Getting the electrical box built:
The details of the electronics for the scanner control circuitry are available on Mike Sanderson’s website. We also had some help in implementing these from the guys at Sutter—for electronics neophytes, a similar query may help you build this box. Once this is done, cables for mirrors plug into this box and Hsync and Vsyncs get plugged into the Bitflow card using the coaxial cable provided with the card/computer by Video Savant. Plug PMTs into their sockets and plug their inputs into the power supplies and outputs into the appropriate BNC cable end going into the Bitflow card. Stage should be plugged into the control box and the control box is cabl-ed to COM1 on the computer. The Z motor should be installed between the scope body and the objective and the Z-controller should be cabled to COM2 port on the computer.

Inserting galvos and lenses:
The parts that Sutter makes creates a scanning system suitable for resonant scanning, FRAP, confocal scanning, etc. To align a scanner, you need to first place the 10x eyepiece (see image under 'Overview of Construction'). The 10x eyepiece should be in such a position such that a sample (the stained pollen grains are very useful here) are in focus when you look through the eyepiece from a position to the left of the scope (i.e. move the eyepiece right-left until a real image is in focus when the objective lens is place on the pollen grain and is also in focus in the microscope eyepiece.

You also need to loosely insert the CRS/M3S scanners into the mounts and roughly position them. Also, it is necessary to place the variable neutral density wheel into the light path and make sure you have the dichroic (above the objective) in place. Leave the shutter out for now.

Installing the laser and routing optics and rough alignment:
Essentially this is a stepwise process in which you need to first get the laser into the scan box such that it is parallel to the breadboard drilling pattern and also parallel to the table. Then, you need to approximately get the mirrors positioned so they deflect the beam in the right path into the tube lens. The right tool here is actually the back of a business card. A 2 photon beam at ~780-~820nm makes a red spot on a white card and you can mark a grid onto this card as a measure of location while keeping the card in contact with the bottom of the breadboard (a strong laser will actually cause a dark ink spot on a card to catch on fire!)

1. With the mirrors oscillating use the beam-routing periscopes on the table to send the beam into the back of the scanner and hit the mirrors. Now, align the laser into the back of the scope so that it hits the large (CRS) mirror dead center. To walk the laser beam such that it is parallel to the breadboard, use a card that you shift along the scan box from front to rear. First, mark the height where the beam hits when the card is close to the CRS and check that the beam is no gaining or losing height as it heads toward the scanner. Where necessary, you will have to adjust the periscope mirrors (walking the beam) to get this right. Similarly,verify that the beam is coming in parallel to the breadboard pattern on the scanner (coming in perpendicular to the path that it will take going from mirrors into the tube lens—again using a card moved from front to back of the breadboard keeping the card always in a similar spot relative to each successive hole. Once this is the case, you are in pretty good shape.

2. The beam should be hitting the mirrors and, depending on their angle on the breadboard, should be progressing about 90 degrees into the 10x eyepiece and tube lens. However, this mount is flexible in many ways so you may need to adjust it as appropriate so that the beam passes through the 10x eyepiece and into the tube lens. This may take minor tweaking. A card placed into the lightpath between the 10x eyepiece lens and tube lens should reveal a square scan pattern going into the tube lens when the mirrors are turned 'on'. If this pattern is not centered on the front of the tube lens, adjust your mirrors slightly.

Fine-alignment of the laser:
From here, it is a matter of getting the beam to pass cleanly into the back aperture and then parallel through the lens. I found that Steve Block had a nice description of fine-alignment in his chapter on building a laser trap (for the CSH series). I suggest this for the absolute novice. In brief, fine tune the position of the mirror (by changing the mount subtly or using the ‘Position’ potentiator knob on the electronics box you’ve built) . In parallel, move the beam using the steering mirror on the periscope. By successively moving one and then dialing back the other (“walking” the beam) you can get alignment perfect. The goal here is to have a diffuse circular pattern when the objective lens is removed from the microscope and to have this large circular pattern centered on a crosshair that you previously drew on a piece of tape on the stage at the point where the exit lens of the objective would touch the stage (essentially, the infinity beam is parallel to the path defined by the objective.

Paul Herzmark at UCBerkeley has compiled the following documents providing nifty and details from his own experiences aligning this path (click here).

Viewing Real-time 2-Photon images through the eyepiece:
Note: Before doing this step, BE VERY SURE that you have placed the 2 photon blocking filter in the light-path leading to the eyepiece!! With the laser ‘on’, place a pollen slide onto the stage and focus using transmitted light. Once you turn this off, you should see an image in the eyepiece.

Installing the shutter:
Place the shutter in place such that the beam passes directly through it when in the ‘open’ position’.
At this point, you can close your scanner box. If you have placed the 2P blocking filter in the eyepiece lightpath, the only way to get exposed to 2P laser light is to get in under the ojective—practically it is hard to get your head under there so unless you place a reflective surface under the objective, you are pretty safe.

Viewing Images with RvView:
Configure camera files using RvSetup
Open RvView
Choose Camera 0 (presuming you only installed a single four channel camera file (link to this) using SysReg when you initialized Bitflow.
An image (at 30fps and representing full-lines) should be visible. See Mike Sanderson’s site for more details on what this looks like and how/why it is generated.

Configuring VS/Confocal to View Images:
Correction and reversal of every other line is achieved through the‘Confocal’ GUI in conjunction with Video Savant. (More information is available in the Video Savant Documentation, particularly ‘Confocal Application’) Click this icon and you should see a window that looks like this:

Note that most of the items from within this dialog can be saved and retrieved later using 'Save Settings' and 'Load Settings'. In addition, you will see the VS main window: FYI, the tabs at the bottom are used to navigate through the different functions of VS.

Configuring the Camera File:
Distinct camera files enables the use of multiple PMTs in Video Savant (through the Bitflow card). This is achieved by the use of distinct ‘camera file’.

To choose the camera files and the details of data input into VS, click the Camera Interface Tab.