Augmented Microscopy

Agilent BioTek instrumentation and software create the unique augmented microscopy experience with the integration and automation of all steps from image capture to publication-ready data. There is no need for other software as the Agilent BioTek Gen5 does it all quickly and simply:

  • Images are automatically captured: end point, time-lapse, montage, z-stack

  • Automated processing, including background flattening and deconvolution, optimizes images for downstream analysis

  • Nuclear and cytoplasmic components are identified with primary and secondary masks

  • Image analysis offers qualitative and quantitative data

  • Annotation of single or sequential images is available on-screen, in real time

  • Video clips can be generated from time-lapse sequences with a single click

Augmented Microscopy creates a 'map' with key analytical information, helping you to make informed decisions faster.



With Gen5 software, images are captured by a manual mode for lower throughput, typically imaging a few samples at a time. Experiment mode automates image capture and analysis in multiple wells. This mode can also include both imaging and plate reading, plus other steps required for some live cell applications, like reagent injection, shaking, and incubation. Both modes offer the intuitive interface and automated functions like find image, auto exposure, auto LED intensity, autofocus (including image-based and laser autofocus). These functions ensure that image capture is simple and efficient. Live cell kinetic sequences can also be completely automated, from initial image capture through movie creation. Images captured in Gen5 are ready for automated processing and in-depth quantitative analysis.

Manual mode screen
Experiment screen

Gen5’s manual mode interface (left) and experiment mode (right) 96-well image display.


Image preprocessing, including background flattening and image smoothing, optimize images for downstream analysis. Image-processing features in Gen5 include:

  • Image stitching, which allows multiple tiled images (montages) to be software aligned, creating a complete picture of the entire sample with great accuracy, to provide more meaningful data
  • Z-projection is essential for 3D imaging applications like spheroid, tumoroid, and hanging droplet assays where biology cannot be captured adequately with an objective typical depth of field.
  • Digital phase contrast overcomes the meniscus effect often seen in 96- and 384-well plates during brightfield imaging. Digital phase contrast is an enhancement technique that minimizes uneven illumination and creates a more consistent contrast across the image for better visualization.

Stitched image of 18 row by 12 column montage
Figure 1. Stitched image of 18 row by 12-column montage shows entire well of a 6-well plate, with hanging drop insert and spheroid. See Application Note for details.
HCT 116 tumoroids, single plane images (A), (B), (C) are z-stacked and z-projected (D) in Gen5
Figure 2. HCT 116 tumoroids, single plane images (A), (B), (C) are z-stacked and z-projected (D) in Gen5. 


Gen5 offers powerful image analysis for a broad range of live and fixed cell imaging. Automated cell counting, label-free cell counting, transfection efficiency, and nuclear translocation analyses are facilitated. Features include dual masks for nuclear and cytoplasmic measurements, dynamic thresholding, and custom object-level metrics.

Nuclear and cytoplasmic masks in Gen5

Figure 3: Nuclear and cytoplasmic masks in Gen5.


After image analysis, it is common to annotate specific details in the image for publication or other documentation purposes. Gen5 has a built-in annotation tool, so there is no need to export image files to another software package. Live cell kinetics can be captured and a .wmv or .mp4 file generated automatically right within Gen5. Still images can be exported in many common formats. These features make publication-ready images and data from Gen5 available with a few clicks.

Hella video
Gen5 automatically generates video of time lapse images, like this from a label-free wound healing assay.
Confluency graph
Figure 4: Quantitative analysis in Gen5.

Figure 5. Annotate images directly in Gen5.


See what other Agilent BioTek imaging users are doing! Search the Agilent BioTe technical resources for citations, application notes, webinars and more, and peruse our Customer Spotlights for all the latest research using augmented microscopy.

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