Zapit: Open Source Laser-Scanning Photostimulation For Neuroscience

Reviewer #1 (Public review):

Lohse et al. describe an open-source system for laser scanning photostimulation (LSPS) in head-fixed animals. Although similar systems have been developed and used by different groups, Zapit provides an open-source solution requiring few custom parts and minimal coding. This tool can clearly facilitate and speed the adoption of LSPS, particularly for the increasingly used purpose of mapping the effects of focal cortical silencing during behavior. Other potential uses include mapping optogenetically evoked movements and selectively activating genetically labeled neuronal subtypes of interest in the cortex. The design is well thought through, and the presentation is mostly clear and well written.

In general, the more modular such a system is, the better, in terms of compatibility with existing hardware and software that potential users may already have purchased - laser, galvo, and camera in particular. The system has struck a reasonable balance between allowing modularity and providing an integrated complete package, but even more flexibility would be welcome for potential users looking to cut costs, as would clearer presentation of such flexibility as already exists.

Comments and suggestions are mostly minor, as follows.

(1) Command signals:

How is the relationship between analog voltage commands and laser power determined? Is this assumed (or required) to be linear (as Figure 7F implies)? Usability and modularity would be improved by an option to measure or provide a calibration curve for systems with a nonlinear mapping between command voltage and laser power.

For the grid calibration step, how is the initial mapping from galvo voltage commands to image position determined? Presumably, some sort of initial guess or calculation based on the hardware specifications is needed for the grid calibration to be feasible. Also, how are the number of grid lines and the distance between them determined?

Why is the mapping between analog outputs and hardware (galvos, laser, masking light) fixed? This would be trivial to make configurable and allow labs with existing setups to adopt Zapit without rewiring existing hardware.

(2) Laser and optics:

In Figure 1, the authors should consider explaining the scanning principle schematically, i.e., depicting how tilting of the scan mirrors translates via the scan lens into beam displacement in the specimen plane. Perhaps Zemax can be used for accurate rendering.

Since the unexpanded beam greatly under-fills the back aperture of the lens, the z resolution is presumably terrible - which is good! That is, for the purposes of LSPS, this advantageously avoids focus-dependent effects, which might otherwise arise due to (e.g.) skull curvature. The authors should consider pointing this out, as well as providing an estimate of the z resolution.

What is the working distance?

Reviewer #2 (Public review):

Summary:

In this work, Lohse and colleagues develop a system for doing targeted photostimulation in mouse cortex. The system uses a camera image to target laser stimulation to stereotactically defined locations in mouse dorsal cortex.

Strengths:

The hardware is well designed, and the software is well documented and supported. The build guide and well-documented software package should allow for simple implementation of the technology. Without a doubt, this is a valuable community resource for the circuit neuroscience field.

Weaknesses:

No weaknesses were identified by this reviewer.