> For the complete documentation index, see [llms.txt](https://bakingtray.swcmicroscopy.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://bakingtray.swcmicroscopy.com/getting-started/tissuecyte_upgrade.md).

# Upgrading a TissueCyte

This page summarises how to upgrade a TissueCyte 1000 to run BakingTray.

![A TissueCyte 1000 commercial serial section 2-photon microscope converted into a BrainSaw system that runs BakingTray. The system is shown with a modified water bath that uses a clamp to reproducibly position the slide. The stock vibratome is swapped for a Leica VT1000. The collection path has been moved closer to the objective and enclosed. The coarse focus control is manual in this image, but the system was later upgraded to a motorised coarse focus module using parts from Thorlabs and Zaber.](https://3832012848-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2F-MAVhQ1tbEzz1UZd9NDb%2Fuploads%2Fgit-blob-afb98928bf6d84918234e66c57cfccce158bdb54%2Fneurovision_early_photo.jpg?alt=media)

BakingTray is a viable way to upgrade and extend the life of legacy TissueCyte 1000 systems. The upgrade has been performed on two systems: one at the SWC ("NeuroVision") and one at the École Polytechnique ("ChroMS"). Conversion to BakingTray provides a variety of benefits to end-users (depending on the exact version of the microscope being upgraded).

* Acquisition on a single Windows 10/11 PC instead of two Windows XP PCs.
* The option to add a fourth detector channel.
* Easier sample setup with a preview tile scan.
* Automatic sample finding (autoROI).
* The ability to pause and gracefully stop acquisitions.
* The ability to easily resume stopped acquisitions.
* Acquisitions will stop if laser mode lock fails or no tissue is detected.
* Laser and PMTs turn off at the end of acquisition.
* Acquisition does not start if there is insufficient disk space.
* All channels and depths from one tile position are stored in a single file with rich meta-data.

Overall, the system becomes easier and more pleasant to use, which simplifies user training. We noticed a reduction in both training time and operator errors after BrainSaw conversion. Furthermore, since BakingTray operates alongside ScanImage, users can do live "Focus" previews whilst panning around the sample, quickly change zoom or image size, etc. Our version of TissueVision's Orchestrator software was unable to perform continuous scanning, making quality control tasks, such as acquiring point-spread functions from fluorescent beads, almost impossible. Even without switching to a resonant scanner, overall acquisition speed will improve substantially because of autoROI.

The upgrade should be performed carefully: some hardware changes are tricky to make (in particular the coarse Z motor upgrade) and these changes are hard to revert once made. Once the upgrade is complete, the end-user becomes responsible for their own support. In our experience, maintenance of these systems is no more involved than maintenance of any other custom two-photon microscope.

### Hardware requirements for a minimal upgrade

The BakingTray upgrade will re-use all the original stages and motor controllers. If desired, the vibratome, detectors and pre-amplifiers, and scanners can also be re-used. The PI C-891 linear stage controllers will need a free firmware upgrade. The linear scanners from Cambridge Technology used in the TissueCyte 1000 have unique tuning parameters and scan significantly faster than stock Cambridge galvos. Should a scanner fail, replacements with the same specifications cannot be purchased from the original manufacturer (Cambridge Technology). This is not a significant constraint, as the system will perform better overall if converted to resonant scanning, which provides much faster scan speed whilst retaining the ability to average frames when desired. Bidirectional scanning artefacts are also easier to manage with a resonant scanner. With bright labelling, resonant scanning is not a compromise in image quality: the high-resolution single-axon tracing work at Janelia was performed on a resonant-scanning STPT rig ([Economo et al., 2016](https://doi.org/10.7554/eLife.10566); [Winnubst et al., 2019](https://doi.org/10.1016/j.cell.2019.07.042)), as was the work of [Liu et al. (2022)](https://www.frontiersin.org/journals/neuroanatomy/articles/10.3389/fnana.2021.791015/full) on BrainSaw.

#### Required new components

You will need a new acquisition PC, a ScanImage Basic licence, and a new DAQ. You can likely recycle an existing NI DAQ, should you have one; alternatively, a new ScanImage vDAQ is probably the easiest option.

The TissueCyte comes with a Thorlabs [liquid crystal variable retarder](https://www.thorlabs.com/half-wave-liquid-crystal-variable-retarders-wave-plates?tabName=Overview) to modulate laser power. This device is not supported by ScanImage, is rather slow, and won't be able to blank resonant scanner turn-arounds should you want to replace the scanner. You will need to replace it with an EOM, such as a Conoptics Pockels cell, or an AOM.

The Z drive that pushes up the stage platform is a stepper motor. It is driven by an mSTEP-407-50 from Advanced Micro Systems (AMS). Using this device with a modern Windows install requires a new USB dongle from the supplier but, unfortunately, the supplier is no longer in business. A more future-proof alternative is to replace the AMS board with a Zaber X-MCA controller (see image below). This not only controls the motor but can integrate with the limit switches on the stage.

![Zaber X-MCA controller integrated into the TissueCyte motor controller box](https://3832012848-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2F-MAVhQ1tbEzz1UZd9NDb%2Fuploads%2Fgit-blob-af30fbea5436d5b03ea7379400e40a2d7c6b0196%2Fzaber_TissueCyte_Z.jpg?alt=media)

The Zaber settings file used at SWC for driving the TissueCyte's Z stage [is in the example config files on the BakingTray repo](https://github.com/SWC-Advanced-Microscopy/BakingTray/tree/master/ExampleConfigFiles).

### Optional hardware upgrades

The TissueCyte 1000 is, of course, an STPT system so has suitable stages and a vibratome. However, the microscope has some unusual features which are not found in a conventional generic two-photon and you may want to change.

The TissueCyte's collection path is a sprawling, open, design with huge lenses and vulnerable PMTs. It is possible to move the collection optics closer to the back aperture and re-package the whole path into a more compact, closed, design which does a much better job protecting the PMTs and the optics whilst either retaining the original PMTs and pre-amplifiers or upgrading these also. The image at the top of this page shows the SWC's "NeuroVision" system, with three 10699 PMTs in Thorlabs [PXT1 PMT housings](https://www.thorlabs.com/pmt-housing-for-side-on-hamamatsu-pmts?tabName=Overview). It has since been updated to Thorlabs [PDA45 silicon photomultipliers](https://www.thorlabs.com/silicon-photomultiplier-sipm-amplified-detectors?tabName=Overview). We do not have instructions for these upgrades, but we can provide advice and share Zemax diagrams and CAD models to help those wishing to go down this path.

Regardless of whether the path is enclosed, it is a good idea to add bandpass filters (the TissueCyte did not come with these) since doing so should reduce spectral cross-talk.

The TissueCyte also did not come with a motorised Z stage. The workaround was to use the PIFOC bias knob and for larger adjustments periodically adjust the objective with a fine-focus knob. However, the microscope is easier to use if motorised coarse focus control is added. We did this using a [Thorlabs PLSX](https://www.thorlabs.com/item/PLSX) stage and a [Zaber X-MCB1](https://www.zaber.com/manuals/X-MCB1) controller (image below). Again, we can share drawings but do not have install instructions.

![TissueCyte with coarse Z added via a Thorlabs PLSX controller](https://3832012848-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2F-MAVhQ1tbEzz1UZd9NDb%2Fuploads%2Fgit-blob-22d8c0861e99d3b48a2964e85449302f2c6f9346%2Fneurovision_coarse_z.png?alt=media)