Introduction

This manual will help you understand the Neurolabware resonant scanning microscope, in order to minimize the amount of time, effort and additional complications during data acquisition.  As this is primarily an instrument for performing optical physiology and calcium imaging, we also hope we can aid in streamlining the analysis and interpretation of imaging data.

Topics will cover some imaging and two-photon excitation background, initial installation, hardware and optical maintenance and diagnostics, electronics diagnostics, upgrades, various assemblies, and some basic software functions.  A crucial parallel resource is Dario Ringach's ScanBox blog, which contains detailed procedures for specific tasks and calibrations involving the ScanBox software, various modular functions, and two-photon calcium imaging generally.

Unpacking and Installation

In order to install the microscope you will need some basic lab tools, a laser, optical table, laser power meter, safety glasses, table laser shields, and so forth.  Some recommendations:

You should have received the following components:

  • Aluminum extrusions (3)
  • Translation stages (3)
  • Microscope body and optics, including custom 2" filter sets
  • Microscope detection unit, perhaps including epifluorescence imaging option
  • Acquisition computer and digitizer
  • Perhaps eye/pupil and disk/ball tracking cameras and accessories
  • PMT amplifiers and power supplies
  • Pockels cell and amplifier
  • Table Shutter unit
  • Table optics, potentially including steering mirrors, kinematic mounts, posts and post bases, polarization optics

You may unpack the above items carefully.

 

Computer and Electronics

Your Dell server/workstation computer should have Windows 10 installed, and Alazar digitizer installed into a suitable PCIe slot.  Please update your Dell bios firmware (available via one-click from Dell support pages) and Alazar firmware to latest versions.  This is an important step.

TeamViewer should also be installed so we aid in various set-up tasks and troubleshooting.  We can't stress enough how vital TeamViewer remote access is.

Matlab 2015b should be installed, along with the following toolboxes:

  • Computer Vision System Toolbox
  • Image Processing Toolbox
  • Optimization Toolbox
  • Parallel Computing Toolbox
  • Signal Processing Toolbox
  • Statistics and Machine Learning Toolbox
  • Data Acquisition Toolbox
  • Instrument Control Toolbox

Cabling

Attach X Y and Z DB9 cables from motor control box to motors.  Attach Theta connector to motorized rotation stage.  Connect the motor control box to the computer via USB.  Connect to power and turn on.

Connect SMA cable from sample trigger on ScanBox to Alazar External Trigger (TRIG IN) with provided SMC to SMA adapter.  Connect Channel A to PMT 0, Channel B to PMT 1, etc.  Connect laser clock to Optional External Clock (no SMC to SMA adapter required).

Connect scan mirrors to ScanBox.  Connect PMT high voltage control to ScanBox.  Connect linear actuator connector to ScanBox.  And Connect ETL/Optotune to ScanBox current source output.

 

PMT Amplifiers:

For variable gain amplifiers: set to non-inverting, 10^3/10^4 V/A, high gain (10X), full bandwidth (FBW).  Connect amplifier output to oscilloscope and turn the offset potentiometer (not bias) to set output offset to +380mV.

Initial alignment and maintenance

The laser beam should exit the laser and pass through conditioning optics.  Use low power alignment modes to align the laser, using kinematic mirror mounts, posts and post bases.  A beam height reference should be used to maintain laser height around the table.

When steering up into the system, the beam should be parallel to the vertical translation of the microscope by the large steel translation rail.  This will ensure constant alignment during vertical height adjustment.

Along the system, the beam should hit approximately center at all steering mirrors.  This will ensure constant alignment during translation of the microscope system.  (+/- 1mm)

Once the scan mirrors are hit, they can be rotated carefully to center the scan square (after the scan lens) on the optical axis.  Use the provided phosphorescence laser viewing card, as short wavelength light will not pass through the main dichroic.  Before screwing in the objective, verify the stationary spot location and size at the objective back aperture.  Proper filling of the back aperture directly determines excitation NA and hence both resolution and brightness of signal.  If the beam is not stationary, the tube lens may need adjustment via the translation set screws.  A beam that is not stationary on the back aperture may cause some vignetting. 

Basic software operation

Welcome to ScanBox

Basic Analysis

The functions to access the data are documented in the scanbox blog:

 

https://scanbox.wordpress.com/

 

 

How to read the data stored by Scanbox is described here -- 

 

https://scanbox.wordpress.com/2014/08/04/reading-raw-data-with-sbxread/

 

The basic processing pipeline is described here -- 

 

https://scanbox.wordpress.com/2014/07/31/basic-processing-pipeline/

 

How to integrate your system with other external events/stimulation is described here -- 

 

https://scanbox.wordpress.com/2014/06/09/scanbox-system-integration/

https://scanbox.wordpress.com/2016/01/22/ttl-synchronization/

 

Non-rigid deformation for image registration/alignment

https://xcorr.net/2014/10/03/sorting-calcium-imaging-signals/

Pockels Cell

Place the pockels cell and pockels cell mount in the beam path.  The beam should enter and exit at the center of the apertures.  Connect the P1/P2 BNC cables to the pockels cell electrodes and corresponding amplifier outputs.  While powered off, switch the amplifier command input settings to unipolar mode and 1kOhm input impedance.

Additionally you can use a 0.050 inch allen wrench to rotate the pockels exit cap to block the rejected portion of the beam.  This removes the need to have an external safety beam dump or block.

Rotate the pockels cell for minimum transmission of the beam at the desired wavelength (910nm-960nm), while the bias voltage is set to 0V.