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.

We also recommend to examine the signal in the imaged specimen and tweak the light sheet using the bottom knob on the lower left corner mirror of the set-up to optimize the image quality. The knob moves the light sheet roughly parallel to the focal plane of the detection lens and thus moving it back an forth one focuses the lens into the middle of the light sheet.

= Software =



Stage Control
Controlling the stage is accomplished primarily through µManager's live window: after a pixel size has been provided, the user can simply click and drag across the display to change the view accordingly. The scroll wheel is used for depth/focus movement. The OpenSPIM software provides an additional dimension of control in the rotational motor: holding the Alt key while moving the mouse side-to-side will cause the sample to be rotated about a previously-determined axis.

Alternatively, the stage controls tab of the OpenSPIM plugin can be used to move to specific locations.

Time Lapses
The acquisition tab makes recording a time lapse of any number of views simple: the user navigates the 4D stage to each view they wish to record and marks that location, specifying the depth and step size of the stack (if applicable).

After choosing any desired options (i.e. enabling anti-drift or continuous acquisition) and specifying a directory in which to save the acquired OME images, a checkbox will enable the time-lapse controls.

There, the user specifies a desired interval between the beginning of each series, and the number of series to acquire.

Once everything is set up, clicking Oh Snap! will begin the acquisition.



= Acquisition =

Taking a single image with the OpenSPIM.

Taking a stack of images with the OpenSPIM.

Taking a series of single planes over time in the movies mode.

Setting up a multi-view imaging of a single fixed sample.

Setting up a multi-view time lapse of live sample.

= Data processing =

Preprocessing of OpenSPIM ome.tiff stacks for multi-view reconstruction.

Registration of multi-view OpenSPIM acquisition using bead based registration pluign in Fiji.

Fusion of registered multi-view OpenSPIM data into s a single output image using content based fusion or multi-view deconvolution.

Processing of long-term time lapse data.

Viewing the registered fused data and rendering of movies using Fiji's 3D viewer.

Tips and tricks.