Operation

Now that we have assembled the OpenSPIM microscope it is time to start using it. We will need to prepare a sample, align the light sheet (Calibration), set-up the acquisition and process the data.

= Sample Preparation =

The basic principles of sample preparation for SPIM differs from traditional microscopy technologies. There are no glass slides and coverslips. Instead the sample needs to be suspended in a water filled chamber in front of the lens so that it can be rotated. This is usually achieved by embedding the sample in a low melting point (LMP) agarose inside a glass capillary equipped with a plunger. For imaging the agarose column with the specimen is pushed out of the capillary using the plunger to hang in front of the lens.

There are many other strategies to mount SPIM samples using FEP tubes, hooks or agarose baskets.

For the purpose of making registration easier, we mix the sub-resolution fluorescent beads into the specimen containing agarose slurry and use them as fiduciary markers to align the different SPIM views. The software to do that is available in Fiji.

Preparation of Drosophila embryos for SPIM imaging.

= Calibration =

Before using the OpenSPIM the light sheet needs to be aligned. It means that the light sheet needs to be shaped by the optics of the system to be parallel to the imaging plane of the camera, perpendicular to the detection axis, as thin as possible, uniform across the field of view and most importantly in focus with the detection objective. Since the procedure is rather involved we provide a series of detailed videos that illustrate the process. Innovations are welcome.

Although once aligned the light sheet is rather stable it should be aligned in regular intervals or whenever the image quality or point spread function of the beads looks suboptimal.

= Acquisition =

= Data processing =