission wavelengths. Image analysis involving top-hat segmentation on both nuclear and PPIX staining was performed by using the InCell Analyzer Workstation software. This assay may be adapted to screen for PPIX formation, degradation, and transportation effectors. Indeed, the inclusion of PPIX transport inhibition would be expected to further widen the linear range of fluorescence and improve the method.