Krieger: BioPipeline

Summary

Krieger is an imaging setup dedicated for live cell imaging. The BioPipeline is fully automated and includes an incubator that can store 44 multi-well plates and a robotic system that automatically mounts the plate on the microscope stage. The microscope is equipped for the image modalities: brightfield, differential interference contrast (DIC), widefield fluorescence imaging and spinning disk confocal imaging. For example, your samples can be stored in the incubator for weeks and can be monitored daily with your preferred imaging modality. We create an automated acquisition pipeline (JOBS) for you and train you how to use the JOB independently.

Image modalities

• Brightfield
• DIC
• Widefield fluorescence imaging
• Spinning disk confocal imaging

Applications

• Prolonged fluorescence live cell imaging. There are 6 different laser lines available for the widefield fluorescence and spinning disk confocal mode. A fluorescence mode can also be combined with brightfield or DIC.
• Highthrough put screening / high-content screening. For live or fixed samples in multi-well plates with any of the imaging modes.
• Organoid development imaging. Organoids can be monitored using any of the imaging modes (or a combination of imaging modes).
• Live cell drug screening. Effects of the drugs can be monitored with any of the imaging modes (or a combination of imaging modes).

Technical description

The Nikon Eclipse Ti2-E with 25 mm (d) large field of view, automated stage, perfect focus system, cage incubator (Okolab) to control the temperature (37 ⁰C) and CO₂. A plate incubator with robotic plate mounter is connected to the cage incubator. The following Nikon objectives are available for this system: Plan Apo lambda 4x NA 0.20 air, Plan Apo lambda 10x NA 0.45 air, Plan Apo lambda 20x NA 0.75 air, Apo LWD Lambda S 20XC WI NA 0.95, Plan Apo lambda 40x NA 0.95 air, Plan Apo VC 60x WI NA 1.2. An automated water dispenser is installed to use in combination with the water objectives. Loading and incubating the plates is controlled by the NIS-scheduler software and all imaging functions are integrated in the NIS-Elements software (High content analysis package).

For fluorescence imaging, the sample is excited by the lasers of a Lumencor Celesta (7 laser lines: 405 nm, 446 nm, 477 nm, 520 nm, 546 nm, 638 nm, 749 nm). A spinning disk scanner (X-Light V3, CrestOptics) is mounted on the microscope. For spinning disk confocal, the excitation light passes through a spinning disk with 50 µm pinholes. This disk is removed from the light path in widefield mode. The excitation light is reflected to the sample by a quad dichroic (for laser lines 405 nm, 477 nm, 546 nm and 638 nm) or a dual dichroic (for laser lines 446 nm and 520 nm). The emission is filtered with individual emission filters. The images are acquired with an ORCA-Fusion digital CMOS camera (Hamamtsu).

For DIC and brightfield, the sample is illuminated with a diascopic LED (Nikon). DIC is compatible with 10x, 20x, 40x, and 60x magnifications. These images are acquired with the same camera as used for the fluorescence mode.

Location

Leichtag building, Room 470