This comprehensive guide provides detailed instructions for building the one-photon light-sheet system. Whether your ultimate goal is to construct the two-photon system or utilize the one-photon configuration, we highly recommend starting with the installation of the one-photon system. This sequential approach allows you to gain a solid foundation and a better understanding of the system's components and functionality, which will facilitate a smoother transition to the advanced two-photon configuration if desired.
The construction of the system can be done on two levels. At the first level, the light-sheet unit is a standalone light source. This basic configuration allows you to perform light-sheet imaging experiments in the same manner as you would normally acquire epifluorescence images or stacks. At the second level, the concerted motion of the light-sheet and imaging objective is computer-controled and allows you to perform fast, high-precision volumetric scans. This advanced level of control opens up additional capabilities and enhances the versatility of the light-sheet unit for various imaging applications.
If your ultimate goal is to set up the two-photon version, you have the option to directly use optics optimized for infrared transmission in the initial one-photon installation. This strategic choice ensures compatibility and paves the way for a seamless transition to the two-photon configuration at minimal cost by avoiding the need for component replacements when upgrading to the two-photon version.
Click the images to open the model browser.
- Building Instructions
- Purchase List
- Custom Parts
- Screw kit
- Let's Start to Assemble
- Photos of the System in Combination with an Electrophysiology System:
- Example Recordings:
- A Conference Talk Presenting the System:
- Upgrade the One-Photon Unit into a Two-Photon System by using a Hollowcore Crystal Fiber
Table of contents generated with markdown-toc
These parts are milled from an aluminum block. If you do not have a mechanical workshop in-house then you can send the *.step files that we provide below to an online milling service. For parts with threaded holes, join the mechanical drawings to the .step file.
- The light-sheet unit central cube: View the 3D model or download the CAD model as a step file and mechanical drawings.
- The fiber holder : View the 3D model or download the CAD model as a step file and mechanical drawings.
- Adaptor plate 1 : View the 3D model or download the CAD model as a step file and mechanical drawings.
- Adaptor plate 2 : View the 3D model or download the CAD model as a step file and mechanical drawings.
- Custom bracket : View the 3D model or download the CAD model as a step file and mechanical drawings.
- Sample chamber holder : View the 3D model or download the CAD model as step file and mechanical drawings.
- T-bracket to hold the beam trap : View the 3D model or download the CAD model as step file and mechanical drawings.
- Piezoactuator dummy : View the 3D model or download the CAD model as step file and mechanical drawings. This piece is only necessary if you do not want to install the piezo actuator required for the fast scan mode.
- Sample chamber. You can download the CAD model as step file to send it to your 3D printing service.
You will need Hex Head Socket Cap metric screws:
| type | length (cm) | amount |
|---|---|---|
| M3 | 10 | 4 |
| M4 | 30 | 4 |
| M4 | 25 | 2 |
| M4 | 20 | 5 |
| M4 | 10 | 2 |
| M5 | 30 | 2 |
| M6 | 20 | 3 |
E.g. this Srew kit will do it.
(1) Purchase continous lasers whose wavelength are adapted to your favorite fluorescence probes. We use a blue 488nm and a green 561nm laser. Choose the option with prealigned fiber coupler.
(2) To combine the two lasers into a single-mode fiber, attach the two inputs of the fiber optic coupler to the FC/PC connector of each laser. The blue and green lasers will be combined and available at both output fiber ports.
(3-5) Assemble the Fiber support tube: Take the FC/PC connector which will later hold the optical fiber that delivers the laser. Screw the connector into the lens tube (black) but not too far so that you can easily attach and detach the optical fiber. Fix the connector with the two retaining rings.
You will need to unscrew the bracket that holds the objective support plate from the original Scientifica setup.
This bracket will then be replaced with a custom bracket that has holes to allow the light sheet unit to be attached to the Scientifica system in the next steps.
(1-2) Attach the top plate (previously unscrewed from the Scientifica Scope) to the Custom Bracket (custom piece) using the original screws.
(3) Attach the Adapter Plate 1 (custom part) with four M4x30 screws.
(4) Attach the assembly to the topmost Z-stage of the microscope with two original screws.
(1) Start from the Adapter Plate 2 (blue, custom piece).
(2) Attach the Horizontal Manual Stage to the Adapter Plate 2 with the four M3 screws included with the stage.
(3) Attach the Vertical Manual Stage to the Horizontal Manual Stage with the four M3 screws included with the stage.
(4) Attach the Light-Sheet Unit Central Cube (green, custom piece) to the Vertical Manual stage with four M3x10 screws.
(5) Insert the Galvanometer Mirror (light blue), align it at 45° relative to the sides of the Light-sheet Cube, and fasten it in place with two M5x30 screws.
(6) Attach the Piezocrystal (PZ 400 SG OEM). If you want to start with a simple low-cost version that uses only the slow scanning mode then install instead the Piezo Dummy (custom piece) to the Light-sheet Cube with two M4x20 screws.
(7) Attach the Fiber Holder (red, custom piece) to the Piezo with two M4x25 screws.
(8) Insert the Fiber support tube (see points 3-5) into the Fiber Holder, fasten it with one M4x20 screw and attach one of the output fibers of the fiber optic coupler to the connector (2).
(9) Screw in the Zeiss Plan-Neofluar Objectives (5x/0.16 M27). If your ultimate goal is to build the two-photon version then you should use instead the Olympus LMPLN5xIR/0.1 objectives, which is optimized for near-infrared transmission.
(10) Attach the Light-sheet Unit to the Scientifica Scope by fixing the adapter Plate 2 onto the Adapter Plate 1 using three M6x20 screws.
Before turning on the laser, prepare the room for laser safety (remove any jewelry, make sure no one is in the path of the laser, and that the laser does not hit any reflective surfaces). Unscrew the focusing objective (Obj 2) of the light-sheet unit. Switch on the galvanometric mirror and then switch on the laser at low power. By moving the fiber support tube (black) relative to the fiber holder (red), adjust the distance of the fiber outlet to the collimation objective (Obj 1) until the beam exiting the cube is well collimated.
Next, carefully center the beam in the opening where Obj 2 will be located by adjusting the orientation of the galvanometer mirror. Once this is done, secure the mirror position with the fixing screw. Secure Obj 2 in place again by screwing it in.
To configure the detection path, use the standard ports and configuration of the Scientifica scope.
- Use a detection objective that is best adapted to your application. If you want to install the fast z-scan mode then attach the objective via an objective scanning piezo system.
- Position the filters into the filter wheel adapted to your application. For GFP imaging:
- Attach the camera to the camera port. In our systel, we use the Hamamatsu ORCA-Flash4.0 V3 camera
The light-sheet unit can be operated in two modes:
To drive the galvanometer, a simple and affordable function generator is sufficient. Set the mirror to oscillate at approximately 400Hz with a triangular or sawtooth waveform.
If your image acquisition software allows the acquisition of z-stacks by controlling either the motorized stage of the z-focus or the piezo objective scanner, then you can operate the light-sheet unit in two modes.
Slow Scan Mode:
Even if you have not installed the piezo-control of the objective and optical fiber, you can still perform volumetric recordings. Simply control the movement of the objective using the motorized stage of the Scientifica scope, similar to a standard epifluorescence recording. Since the light-sheet unit is attached to the same stage as the objective, it will move along with the objective, ensuring that the light-sheet always illuminates the focal plane.
Fast Scan Mode:
If you have installed a piezoactuator for both the imaging objective and the fiber, you can achieve high precision and fast volumetric recordings. Synchronizing the movement of the fiber with that of the objective is crucial to ensure that the light sheet remains in the focal plane of the objective throughout the volumetric acquisition.
- To control the light-sheet volumetric scanning you can use our custom developed control software written in Matlab. Via a national instrument card PCIe-6363, this software simultaneously drives the objective piezo scanner, the galvomirror and the piezo that moves the fiber in order to scan the light-sheet through the sample. The software is open source.
- Alternatively, you can use:
- ScanImage, the control software provided by Scientifica
- Micromanager, which supports the control of all Scientifica stages as well as of most common objective scanners from e.g. Physical Instrument or Piezo Jena.
- or the open-source python software presented here: https://www.frontiersin.org/articles/10.3389/fcell.2022.875044/full
- Install the sample chamber holder (custom part).
- 3D print the sample chamber.
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Take the 3D printed sample chamber and use cyanoacrylate or UV-curing adhesive to glue a glass window on each side as well as an O-ring into the hole on the short side, which will allow you later to introduce and position the sample via a capillary (inner diameter 0.85mm, outer diameter 1.47mm, length 115mm) for the imaging sessions.
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Place the sample chamber into the predefined opening of the sample chamber holder.
Protect the experimenter from the laser by installing a beam blocker in front of the sample chamber. Fix the T-bracket (custom piece) with two M4x20 screws to the sample chamber holder and then fix the beam blocker with two M4x10 screws to the T-bracket.
- Fill the sample chamber with water and add a few drops of a 1.5mM fluorescein solution into the water-filled sample chamber to visualize the laser. Switch on the laser at low power until you can see the fluorescence laser profile. Be careful not to look directly into the laser and to keep working at low power (<1mW after the second illumination objective) for alignment.
- The use of fluorescein requires cleaning with ethanol or isopropanol after alignment to remove residual fluorophores that may cause imaging noise.
- It is also possible to use an agarose cylinder and to observe the scattered light from the laser, to do this remove the filter between the detection objective and the camera and follow the same procedure.
- Align the laser waist under the detection objective by moving the entire light-sheet forming unit with the x-translation stage
- Switch on the camera and align the laser into the focal plane of the detection objective by moving the light-sheet forming unit with the z-translation stage until you see a sharp image of the laser beam with the camera. If you do not see the laser beam, move the galvanometer mirror to bring it within the field of view.
- Precisely align the laser waist in the center of the field of view using the x-translation stage
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Remove the fluorescein solution from the sample chamber and clean it.
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Now generate the light sheet by driving the galvanometer with a saw tooth pattern either with a function generator or via the control software (see above). Adjust the amplitude of the movement such that the light sheet covers the field of view.
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Now you can place your preferred sample into the light sheet and image it. Fine-tune the z-position and x-position of the light-sheet until you get the sharpest image possible.
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you can now produce 3D scans of your sample by manually moving the objective focus of the microscope or by recording 3D time-lapse movies using your microscope control software.
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Troubleshooting: if your image is not uniformly sharp throughout the field of view, the light sheet might be tilted with respect to the focal plane of the detection objective. In this case, you can correct this by tilting slightly the entire unit. For this, untighten the screws that connect the unit via the adapter plate to the microscope translation stage. Use a sheet of paper as a spacer between the adapter plate and the stage to tilt the unit in the correct direction once the screws are tighten. Repeat this procedure until your image is in focus across the entire field of view.
Using the green laser. Shown is a high-resolution recording of a zebrafish brain (6dpf) with a pan-neuronally expressed red calcium indicator (elav3-jRGECO):
Movie_StackHD_jRGECO.mp4
