High-content imaging plays a key part in drug discovery today, facilitating the simultaneous capture of multiple cellular features and providing a more in-depth view of cellular responses to perturbations than single-parameter assays.
Molecular Devices developed the next-generation ImageXpress™ HCS.ai High-Content Screening System to advance this capability.
The HCS.ai high-content screening system boasts a completely reengineered hardware and software platform, with adaptive autofocus and enhanced x, y, and z stage movement delivering greater throughput.
Its state-of-the-art optical light path features a high-quantum-efficiency camera and optimized illumination, enabling lower background, reduced exposure times, and a considerably improved signal-to-noise ratio.
This article demonstrates how the newly engineered imager and its software interface facilitate a streamlined, end-to-end workflow, ranging from acquisition setup to advanced data analysis.
Two representative assays are shown to yield Z-prime scores over 0.5, highlighting the system’s robustness and suitability for high-content screening:
- G-protein coupled receptor (GPCR) assay
- Cell painting assay
A 100% average improvement was observed in image acquisition speed with the ImageXpress HCS.ai system versus other leading high-content imaging platforms.
The system’s combination of improved throughput and exceptional image quality positions it as an ideal solution for high-throughput imaging applications in both 2D and 3D.
The ImageXpress HCS.ai system offers a range of benefits, including:
- Increased image acquisition speed up to 100% with the ImageXpress HCS.ai High-Content Screening System next-generation imager (vs a market leading high-content imaging system)
- High z scores across assays, validating the system’s performance in high-throughput screening workflows
- Enhanced cell segmentation accuracy with AI-driven image analysis workflows
Methods
Cell culture and staining
G-protein coupled receptor assay
U2OS cells expressing GFP-tagged ß-arrestin were initially seeded into a 384-well flat-bottom plate (Greiner) before being cultured in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific) for one day.
The cells were then treated with isoproterenol, a GPCR agonist (SelleckChem), for an hour before being stained with Mitotracker Deep Red (Thermo Fisher Scientific) and Hoechst (Thermo Fisher Scientific).
Next, cells were fixed with 4% paraformaldehyde (PFA, Thermo Fisher Scientific) before removing fixation solutions and washing twice with PBS to ensure the cells were ready for imaging.
Cell painting assay
MCF-7 human breast cancer cells (ATCC) were passaged and maintained in line with the manufacturer’s recommendations. The cell painting assay was performed according to protocols developed by Bray et al. and Cimini et al.1,2
This process saw MCF7 cells seeded in Greiner 384-well μClear plates, with 2000 cells per well in 40 μL of MEM media. This medium was supplemented with 10% FBS and 10 μg/mL of insulin.
Cells were incubated at 37 °C for a total of 24 hours prior to compound treatment. The culture medium was then replaced with 2% (vol/vol) FBS in MEM 24 hours following seeding and before the addition of compounds.
Eleven compounds were used: 5-Fluorouracil, CCCP, Ca-074-Me, chloroquine (Enzo), cytochalasin D, latrunculin B, rotenone (Enzo), staurosporine, etoposide (Calbiochem), and tetrandrine. All compounds were purchased from SelleckChem unless indicated.
Compounds were tested in quadruplicate wells using a seven-point, 1:3 dilution series. Negative, positive, and DMSO controls were included in the same plate.
Cells were incubated along with the compounds for 24 hours.
Live cells were stained with MitoTracker DeepRed (500 nM) for 30 minutes in the dark at 37 °C before being fixed with PFA (3.2% vol/vol) for 20 minutes. Cells were then washed and permeabilized using triton-100 (0.1%) at room temperature for 20 minutes.
Staining solution was prepared at different concentrations: 1.5 μg/mL WGA, 100 μg/mL concanavalin A, 5 μg/mL Hoechst, 5 μg/mL Phalloidin, and 3 μM SYTO 14 dye in blocking solution (1X HBSS and 1% wt/vol BSA).
The staining solution was used to wash and incubate cells for 30 minutes at room temperature before the staining solution was removed and cells were washed three more times before sealing with adhesive foil. Every wash step was performed with 1X HBSS.
Image acquisition and analysis
G-protein coupled receptor assay
The ImageXpress HCS.ai High-Content Screening System was used to acquire images (Figure 1 and Figure 2) at 20X magnification with a confocal option 60” disk. The DAPI, FITC, and TL channels were used in this instance.
Maximum projection images from Z-stacks of five planes with a 1 μm step size were acquired. The IN Carta® image analysis software’s built-in segmentation tools were used to segment nuclei and GPCR puncta, and these were counted to generate a dose-response curve.
Cell painting assay
Images were acquired using the ImageXpress HCS.ai High-Content Screening System (Figure 1 and Figure 2). This was done using 20X magnification and the confocal option 60” disk.
Maximum projection images from Z-stacks of 13 planes with a 2 μm step size were acquired. The IN Carta image analysis software was used to analyze these images, with SINAP used for nuclei segmentation.
A total of 246 measurements per cell from the assay were uploaded to the StratoMineR™ software to facilitate additional analysis.
Figure 1. The ImageXpress HCS.ai System is redesigned with improved optics and hardware to support fast image acquisition without compromising image quality. 1) It supports standard SBS format labware and slides, 2) offers either laser or LED light source, 3) has four spinning disk geometry options available, 4) automated magnification changer offers up to 12 effective magnifications in one system (including four water immersion options). Image Credit: Molecular Devices UK Ltd
Figure 2. Redesigned image acquisition software (MetaXpress® High-Content Image Acquisition and Analysis Software) offers an intuitive, user-friendly environment that is quick for novices to master while remaining flexible and customizable for advanced users. There are also several new features, including easily controlled transmitted light acquisitions and identification and the centering of “rare” objects in a well for high-magnification acquisition. Image Credit: Molecular Devices UK Ltd
Results
Delivering up to 50% shorter image acquisition times
The ImageXpress HCS.ai System has been developed to deliver assay flexibility and speed.
A comparative evaluation of image acquisition times was conducted across multiple settings to assess imaging speed and benchmark the system against other leading high-content screening imagers.
Acquisition time was found to be 50% shorter for images acquired in widefield on average, meaning this was 100% faster. Acquisition time was found to be 38% shorter for images acquired in confocal mode, meaning this was 61% faster (Figure 3).
For the cell painting assay, acquisition was completed in under 96 minutes (water immersion, confocal, 4FL channels, and TL) versus other similar systems, which took around 162 minutes.
Figure 3. Average acquisition speeds of three runs for each image acquisition setup shown (four instruments, 20X). For 3D imaging, five Z-planes were acquired, while for 2D imaging, one focused plane was acquired. For widefield speed tests, only 2D imaging was performed. Speed comparisons were made using the same exposure time between systems; under these settings, the ImageXpress HCS.ai System had at least 2X brighter pixel intensities and at least 2X signal-to-noise ratio improvement. Image Credit: Molecular Devices UK Ltd
Assay performance: GPCR assay (Transfluor®)
G-protein coupled receptors (GPCRs) are widely employed in cell-based screening assays, representing the largest class of pharmaceutical targets.
ß-arrestin in the cell cytoplasm translocates to endocytic vesicles and pits upon GPCR activation (Figure 4A). In the example presented here, U2OS cells expressing GFP-tagged β-arrestin were treated with isoproterenol (a GPCR agonist) to enable imaging and analysis of internalized GPCR vesicles via high-content imaging.
Precise cell segmentation is key to accurate vesicle puncta counting and dose-response quantification. The IN Carta Image Analysis Software’s Cellpose 3.0 learning-based algorithm enables accurate cell segmentation, displaying masks in random colors to distinguish adjacent cells (Figure 4B).
Cellpose offers cellular segmentation from a wide range of image types without retraining. This feature allows it to work across different channels and varying sizes while accommodating images with varying blur, noise, and contrast.
Figure 4. Evaluation of the GPCR assay. (A) Cells with activated GPCR (treated with isoproterenol) shown on the left, negative control on the right. Note the presence of ß-arrestin puncta (green) in the treated samples. (B) Cell masks in random colors, generated using Cellpose 3.0 in the IN Carta image analysis software, were based on DAPI staining and vesicle staining. Image Credit: Molecular Devices UK Ltd
The number of GFP puncta was used when calculating the assay’s Z-prime score (0.75 and 0.67) (Figure 5B).
Figure 5. Images were analyzed using IN Carta image analysis software. A) The image analysis protocol: Nuclei were segmented with Robust on the DAPI channel; vesicles were segmented with Robust Puncta on the FITC channel; cells were segmented with Cellpose 3.0 on the FITC channel with reference to the DAPI channel. B) The average of eight technical replicates from two HCS.ai systems is shown. C) The dose-dependent increase in the number of puncta, showing activation of the G-PCR pathway in response to isoproterenol. EC50 = 0.014 (1), 0.011 (2). (Dose-response curve generated using Quest Graph™ Four Parameter Logistic (4PL) Curve Calculator. AAT Bioquest, Inc., 7 Jan. 2025). Image Credit: Molecular Devices UK Ltd
Cell painting
Cell painting is a high-content, multiplexed imaging assay that employs up to six fluorescent dyes to label and visualize eight distinct cellular components.
This method has proven to be very effective in the characterization of cellular phenotypes, functioning as a proxy for gene expression, cell state, and even drug mechanism of action.
The example presented here saw MCF7 breast cancer cells treated with a small compound set before analysis via the cell painting assay (Figure 6).
Cell painting and other multiplexed assays require imaging across a range of fluorescent channels. The ImageXpress® HCS.ai system (Advanced Model) is fitted with a seven-color laser and a total of eight fluorescent channels, making it ideally suited to use in highly multiplexed studies.
Images in this example were acquired using the HCS.ai system before being analyzed with the IN Carta image analysis software.
Measurements from the IN Carta image analysis software were then uploaded to the StratoMineR™ software. This cloud-based platform has been specifically developed for the analysis of phenotypic profiling assays and other multiparametric data.
Data analysis was performed for multidimensional phenotypic profiling. Data reduction via uniform manifold approximation and projection (UMAP) showed clustering of cells treated with the same compounds.
Phenotypic distance scores further highlighted that cells treated with latrunculin, staurosporine, doxorubicin, and tetrandrine exhibited notably different profiles versus untreated controls.
Figure 6. Cell painting assay on the ImageXpress HCS.ai System. A) Representative images of control and treated MCF7 cells. Scale=50 μm. Images were analyzed using IN Carta Image Analysis Software. SINAP was used for nuclei segmentation. 246 measurements per cell from the assay were uploaded to the StratoMineR software for further analysis. B) Graph showing the average physical distance score for compounds used. C) UMAP representation of the phenotypic profiles. Image Credit: Molecular Devices UK Ltd
Conclusion
The newly designed ImageXpress HCS.ai high-content screening system was shown to achieve up to 100% average increase in acquisition speed, considerably accelerating screening workflows versus other leading systems.
The IN Carta image analysis software features the Cellpose 3.0 learning-based algorithm, enabling accurate cell segmentation with varying blur, noise, and contrast.
The ImageXpress HCS.ai system was also shown to support highly multiplexed assays, facilitating clear clustering based on specific phenotypic profiles.
It was observed that both assays produced high Z’ scores (>0.5), highlighting the system’s ideal positioning for use in robust, high-throughput screening applications.
References and further reading
- Bray, M. A., et al. (2016) Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes, Nature Protocols, 11(9), pp. 1757–1774. DOI:10.1038/nprot.2016.105. https://www.nature.com/articles/nprot.2016.105.
- Cimini, B. A., et al. (2023) Optimizing the Cell Painting assay for image-based profiling, Nature Protocols, 18, pp. 1981–2013. DOI:10.1038/s41596-023-00840-9. https://www.nature.com/articles/s41596-023-00840-9.
Acknowledgments
Produced from materials originally authored by Angeline Lim and Zhisong Tong from Molecular Devices.
About Molecular Devices UK Ltd
Molecular Devices is one of the world’s leading providers of high-performance bioanalytical measurement systems, software and consumables for life science research, pharmaceutical and biotherapeutic development. Included within a broad product portfolio are platforms for high-throughput screening, genomic and cellular analysis, colony selection and microplate detection. These leading-edge products enable scientists to improve productivity and effectiveness, ultimately accelerating research and the discovery of new therapeutics. Molecular Devices is committed to the continual development of innovative solutions for life science applications. The company is headquartered in Silicon Valley, California, with offices around the globe. For more information, please visit www.moleculardevices.com.
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