About This Documentation

Purpose

This documentation provides comprehensive information for operating and maintaining the Sutter 2P microscopy system for in vivo 2-photon calcium imaging (2PCI). It serves as a reference for system setup, daily operation, troubleshooting, and custom software configurations.

System Overview

Hardware Components

The rig is built around a Sutter MOM (Moveable Objective Microscope), which features a moveable objective rather than a moveable stage.

Key components:

  • Sutter MOM microscope with 40× 0.8 NA objective (Olympus)
  • MaiTai HP laser (Newport) for 2-photon excitation
  • Two National Instruments DAQ boards:
    • NI USB-6229 (Dev2) - Ephus acquire/sound stimulation
    • NI PCI-6110 with BNC-2090A breakout (Dev1) - ScanImage 2P
  • Hamamatsu H10770PA-40 PMT with Sutter PS-2LV controller
  • Tucker Davis ES1 freefield speaker with ED1 driver
  • Sutter ROE-200 micromanipulator
  • ThorLabs KDC101 motor controller with PRM1Z8 for laser power control
  • Dell Precision Tower 5810 workstation (Windows 10)

Software Stack

  • Operating System: Windows 10 (21H2 x64)
  • Ephus 2.1.0: Widefield imaging and sound stimulus delivery (MATLAB 2013b 32-bit)
  • ScanImage 5.3.1 (2017): 2-photon microscopy control (MATLAB 2015b x64)
  • Custom user functions: Automated workflows and experiment control
  • Acquisition specs: 145×145 μm images at 256×256 pixels, 5 Hz effective frame rate

Key Features

Synchronized Dual-DAQ Acquisition

The system synchronizes two DAQ boards for coordinated 2P imaging and stimulus delivery:

  • Trigger routing from ScanImage (Dev1/PFI13) to Ephus (Dev2/PFI0) via USER2 BNC connection
  • Sample clock sharing for precise temporal coordination
  • Custom user functions respond to ScanImage events (acqModeArmed, acqModeDone, acqAbort)

Custom User Functions

Ephus user functions:

  • Save stimulus pulse details to .mat files
  • Random pulse selection (with/without replacement)
  • Automatic QCam reset for looping acquisitions

ScanImage user functions:

  • digtrig_stimPulse_train: Triggers Ephus stimulus with configurable delay
  • digtrig_stimPulse_train_withCam: Version with pupillometry camera support

Safety Protocols

  • Laser safety glasses: LG8 (630-695nm) for 690nm alignment, LG9 (610-695nm + >740-1070nm) for 920nm
  • Proper alignment procedures with protective equipment
  • Power monitoring and control via motorized rotation mount

Known Issues

  • Herringbone artifact: Normal for End-On Hamamatsu PMT (H10770PA-40) configuration
  • Timing delay: ~97-99ms (occasionally up to 700ms) between ScanImage acquisition start and acquisitionStarted timestamp in user function

Maintenance & Support

Primary Technical Contact: - Rick Ayer, Sutter Instruments - Email: rick@sutter.com

Contributing

This documentation is maintained as a living document. Updates should include:

  • Configuration file changes with date stamps
  • New procedures or workflow improvements
  • Troubleshooting solutions for common issues
  • Hardware or software modifications

Last updated: December 11, 2025