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Related Experiment Video

Updated: Jan 25, 2026

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy
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High-Throughput Imaging of PPIX Using Confocal Microscopy.

C Trent Brewer1, Jonathan Low1, Taosheng Chen2

  • 1St. Jude Children's Research Hospital, Memphis, TN, USA.

Methods in Molecular Biology (Clifton, N.J.)
|May 2, 2019
PubMed
Summary
This summary is machine-generated.

This study introduces a high-throughput imaging method to detect protoporphyrin IX (PPIX) levels, aiding in screening drugs that modulate aminolevulinic acid synthase 1 (ALAS1) and potentially impacting cancer treatment.

Keywords:
Confocal microscopyFluorescence imagingHigh contentHigh-throughputProtoporphyrin IX

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Drug Discovery

Background:

  • Elevated protoporphyrin IX (PPIX) levels can result from xenobiotic induction of aminolevulinic acid synthase 1 (ALAS1).
  • ALAS1 is crucial for heme biosynthesis, and its upregulation is linked to CYP450 enzyme activity.
  • PPIX accumulation has implications for toxicity, cancer diagnosis, and treatment.

Purpose of the Study:

  • To develop a high-throughput fluorescence spectroscopy method for detecting cellular PPIX.
  • To enable screening of drugs that induce ALAS1 via nuclear hormone receptors.
  • To facilitate research into PPIX's role in cancer and potential therapeutic strategies.

Main Methods:

  • Utilized a GE Healthcare InCell 6000 confocal imaging system for PPIX detection and localization.
  • Employed laser excitation (405 nm) and a scientific CMOS camera for rapid, low-photobleaching measurements.
  • Applied top-hat segmentation for image analysis of nuclear and PPIX staining using InCell Analyzer Workstation software.

Main Results:

  • Successfully established a plate-based imaging technique for quantifying cellular PPIX.
  • Demonstrated method's adaptability for screening PPIX formation, degradation, and transport modulators.
  • Indicated that PPIX transport inhibition could enhance assay linearity and sensitivity.

Conclusions:

  • The developed high-throughput PPIX detection assay is valuable for drug screening and understanding PPIX-related biological processes.
  • This method supports the identification of compounds affecting ALAS1 and nuclear hormone receptor pathways.
  • The assay has potential applications in cancer diagnostics and therapeutics by monitoring cellular PPIX levels.