Large field of view (FOV) 2D slices of a 1 mm diameter midbrain neural organoid stained with DAPI (blue, nuclear stain), β-tubulin (green, neuronal stain), and GFAP (red, astrocyte stain). Acquired on THUNDER Imager Cell with 10x magnification. Left: Raw widefield image, Right: THUNDER cleared image. Scale bar 400 μm. Sample courtesy of Dr Tanya Singh, University of Oxford, UK.

Fast, High-Contrast Widefield Imaging of Optically Challenging Samples

How to investigate large 2D cell populations, complex organoids, and sensitive zebrafish using THUNDER Imager Cell and the Kinetix22 sCMOS camera

Large field of view (FOV) 2D slices of a 1 mm diameter midbrain neural organoid stained with DAPI (blue, nuclear stain), β-tubulin (green, neuronal stain), and GFAP (red, astrocyte stain). Acquired on THUNDER Imager Cell with 10x magnification. Left: Raw widefield image, Right: THUNDER cleared image. Scale bar 400 μm. Sample courtesy of Dr Tanya Singh, University of Oxford, UK. 2D_slices_of_midbrain_neural_organoid.jpg

Summary

This application note demonstrates how combining THUNDER Imager Cell with the Teledyne Photometrics Kinetix22 sCMOS camera enables fast, high‑sensitivity imaging of challenging 2D and 3D samples—while preserving the key advantages of widefield microscopy, including speed, simplicity, and gentle illumination.

Widefield imaging is widely used for live‑cell and large-area imaging. However, imaging optically thick or highly scattering samples – including dense 2D cultures, 3D organoids, and whole organisms – is fundamentally limited by out‑of‑focus background, reduced contrast, and phototoxicity when higher illumination intensities are required. This workflow shows how these challenges can be addressed by pairing the THUNDER Imager Cell system with the Teledyne Photometrics Kinetix22 sCMOS camera for high‑sensitivity, large‑field detection.

The result is high‑contrast, high-speed, sub‑cellular resolution imaging across large sample areas at low light doses. Performance is demonstrated across a range of challenging fixed and live biological samples, including large 2D cell populations, complex neural organoids, and sensitive live zebrafish embryos.

Key learnings

  • Overcome widefield limitations in thick and challenging 2D and 3D samples: Learn how to remove out of focus blur and improve contrast in organoids, large cultures, and whole organisms using different THUNDER Computational Clearing methods to fit your sample.
  • Achieve high sensitivity imaging at low light doses: Discover how the Kinetix22 sCMOS camera supports low light sensitive imaging, helping to minimize photobleaching in fixed samples and phototoxicity in live-cell experiments.
  • Capture large fields of view without compromising resolution: See how the THUNDER Imager Cell and Kinetix22’s matching 22 mm field of view (FOV) supports large area imaging while maintaining sub cellular resolution - ideal for large scale biological studies.
  • Image fast biological dynamics with confidence: Understand how high speed acquisition paired with enhanced contrast enables accurate imaging of dynamic processes in live samples.
  • Simplify complex imaging workflows with automation: Explore how intelligent automation tools such as Adaptive Immersion and SmartCORR, and simplified experimental setup and navigation, result in an easy-to-use platform for all users.

Introduction

Live‑cell imaging of large, complex biological samples often requires large fields of view, sub-cellular resolution, high-sensitivity, and fast acquisition – all while maintaining low illumination doses for gentle long-term observation. Widefield fluorescence microscopy can meet these demands, but signals can be buried in its inherent out of focus blur. Optical sectioning microscopy techniques can mitigate this, but can introduce trade-offs in speed, phototoxicity, or experimental complexity.

How can you achieve high-speed, high‑contrast imaging of large and optically challenging samples with low phototoxicity?

Widefield microscopy remains a powerful approach for live-cell imaging—provided that background fluorescence is effectively suppressed. This application note demonstrates how THUNDER Imager Cell, paired with the Teledyne Photometrics Kinetix22 sCMOS camera, delivers a balanced, live cell friendly imaging solution for a broad range of discovery biology applications. After outlining how to optimize imaging parameters for live-cell experiments, we highlight performance across diverse sample types.

Case studies include:

  • 2D imaging of large field of view (FOV) U2OS and HeLa cells
  • Imaging of neural organoids
  • Live imaging of zebrafish embryos

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