Tutorial

Press [ESC] at any time to open the help and settings menu.

Plan an insertion in six minutes

If you want the quick introduction to Pinpoint, this video will get you all the critical information for planning your first recording. The rest of the tutorial sections go deeper into each topic and explain some of the hidden settings that you can tweak to make Pinpoint work for your specific use case.

Overview

Pinpoint presents you with an interactive 3D scene where you can plan and test different probe insertion trajectories. The scene view shows the mouse brain (1) composed of 3D models built from the Allen Common Coordinate Framework annotations and (2) the probe 3D model, showing in white the active recording region. The scene view and probe can be rotated and moved by clicking and dragging with your mouse, see the section titled Controls, below. (3) The rotation gizmo helps you understand the orientation of the brain, with Yellow representing the Dorsal-Ventral axis, Red the Anterior-Posterior axis, and blue the Left-Right (or Medial-Lateral) axis. Double-clicking the gizmo will snap the camera view to the corresponding planar view (i.e. Axial, Coronal, and Sagittal).

Pinpoint interpolates the recording region on the probe in two viewports to help you understand where your electrodes are placed in the brain. The probe UI ( 4) shows you a linear interpolation through the recording region. The in-plane slice (5) shows you a view of not only the probe but also the surrounding regions. You can click regions in the in-plane slice to highlight them in the 3D scene. You can also highlight regions of interest by searching for them in the search panel (6). Click a region turns it on and off and pressing the * Clear* button removes all highlighted regions. The snap-to-area button ( downward arrrow) moves the probe tip to the center of that region.

To set up a new probe, select the corresponding button the new probe panel ( 7). By default, probes are spawned at Bregma. You can change the reference coordinate in the Atlas settings. When you have multiple probes in the scene you can click on the probe objects to switch the active probe, or you can click on the probe panels on the left side of the screen.

The coordinates of a probe are shown in the last panel (8) and can be copied to the clipboard using the download icon at the top right. When the probe is outside of the brain we display the tip coordinate and angles. When the probe is inside the brain Pinpoint computes for you the brain surface coordinate and * depth* of the probe, which are the coordinates you would need to re-create an insertion. See the surgery section for a few important details.

Probe panel

The Probe panel provides easy access to the information about the active probe in the scene. Using the panel you can tweak the entry coordinate by changing the AP/ML/DV position data or change the insertion depth. You can also adjust the insertion angles.

The three buttons on the top right lock the probe (preventing accidental keyboard or mouse movements), duplicate the probe in the scene, and download the probe data to the clipboard. You can paste the copied data into any text editor.

The bottom row has the Accounts dropdown where you can choose which experiments this probe is saved in, as well as the snap to dura button and home button.

The home button moves the probe back to the current reference coordinate (defined in the Atlas settings). The snap to dura button snaps the probe back to the surface of the brain, moving only along the depth axis.

Camera controls

Probe controls

Click + Drag

To control your probe with mouse clicks and dragging:

1. Left-click and hold on the probe object

2. Press and release any of the axis keys on your keyboard, the axis should become visible in the 3D scene

3. Continue holding the mouse and drag the probe along that axis.

The axis keys are [W/S] for AP, [A/D] for ML, [Q/E] for DV, and [Z/X] for depth. Note that in some views it may be hard to drag along a particular axis (e.g. in the default axial view you can’t drag a probe along the DV axis, because the axis is not visible).

Keyboard

You can also press the keyboard keys to move the probe along that axis in 1/10/100/1000um increments. Pressing and holding a key moves continuously.

Press [SHIFT] to increase the speed of each movement. Press [CTRL] to reduce the speed.

Use [W/A/S/D] to move the probe along the AP or ML axis. Use [Q/E] to move the probe along the DV axis and [Z/X] to move the probe along the depth axis.

You can also rotate the probe with keyboard clicks.

Use [1/3] to yaw the probe around the vertical axis. Note that NP2.4 probes spin around shank 0 (the leftmost probe shank when looking from the front).

Use [R/F] to change the pitch angle of the probe on the manipulator (restricted to the vertical to horizontal range).

Use [,/.] to roll the probe along its own axis.

Probes can be deleted with [Backspace]. To recover a probe you didn’t mean to delete press [CTRL + Backspace].

You can also quickly switch between probes in the scene by pressing the [N] and [M] keys.

Controller

You can also use a USB controller to move probes.

Channel map

Once the probe is at the position and angles you want you can change the active set of channels in the Probe menu. The default option is the first bank of electrodes (first 384 channels for Neuropixels 1.0, or the bottom 96 channels on each shank for Neuropixels 2.4). Use the “all” option to see the entire probe at once.

Designing a trajectory and probe coordinates

Probe coordinates

A probe insertion is defined by a target coordinate in the brain and a set of angles to reach that point. When you export a planned insertion Pinpoint will compute the brain surface coordinate and depth of the probe, which you will need to actually perform your experiment.

Note that insertions are defined in a Coordinate Space with a particular Transform. The space defines the rotation of the axes relative to the reference point (i.e. which way do AP/ML/DV go relative to Bregma). By default all insertions in Pinpoint are defined in the common coordinate framework space, using the IBL axis conventions, these can be changed in the settings. The standard transform is the Qiu 2018 transform, which was based on the average of 12 adult C57 mice. You can read more about the transform options on the in vivo alignment page.

Settings

The Settings menu is accessed by pressing [ESC]. Press ESC again to close the window. The tabs (1) on the left side switch between the different sub-menus.

Rig components

Areas & Atlas options

API

Pinpoint can send anatomical data about probes in the scene to the Open Ephys GUI and SpikeGLX during experiments. This is an independent feature from the electrophysiology link features (below) and they can be used together, or separately.

These features are only available on the Windows Desktop build of Pinpoint they will not run in the web browser.

To stream the channel data to Open Ephys, follow these instructions:

1. Launch Open Ephys and connect to or simulate one or more probes using the Neuropix-PXI plugin.

2. Make sure at least one Probe Viewer plugin is in your signal chain. If you don’t see the Probe Viewer in your processor list, you can add it via the Plugin Installer (Probe Viewer version ≥0.3.1 is needed to interface with Pinpoint).

3. Make sure the HTTP Server is enabled in Open Ephys (“File > Enable HTTP Server” should have a checkmark next to it).

4. In Pinpoint, press [ESC] to open the settings menu and navigate to the “API” tab

5. In the “OpenEphys API” section, make sure the IP address matches that of the machine running Open Ephys. If Pinpoint is running on the same computer, this will be “localhost”. The port number will always be 37497.

6. Toggle the API on

7. In the probe matching list on the right, you should see each of your Pinpoint probes. Use the dropdown menus to link each probe to its corresponding name in Open Ephys.

8. You should immediately see should see an update to the anatomy data in Open Ephys

For SpikeGLX, follow these instructions:

Make sure that your SpikeGLX version is updated, you’ll need a 202306 or later version number. You can update to the latest SpikeGLX here.

1. Download HelloSGLX and place the folder in a convenient location, e.g. C:/HelloSGLX

2. Launch SpikeGLX

3. In the Options > Command server Settings menu, make sure that Enable Remote Command Server option is checked.

4. Start a new acquisition, using existing data or a live probe

5. In Pinpoint, press [ESC] to open the settings menu and navigate to the “API” tab

6. Make sure the location of the HelloSGLX file is accurate.

7. Toggle the API on. If you see an API Status error, it probably means you didn’t turn the command server on, see step 3.

8. In the probe matching list on the right, you should see each of your Pinpoint probes. Using the dropdown menus, select which SpikeGLX probe ID each probe should be linked to

9. You should immediately see an update to the anatomy data in SpikeGLX (make sure to open the probe shank view to see this)

Ephys Link

Pinpoint can be connected to electrophysiology manipulators enabling experimenters to automate the placement of manipulators as well as observe live where probes are placed inside the mouse brain.

We currently (July 2023) support Sensapex uMp-4 and New Scale 3-axis manipulators.

These features are only available on the Windows Desktop build of Pinpoint they will not run in the web browser.

To enable this feature, an instance of an Ephys Link server must be running on the local network. Please see the instructions here on how to install and run a server.

Connecting to a server

1. Press [ESC] to open the settings menu

2. Switch to the “Ephys Link” tab

3. Fill in the server’s IP address

4. And the port number

5. Click “Connect”. Upon success, the connect button will read “Disconnect”

6. Panels for each manipulator will appear here. These panels allow for the configuration of manipulators and linking them to probes in the scene.

7. Press [ESC] again to close the settings menu

Linking a probe in the planner to a manipulator in vivo

This step is not required for New Scale Pathfinder connections. See below for details.

1. Spawn the corresponding probe in the planner scene.

2. Press [ESC] to open the settings menu.

3. Switch to the “Ephys Link” tab.

4. Scroll down to the panel that represents the in vivo manipulator.

5. Use the dropdown to configure the manipulator’s handedness. Currently (July 2023), configuration is necessary for Sensapex uMp manipulators only. Configuration is hidden for New Scale manipulators. See the section below for more information.

6. Use the dropdown to select a probe to link to this manipulator. Probes are identified by their unique ID (UUID) and their color in the scene.

7. Upon connection, the probe’s stereotaxic position will be used as the zero coordinate that all movements will be relative to. This zero coordinate can be edited manually through the text boxes or by clicking the home button to reset the position to the current probe position.

8. The probe’s offset from the brain surface and which direction it is calculated can be adjusted here. The offset is the distance from the probe tip to the brain surface. This offset can be determined as the distance from the tip to the brain surface along the DV axis or the probe’s depth axis.

9. Manual control of the manipulators can be enabled here. This allows manipulators to be controlled with the same keyboard shortcuts used to move probes in the planner around. Manipulators can also be driven back to their zero coordinate.

10. Press [ESC] again to close the settings menu.

Sensapex Handedness

Handedness determines the orientation of the manipulator and which direction it moves. Handedness is classified by which side of the manipulator the probe will be mounted on. Ephys Link will use this to correctly orient the manipulator’s direction.

Below is a diagram of the handedness of Sensapex manipulators. Use this to determine the handedness of your manipulator.

New Scale Pathfinder connections do not require configuration

Launching Ephys Link requires specifying the type of manipulator that is being used. Configuration in Pinpoint will differ depending on the type of manipulator. New Scale Pathfinder connections do not require any configuration in Pinpoint as all manipulators are configured in Pathfinder. Pinpoint will automatically create probes (colored in magenta) for each manipulator from Pathfinder and automatically echo their positions and angles.

Enabling experiment automation (Ephys Copilot)

Automation is currently a beta feature that is still in development. Please contact Dan or Kenneth if you would like to try it out.

Account

The Accounts tab in the settings allows you to save insertions across computers. Insertions are stored in experiments. One insertion can be shared across multiple experiments.

1. Register a new account, we kindly ask that you use an email address so that we can keep in touch about major updates to Pinpoint in the future. We won’t give away your email or send a message more than once or twice a year.

2. Log in to your account by providing the username and password. Leave the “stay logged in” toggle checked if you are on a private computer.

3. Open the experiment edit panel to add and remove experiments and re-name them. Experiments are just buckets, you can use them in any way you want: to represent mice, to actually represent different experiments, to represent differents sets of insertions targeting particular brain regions, etc.

4. Select the current active experiment from the dropdown. This will change the visible list of insertions below.

5. The insertion list shows the insertions in the active experiment. You can rename insertions, delete them from the experiment, and enable/disable them in the 3D scene. Coming soon: we will add options to mark insertions as “recorded” so that we can calculate coverage maps for experiments.

6. Any time your local data is out-of-sync with the data stored on the server the “unsaved changes” message will appear. If you are using Pinpoint in a browser please note that unsaved changes do not get saved if you exit the browser, the program auto-saves changes about once a minute. If you are using Pinpoint in a desktop app, exiting will automatically save all changes.

Surgery

To use your coordinates for a surgery, rotate the manipulator to match the azimuth angle (phi) and set the probe elevation angle (theta). Then move your probe tip to Bregma and zero your manipulator. (Optional: move your probe tip to Lambda and record the distance from Bregma to Lambda, then enter that into the Atlas settings in Pinpoint). We recommend using the technique shown in when estimating the position of Bremga and Lambda on the skull (i.e. align the “curve” between the sutures, not the exact point where they attach).

Translate your probe using the manipulator to match the AP/ML/DV brain surface coordinate provided by Pinpoint. If your probe is rotated off-axis use the * convert AP/ML to probe axis* setting so that you can match the Forward/Side/DV position instead. Use caution! You may reach the brain surface before you reach the DV value specified by Pinpoint, don’t break your probe!

Once the probe is touching the dura, zero the depth axis and insert until you reach the specified depth.