Hardware requirements
This page describes the hardware you ought to have to run BakingTray using ScanImage.
Last updated
This page describes the hardware you ought to have to run BakingTray using ScanImage.
Last updated
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Hardware of course goes out of date quickly, but the following is an example of a successful configuration for resonant scanning.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 with a warranted life of 3 years or 2,880 TB.
BakingTray works with any scanning hardware and acquisition cards , 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.
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 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).
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.
The sample sits in a water bath atop an X/Y/Z 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.
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.
Your microscope should be fixed in X and Y. Do not use microscope that can translate in these axes, as this could result in a hardware crash. A coarse Z motor is very useful, however. Zaber are a good source of these. Zaber controllers are adaptable and can be used with motors from other vendors, such as ThorLabs. The Zaber control wheel by default switches between position and velocity mode if you press the wheel button. You should disable this for safety reasons. This can be done in Zaber's software using the trigger feature as follows:
This assumes your X-MCB1 controller is set to device 1, so if not, just replace the first 1 in each command with the actual device number.
We have run galvo/galvo using both and acquisition cards. However, PXIe devices are recommended as they're easier to manage in a chassis. The has also been tested but higher sample speeds don't work on all motherboards.
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 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 ) 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 to ramp laser power with depth. Choose one with a low dispersion crystal and the BK option to reduce resonances.
You can image an EM grid such as the 2145C from to assess FOV and distortion. For field flatness tou can take a z-stack through , or make your own by cover-slipping a small drop of fluorescein solution.
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 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 .
Known to work well are the 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 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 best option for the vertical stage (Z-jack) right now is the . This has 38 mm of travel and a 14 kg load limit. Buy it with a basic controller with no joystick. A hardware-based joystick is not needed and could even cause problems. Zaber's 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. Avoid Aerotech: they dropped MATLAB support in 2024 when they upgraded their motion control library.
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 for tilt correction.
You can use any vibratome. The vibratome can be gated either or with a 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 .