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Hardware requirements

This page describes the hardware you ought to have to run BakingTray using ScanImage.

Acquisition PC

If you're building from scratch, buy the fastest Intel-based computer you can. Prioritise CPU speed over number of cores, since ScanImage runs single threaded. Otherwise, any moderately fast PC should work.
We originally acquired images to a local RAID array: four platter drives in RAID 1+0. The striping is necessary when using a resonant scanner. Unless you anticipate very large datasets, 4 TB drives should be sufficient. For over a year now we been acquiring to a single 8 TB SSD and this has worked well.
Hardware of course goes out of date quickly, but the following is an example of a successful configuration for resonant scanning.
  • i7-6700K @ 4 GHz, 16 GB DDR4-2133 MHz RAM, H170-Pro motherboard
  • Adaptec RAID 6405E, 4x WD Black RAID 1+0.
  • NVIDIA GeForce GT 730 to drive a pair of DELL U2715H monitors. Otherwise the on-board graphics are fine.
  • Oxford Semiconductor 4 port PCIe serial adaptor for PIFOC and motion control hardware (laser comms via motherboard serial port).
A PCIe serial adapter card will have a lower latency than USB-serial and so is preferable. Install the card in the lowest bus number possible on your motherboard. If you do not do this, Windows will re-assign COM port numbers when you change other hardware (e.g. swap out or move an NI card in the chassis).
The hardware RAID above is necessary as a single platter drive won't provide enough bandwidth for resonant scanning. You don't need RAID for galvo scanning. You can substitute a single SSD for resonant scanning. Samsung currently make 8 TB SSDs with a warranted life of 3 years or 2,880 TB.

Data acquisition devices

BakingTray works with any scanning hardware and acquisition cards supported by ScanImage, including the vDAQ. For resonant scanning with four channels on NI hardware we use:
  • Chassis: NI PXIe-1073
  • Image acquisition: NI PXIe-7961R FPGA and NI-5734 digitizer
  • PIFOC, Pockels, and scan control: 3x PXIe-6341
  • Abalog PMT control: older versions of ScanImage required a four channel NI USB-6343 but now you can use 2x NI USB-6009 devices, which is far cheaper.

Galvo scanning DAQs

We have run galvo/galvo using both NI PCI-6110 and NI PCI-6115 acquisition cards. However, PXIe devices are recommended as they're easier to manage in a chassis. The PXIe-6124 has also been tested but higher sample speeds don't work on all motherboards.

Scanning Hardware

  • We recommend resonant scanning as it is much faster for high resolution images even though there is an increase in shot noise due to the shorter dwell time. Using moderate PMT gains will help with the shot noise and will have no negative consequences. For example, set multialkali PMTs to about 500V rather than the 700 or 800V that are typically used for in vivo imaging. This will result in reduced amplification; there will be no decrease in sensitivity. Averaging frames is also possible, but for bright labeling this not needed. Unlike linear scanning at short line periods, the bidirectional "comb" artifact is virtually gone with resonant scanning as it is constant across the scan line. We have tried 12 kHz, 8 kHz, and 4 kHz resonant scanners and all work. However, we do not recommend the 12 kHz scanner as lower scan angle produces an excessively small field of view which results in more stage motions. The 8 kHz scanner will image samples faster in practice, since fewer tiles are needed to cover the sample.
  • A 400 micron travel range PIFOC (we use a P-725.4CDA) is recommended for optical sectioning as it is the most flexible option. However, shorter travel range PIFOCs are faster and are acceptable if you are certain you will never want to image cleared tissue.
  • You will need a Pockels cells to ramp laser power with depth. Choose one with a low dispersion crystal and the BK option to reduce resonances.

The microscope

You ideally want a microscope capable of imaging a large FOV (>1 mm) that is flat and undistorted. The FOV affects scanning speed: if you have a small FOV the tile scanning becomes slow. For our purposes a flat field would be one with less than 10 microns of sag in focal plane. If you lack this, everything will still work but it can be trickier to get good overlap of features at tile edges. "Distortion" refers to pincushion and barrel distortion: the less of this the better. Again, you can work with a microscope that exhibits it. We care about distortion because it affects tile overlap areas when stitching images (although some degree of correction is possible).
You can image an EM grid such as the 2145C from 2spi to assess FOV and distortion. For field flatness tou can take a z-stack through one of these slides, or make your own by cover-slipping a small drop of fluorescein solution.
You will want at least a manual coarse focus stage with 20 mm of travel for the objective. Ideally a motorized coarse z stage: this is easier to use.
For objectives: a Nikon 16x NA 0.8 objective works well and you don't need to spend more to get good results unless you are planning on routinely imaging fine structures (under about one micron).

Lasers

BakingTray interacts with the laser to turn it off at the end of acquisition and stop acquisition if the laser fails to modelock. The system has been well tested with MaiTai and Chameleon lasers. We've run these rigs with both Spectraphysics and Coherent lasers and don't have a strong preference. You need only worry about a pulse compressor if your pulses are under 100 fs and you have a lot of glass in your system (e.g. optically conjugated scanners).
The system can run with multiple lasers simultaneously, since this is supported by ScanImage. However, BakingTray currently only monitors the modelock state of one laser. There is no facility currently for re-imaging sections at a different wavelength or with a different laser.

Motion Hardware

The sample sits in a water bath atop an X/Y/Z stage.
We have had good results with PI stages and generally use these, however Zaber stages also work well. Any stage from these manufacturers will likely be supported out of the box or will require only very minor code changes. However, choosing a stage with good properties is key: see below. BakingTray is modular, so it's easy to add classes to support stages from other manufacturers.

The stage

You will need a high quality, heavy-duty, 3-axis stage. This stage will translate the sample in X/Y for tile scanning and also raise it in Z and move in X for slicing. The sample stage will be controlled by BakingTray, not ScanImage. Motions in the X/Y plane need to be as fast and accurate as possible, since the microscope spends much of its time just moving the sample.
Known to work well are the PI V-551 direct-drive stage and C-891 controllers. We use 130 mm of travel for X and 60 mm of travel for Y.
Also known to work are PI M-531.DD, and PI M-605.2DD. Tests indicate that Zaber's X-LDA stages should work very well, but no microscope is yet built with them. You would need the 150 mm and 75 mm stages. Using cheaper stages is not recommended: the reliability of the stage is critical.
The vertical stage (Z-jack) is a little more tricky. A known working option is an Aerotech PRO190SV-035 or PRO190SV-050. This stage is powerful enough to lift the X/Y stages plus the water bath. However, Aerotech stages are expensive and a little awkward to set up. PI may be able to supply an equivalent stage that is not listed on their website: contact them to ask. Zaber's X-VSR40A 40 mm lift stage looks good and should pair well with their X-LDA stages (above). We have not yet tried this Zaber stage on a rig, however.
BakingTray is highly modular, and it's fairly easy to modify the software to use stages from other vendors will require a little coding to set them up.

Constructing the XYZ stage

The X/Y stages can be mounted directly on top of an AeroTech lift stage (you will need to machine a coupler). The AeroTech stage can in turn be mounted on a breadboard with three ThorLabs BLP01 adjustable height legs for tilt correction.

Vibratome

You can use any vibratome. The vibratome can be gated either via TTL or with a FaulhaberMCDC serial-based DC motor controller. A nice option is a Leica VT-1000 vibratome head and blade, which can be purchased as spare parts from the manufacturer. The vibratome does not need a linear motor: it will not translate, the slicing is all done by the XYZ stage. Choose a vibratome based around a DC motor, as these work well and are robust. Avoid voice-coil solutions, which can be temperamental when used in a way the manufacturer did not anticipate. You should mount the vibratome in a way that allows you to control the roll axis of the blade.