A new incubator for the lattice light-sheet microscope

The lattice light-sheet microscope is now equipped with an OKOlab stage incubator specially designed for this microscope (IP, Fig 1A). It allows to control with great precision the temperature (+/- 0.1°C), humidity (50 to 95%, 1% resolution) and CO2 (0.1%) at the sample level.

Figure 1 : (A) The stage incubator (IP) is inserted around the objectives. (B) The stainless steel perfusion chamber (CP) is attached to the IP with magnets. It has two perfusion circuits: perf 1 with an inlet in a “gutter” and an outlet in the chamber itself, and perf 2 with an inlet and an outlet placed near the sample. (C) As usual the sample (S) is mounted on a 5 mm diameter coverslip at the end of a sample holder (pink).

The IP is provided with a control box and a touch screen to control all the experimental parameters. Thanks to several heating elements and several sensors placed around the objectives and in the base of the incubator, the IP ensures a very high precision and stability of the experimental conditions. This equipment also allows to choose between different perfusion routes according to the experimental protocol (Fig 1 B).

Previously the sample temperature in the LLSM was based on heated liquid circulation that was not precisely controlled. This led to significant thermal drift during long experiments (>30 min) resulting in defocusing of the light sheet, spatial drift of the sample (up to 1 µm over 1h), and degraded physiological stability for live samples.

The OKOlab IP allows to correct these problems, which is essential mainly for (1) long term imaging on live samples, and (2) image acquisition in single molecule localization mode (SMLM) for which the slightest spatial instability is prohibitive.

Moreover, the perfusion placed near the sample by means of tubes (Fig1 B, Perf 2), which does not exist on our current configuration, will allow a local manipulation of the experimental conditions (pharmacological, mechanical, electrical stimulation).

Finally, this equipment has been recommended to us by our colleagues at the Advanced Imaging Center (HHMI Research Campus, USA) who are pioneers in the use of the LLSM in a microscopy platform.