Selective Plane Illumination Microscopy (SPIM) Light Sheet

Customer Story

A customer wrote: “We need a microscope (single photon) to study the whole  zebrafish’ brain in vivo. We need a 3D rendering of the whole brain, or perhaps in some cases the entire zebrafish. Most likely, light sheet imaging technology, or something similar, is most suitable for us. The spatial resolution requirements are flexible, although of course it would be better to have resolution at the cellular level.

We would like to visualize (3D) the whole brain of the zebrafish at least 1 time per second, or faster. So, a serious question is the motorized stand and the computer control, and/or scanning system. We would like to be able to work with the main fluorescent channels – GFP, YFP and red.
Perhaps you sell just such systems. Could you recommend a suitable system? We would like to purchase individual components and then assemble them ourselves since the budget is important. Is it possible? I think this way we can save the budget and collect exactly what we need.

Can you recommend a suitable system and give an estimated budget?”

Bioimager suggests using low price Biopen SPIM light sheet microscope package which is the commercial version of OPEN-SPIM which was developed by Max-Plank institute initially. We modified and improved it. Its base price for a single channel, single beam is $30k. A dual color with double sided illumination will be near $50k. So you get a fair idea of what functionality and price. You will use ImageJ software. We can offer your desired software with a nice GUI at a reasonable price.

SPIM Principle

The SPIM technology offers fast, optically-sectioning, minimally-invasive 3D acquisition of fluorescing specimen over time. It achieves that by focusing a thin laser light-sheet into the specimen, taking two-dimensional images of the illuminated slice with a perpendicularly positioned detector (CCD camera). Three-dimensional stacks are obtained by moving the specimen orthogonal to the light-sheet between consecutive images. By mounting the sample in a rigid medium, e.g. agarose, and hanging it into the sample chamber in front of the detection lens, it is possible to rotate the sample and collect 3d stacks from multiple angles (views).

SPIM Applications

SPIM promises to revolutionize several fields of biological research, in particular developmental and cell biology, by allowing imaging of large samples with high resolution over extended periods of time. SPIM has been used in a spectacular fashion to record the development of embryos of model organisms such as Drosophila and zebra fish with cellular resolution throughout the developing specimen (the so-called in toto imaging). The ability of SPIM to deliver high signal-to-noise 3D images of large specimen from different angles in an extended time-lapse is currently hard to achieve with any other microscopy technology. Since monitoring biological systems with high resolution over time is the goal of essentially all fields of biological inquiry, SPIM technology is imminently useful to biologists.

Features

• Customizable light-sheet microscope based on your application and budget
• The lowest price and most compact lightsheet microscope in the market
• Automated XY, Z and R (rotation)
• Customized chamber(s) according to the client’s sample(s)
• Big list of  filters and lasers from UV to IR is available
• Objective lenses: 4x, 10x, 20x, 40x
• Camera: CMOS, CCD, sCMOS
• MicroManager Software
• Easy Upgradable from single to two colors, anytime

Configuration

1) Single Channel , one dimensional illumination Single Channel laser can be selected at visible range  and infrared. Alternatively, one can get two or more light sources at different wavelengths and alternate base don the usage.	2) Single Channel, Two dimensional or double-sided illumination Single Channel laser can be selected at visible range  and infrared. Alternatively, one can get two or more light sources at different wavelengths and alternate base don the usage.
3) Multiple Channel, One dimensional illumination Laser wavelengths can be selected at visible range  and infrared,	4) Multiple Channel, Two-dimensional or double-sided illumination
Two Dimensional Two camera System

Installing the OpenSPIM/Fiji package

This is a Fiji package:

OpenSPIM-20150426.zip (32-bit) OpenSPIM-20150427.zip (64-bit)

Installation:

1. Download and unpack the right (32bit/64bit) OpenSPIM-… .zip
2. Launch Fiji: in the OpenSPIM.app/ folder, open ImageJ-win32.exe (or -win64.exe).
3. Update Fiji (and OpenSPIM & µManager): Help->Update Fiji (or simply choose Yes, please if you are asked to update).
4. Install the camera driver if needed (see below).
5. Run Micro-Manager: Plugins->Micro-Manager->Micro-Manager Studio.

Note: if you are interested in building the software from scratch so you can modify it to your heart’s extent, please have a look at this page.

Drivers

Most of the drivers are shipped within µManager, bundled in our OpenSPIM software. However, some µManager drivers require separate installations (such as, in the case of the OpenSPIM version 1.0 setup, the camera driver).

Camera Drive

Theoretically, OpenSPIM works with all cameras supported by µManager. However, OpenSPIM version 1.0 refers to the setup that has a Hamamatsu ORCA camera. For such a setup, you need to download and install the DCAM driver.

If you have a QImaging camera, download and install the QImaging drivers.

Initial hardware configuration

Hardware configuration protocol with screengrabs

When Micro-Manager is first started, it will prompt the user to select a hardware configuration (defaulting to none).

To create a hardware configuration to use, go to Tools->Hardware Configuration Wizard… and select Create new configuration.

1. From the Available Devices list, expand CoherentCube or Omicron and add the ‘CorentCube ‘or ‘Omicron Laser Controller’ device. Select the appropriate serial port and RS 232 settings and click OK. Ensure the BaudRate property is at 19200 for the Coherent Laser or 500000 for the Omicron laser, or the computer will not be able to communicate with the laser.
2. From the HamamatsuHam category in the Available Devices list, add the HamamatsuHam_DCAM device. No further configuration is needed for the camera.
3. Finally, from the PicardStage category, add in turn the twister, Z stage, and XY stage devices. The software will attempt to guess the correct serial numbers for the motors; these should be verified and corrected as necessary.
4. Once these five devices have been added, click Next. On the following page, default devices are specified. The camera and shutter should already be set to HamamatsuHam_DCAM and CoherentCube / Omicron Laser Controller, respectively, but the user will need to set the default focus stage to Picard Z Stage. Leaving Auto-shutter enabled, click Next. The laser does not require a delay, so the setup can advance without any changes on this page. As there are no state devices to set up, the following page can also be skipped. On the final page, the user can specify a file to which the configuration will be saved before finishing the wizard.

Once the hardware configuration has been loaded, the OpenSPIM plugin can be opened using Plugins->Acquire SPIM image. At this point, µManager’s Snap/Live Window will be opened along with the OpenSPIM GUI.