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{% include image src="1I_1D_OpenSPIM.png" width="70%" caption="OpenSPIM rendering with laser on" %}
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Before using the OpenSPIM the light sheet needs to be [**aligned**](Light-sheet_Calibration).
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This 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.
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Although the light sheet is rather stable once aligned, it should be [**aligned**](Light-sheet_Calibration) at regular intervals or whenever the image quality or point spread function (PSF) of the beads looks suboptimal.
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We also recommend examining the signal in the imaged specimen and tweaking 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 and forth focuses the lens into the middle of the light sheet.
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-**gel for bead scans and Matlab script to evaluate them**. Using this you can estimate the size of the PSF of your microscope
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It is also possible to measure the [light-sheet thickness](Light_sheet_characterization) using a small mirror mounted in the sample chamber.
<td>This holder includes two threaded holes so it's easiest to make from metal (eg aluminum). Holder is mounted on the 4D stage arm with M6 screw. Glass slide with 1 mm thickness can be held with nylon or nylon-tipped screw</td>
Copy file name to clipboardExpand all lines: Operation.md
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---
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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.
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But first, what's the magic behind OpenSPIM? For some it might be a good idea to first undertsand some [fundamental principles of optics](https://openspim.org/SPIM_Optics_101) before proceeding.
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# Sample Preparation
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{% include video id="G6P52G06oPw" width="400" height="243" caption="Pulling Drosophila embryos in agarose inside a glass capillary (*with some priceless random audio*)" %}
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{% include image src="1I_1D_OpenSPIM.png" width="70%" caption="OpenSPIM rendering with laser on" %}
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Before using the OpenSPIM the light sheet needs to be [**aligned**](Light-sheet_Calibration).
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Before using the OpenSPIM the light sheet needs to be [**aligned**](xopenspim/alignment_welcome).
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This 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.
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Although the light sheet is rather stable once aligned, it should be [**aligned**](Light-sheet_Calibration) at regular intervals or whenever the image quality or point spread function (PSF) of the beads looks suboptimal.
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We also recommend examining the signal in the imaged specimen and tweaking 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 and forth focuses the lens into the middle of the light sheet.
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-**gel for bead scans and Matlab script to evaluate them**. Using this you can estimate the size of the PSF of your microscope
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Although the light sheet is rather stable once aligned, it should be [**aligned**](xopenspim/alignment_welcome) at regular intervals or whenever the image quality or point spread function (PSF) of the beads looks suboptimal.
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It is also possible to measure the [light-sheet thickness](Light_sheet_characterization) using a small mirror mounted in the sample chamber.
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Click [here](xopenspim/alignment_welcome) to follow the most recent OpenSPIM alignmetn guide (2021) and [here](Light-sheet_Calibration) for the original light-sheet calibration site (2013).
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# Software
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A new [µManager](https://micro-manager.org/) based OpenSPIM plugin called [μOpenSPIM](https://openspim.org/micro-openspim) has been developed. We recommend using [μOpenSPIM](https://openspim.org/micro-openspim) for any current OpenSPIM system that uses [µManager](https://micro-manager.org/).
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-[**Registration**](Registration) of multi-view OpenSPIM acquisition using bead based registration plugin in Fiji.
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-[**Fusion**](Fusion) of registered multi-view OpenSPIM data into s a single output image using content based fusion or multi-view deconvolution.
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-[**Time lapse registration**](Timelapse_Registration) of long-term time lapse data.
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-[**Browsing**](https://fiji.sc/BigDataViewer) of OpenSPIM data with Fiji's BigDataViewer (coming soon).
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-[**Browsing**](https://fiji.sc/BigDataViewer) of OpenSPIM data with Fiji's BigDataViewer.
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-**3D rendering** of fused OpenSPIM data (TBD).
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If you have a cluster available, you could imitate the way the Tomancak group executes [SPIM Registration on their cluster](https://fiji.sc/SPIM_Registration_on_cluster).
Copy file name to clipboardExpand all lines: People.md
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<td><ahref="https://www.mpi-cbg.de/research/research-groups/pavel-tomancak.html">Research Group Leader</a> at the MPI-CBG in Dresden. Provides ideas, concepts, research questions and funding.</td>
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</tr>
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<tr>
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<tdalign="center">{% include image src="Jan.jpg" width="40%" caption="Jan Huisken" %}</td>
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<td><ahref="https://www.mpi-cbg.de/huisken">Research Group Leader</a> at the MPI-CBG in Dresden.</td>
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<tdalign="center">{% include image src="PeterGPitrone.png" width="40%" caption="Peter Gabriela Pitrone" %}</td>
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<td>Light Microscopist for Andor Technology. Designed and built the OpenSPIM.</td>
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</tr>
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<tr>
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<tdalign="center">{% include image src="PeterGPitrone.png" width="40%" caption="Peter Gabriela Pitrone" %}</td>
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<td>Microscopy technician in the <ahref="https://www.mpi-cbg.de/research/research-groups/pavel-tomancak.html">Tomancak Lab</a>. Designed and built the OpenSPIM.</td>
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<tdalign="center">{% include image src="Johannes_Girstmair.jpg" width="40%" caption="Johannes Girstmair" %}</td>
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<td><ahref="https://www.mpi-cbg.de/research/researchgroups/currentgroups/pavel-tomancak/group-members">Postdoc</a> at the MPI-CBG in Dresden. Dedicated to the developmental biology research of flatworms by using and further enhancing OpenSPIM.</td>
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</tr>
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<tr>
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<tdalign="center">{% include image src="HongKee_Moon.png" width="40%" caption="HongKee Moon" %}</td>
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<td><ahref="https://www.mpi-cbg.de/research/research-groups/pavel-tomancak.html">Scientific Software Engineer</a> at the MPI-CBG in Dresden. Programmed and creates features for µOpenSPIM (the current graphical user interface for OpenSPIM users).</td>
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</tr>
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<tr>
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<tdalign="center">{% include image src="Jan.jpg" width="40%" caption="Jan Huisken" %}</td>
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<td><ahref="https://www.mpi-cbg.de/huisken">Humboldt Professor of Multiscale Biology</a> at the University of Göttingen.</td>
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</tr>
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<tdalign="center">{% include image src="Kevin_eliceiri.jpg" width="40%" caption="Kevin Eliceiri" %}</td>
Copy file name to clipboardExpand all lines: Setup_Extensions_and_Modifications.md
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layout: page
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description: Additional setup tweaks
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# Bright-field illumination
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##Bright-field illumination
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Michael Redd from the Cell Imaging Core Facility at the University of Utah put together an Arduino-based solution to get proper bright-field illumination in an OpenSPIM setup: [Brightfield](Brightfield).
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# Shutter
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##Shutter
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Instructions to make an Arduino-controlled shutter compatible with uManager: [paper by Todd P. Meyrath](http://george.ph.utexas.edu/~meyrath/informal/shutter.pdf) and [tutorial on the uManager page](https://micro-manager.org/wiki/Control_laser_shutters_with_Arduino).
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# Stepper Motor Upgrade
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##Stepper Motor Upgrade
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If you would like to swap out one of the Picard stepper motors you can do so with a bit of effort.
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[If you would like to swap out one of the Picard stepper motors you can do so with a bit of effort.](https://openspim.org/Stepper_Motor_Upgrade)
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Instructions about how to go about this for a Thorlabs DRV001 connected via a Thorlabs BSC201 controller on a 64bit OpenSPIM setup are detailed below (by Jerome Boulanger and Ben Sutcliffe):
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1. Download the micro-manager source code as instructed [here](https://micro-manager.org/wiki/Micro-Manager_Source_Code).
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2. Install all the relevant programs for building micro-manager as instructed [here](https://micro-manager.org/wiki/Building_MM_on_Windows).
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3. Setup up Visual C++ 2010 Express as [here](https://micro-manager.org/wiki/Visual_Studio_project_settings_for_device_adapters).
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4. Install the 64bit APT software from [Thorlabs](https://www.thorlabs.com/software_pages/ViewSoftwarePage.cfm?Code=Motion_Control&viewtab=1).
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5. Download and unzip the APTx64 form [here](https://micro-manager.org/wiki/File:APT_x64.zip).
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6. Then build the ThorlabsAPTStage Device Adapter in 64bit on a 64bit Windows 7 machine, using the 'APT.lib' downloaded in point 5.
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7. Rename the resulting .dll file to 'mmgr_dal_ThorlabAPTStage.dll' then place it into the OpenSPIM.app folder along with the 'APT.dll' downloaded in 5).
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8. The OpenSPIM Fiji/MM bundle can then restarted and a ThorlabAPTStage could be configured as described for a 32bit system [here](https://micro-manager.org/wiki/ThorlabsAPTStage).
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More details to follow about incorporating this into the SPIMAcquisition plugin.
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# OpenSPIM for large samples
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## OpenSPIM for large samples
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## Field of view
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Immersion objectives with long working distance are hard to find. However, at low NA and moderate magnification, a dry objective will also work; the higher NA, the more spherical aberrations will be present. Keep in mind that when using a dry objective in immersion, the WD will increase (approximately by a factor equal to the RI of the new medium, eg. 17 mm in air will increase to 22.6 mm in water).
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## Example parts list
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## OpenSPIM for brains setup
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[Click here for a table that lists the parts used by the OpenSPIM for brains setup built by Monika Pawłowska (Nencki Institute of Experimental Biology, Warsaw).](https://openspim.org/OpenSPIM_for_brains_setup)
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The table below lists the parts used by the OpenSPIM for brains setup built by Monika Pawłowska (Nencki Institute of Experimental Biology, Warsaw).
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## X-OpenSPIM example equipped with two laser lines and a cooling chamber
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<table>
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<trclass="header">
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<th>Manufacturer</th>
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<th>Accessibility</th>
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<th>Description</th>
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<th>File or Link/Model #</th>
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<th>Image</th>
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<th>Quantity</th>
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<th>Price (EUR)</th>
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</tr>
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<trclass="odd">
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<td>Nikon</td>
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<tdalign="center"bgcolor="pink">purchase</td>
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<td>4X Nikon Plan Fluorite Imaging Objective, 0.13 NA, 17.2 mm WD, dry</td>
<td>This holder includes two threaded holes so it's easiest to make from metal (eg aluminum). Holder is mounted on the 4D stage arm with M6 screw. Glass slide with 1 mm thickness can be held with nylon or nylon-tipped screw</td>
A [detailed description of how an OpenSPIM can be upgraded](https://openspim.org/x-openspim_welcome) to contain a second illumination axis with two cameras and multiple laser lines under the control of an Arduino board. The system also features a cooling chamber.
Instructions about how to go about this for a Thorlabs DRV001 connected via a Thorlabs BSC201 controller on a 64bit OpenSPIM setup are detailed below (by Jerome Boulanger and Ben Sutcliffe):
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1. Download the micro-manager source code as instructed [here](https://micro-manager.org/wiki/Micro-Manager_Source_Code).
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2. Install all the relevant programs for building micro-manager as instructed [here](https://micro-manager.org/wiki/Building_MM_on_Windows).
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3. Setup up Visual C++ 2010 Express as [here](https://micro-manager.org/wiki/Visual_Studio_project_settings_for_device_adapters).
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4. Install the 64bit APT software from [Thorlabs](https://www.thorlabs.com/software_pages/ViewSoftwarePage.cfm?Code=Motion_Control&viewtab=1).
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5. Download and unzip the APTx64 form [here](https://micro-manager.org/wiki/File:APT_x64.zip).
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6. Then build the ThorlabsAPTStage Device Adapter in 64bit on a 64bit Windows 7 machine, using the 'APT.lib' downloaded in point 5.
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7. Rename the resulting .dll file to 'mmgr_dal_ThorlabAPTStage.dll' then place it into the OpenSPIM.app folder along with the 'APT.dll' downloaded in 5).
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8. The OpenSPIM Fiji/MM bundle can then restarted and a ThorlabAPTStage could be configured as described for a 32bit system [here](https://micro-manager.org/wiki/ThorlabsAPTStage).
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