Probe-Based High-Content Screening (HCS) Service

We first define the biological question, screening objective, target model, and expected readout strategy.

• Define the screening goal, such as mitochondrial toxicity assessment, drug response profiling, mitophagy inducer screening, ROS modulation, metabolic stress evaluation, cardiotoxicity, neurotoxicity, or cancer therapy response.
• Review the target model, including cancer cells, cardiomyocytes, neurons, immune cells, hepatocytes, renal cells, iPSC-derived models, organoids, or client-provided stable reporter cell lines.
• Select the assay scale, such as pilot screening, assay development, focused compound panel, dose-response screening, or larger library screening.
• Define key mitochondrial readouts, such as ΔΨm, ROS, ATP/ADP, NADH/NAD⁺, Ca²⁺, pH, mitophagy, morphology, apoptosis, or cell viability.
• Identify assay constraints, including plate format, imaging platform, fluorescence channels, treatment time, compound properties, cell growth rate, and desired statistical robustness.

We design a probe-based HCS strategy that matches the biological readout and imaging platform.

• Select suitable mitochondrial probes, reporter systems, or co-staining markers.
• Design single-readout, multiplexed, sequential staining, or endpoint imaging workflows.
• Evaluate channel compatibility to reduce spectral overlap and fluorescence bleed-through.
• Match probe readouts to assay endpoints, such as intensity, ratio, puncta count, morphology score, co-localization, or cell-state classification.
• Design positive controls, negative controls, vehicle controls, pathway controls, and rescue conditions.
• Plan assay layout, plate randomization, replicate strategy, dose ranges, and control placement.

We optimize the cell culture and treatment conditions required for reproducible HCS data.

• Optimize cell seeding density, attachment time, culture duration, and confluency window.
• Select plate format, such as 96-well or 384-well imaging plates.
• Optimize treatment time, compound concentration range, vehicle tolerance, and endpoint timing.
• Evaluate cell morphology, viability, proliferation rate, and imaging field consistency.
• Design conditions for live-cell imaging, fixed-cell endpoint imaging, or time-course acquisition.
• Establish plate handling, washing, fixation, and staining steps to reduce variability.

We develop a staining and imaging protocol that produces a stable signal window and reliable image quality.

• Optimize probe concentration, incubation time, wash conditions, and loading buffer.
• Evaluate signal intensity, background fluorescence, staining uniformity, and cell-to-cell variability.
• Test live-cell compatibility, fixation compatibility, photostability, and phototoxicity.
• Optimize microscope settings, including objective, exposure time, laser power, gain, focus strategy, z-stack requirement, and field number.
• Establish acquisition channels and imaging sequence for multiplexed assays.
• Check whether probe signals remain stable across the full plate and acquisition time.

Before screening, we validate the assay window using biological and technical controls.

• Test positive controls such as mitochondrial uncouplers, ROS inducers, ionophores, apoptosis inducers, lysosomal inhibitors, metabolic inhibitors, or mitophagy modulators.
• Test negative controls, untreated controls, vehicle controls, and non-responsive controls.
• Measure assay robustness using signal-to-background ratio, Z-factor or Z′-factor, coefficient of variation, dynamic range, and replicate consistency.
• Assess edge effects, focus variability, compound autofluorescence, cell loss, and staining artifacts.
• Confirm that the selected readout reflects the intended mitochondrial phenotype rather than nonspecific toxicity.
• Refine the protocol before pilot or production-scale screening.

We perform automated image acquisition using standardized imaging settings and plate-level quality checks.

• Acquire treatment plates under optimized automated imaging settings.
• Perform multi-channel acquisition for mitochondrial probes, nuclear markers, cell viability dyes, lysosome markers, apoptosis indicators, or morphology stains.
• Run plate-level QC for focus quality, field coverage, cell number, saturation, debris, edge artifacts, and fluorescence consistency.
• Flag wells with imaging artifacts, compound precipitation, severe cell loss, or autofluorescence.
• Support live-cell time-course imaging when required.
• Prepare image datasets for downstream quantitative analysis.

We build or adapt image-analysis workflows to convert fluorescence images into quantitative mitochondrial phenotypes.

• Perform cell segmentation, nuclear segmentation, mitochondrial segmentation, and object detection.
• Quantify fluorescence intensity, ratio readouts, puncta number, organelle overlap, mitochondrial/cytosolic ratio, and cell-level response.
• Extract morphology features such as mitochondrial length, branching, fragmentation, circularity, network connectivity, and texture.
• Analyze mitophagy puncta, MitoTimer red/green ratio, calcium flux response, ROS intensity, membrane potential loss, apoptosis markers, or viability readouts.
• Support population-level and single-cell-level analysis.
• Generate dose-response curves, response heatmaps, phenotype clustering, and compound ranking.

We summarize screening results and provide actionable recommendations for follow-up validation.

• Identify candidate hits based on predefined thresholds or phenotype signatures.
• Rank compounds according to mitochondrial response, toxicity profile, rescue effect, pathway modulation, or multi-parameter score.
• Compare hit profiles across controls, dose levels, time points, or disease models.
• Flag potential false positives caused by autofluorescence, cytotoxicity, precipitation, or nonspecific staining.
• Recommend secondary validation assays, including functional characterization, orthogonal mitochondrial assays, flow cytometry, plate-reader assays, or live-cell imaging.
• Deliver a final report with assay conditions, QC summary, representative images, feature tables, hit lists, statistical analysis, and interpretation notes.