Imaging and Defining Emergent Behaviors of the Immune Response

Assembly & Fine Tuning

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.

This email address is being protected from spambots. You need JavaScript enabled to view it. 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.

Then click on ‘Properties’.

Choose the appropriate camera file from the dropdown list : PMT_1500x200_4ch_HV_in.rvc refers to the 4 PMTs (4-colors) system loaded into Bitflow using theSysReg utility. You MUST also update the Vertical Image Size to a value equal to 1600. This sets the overall height of the initial images being captured.

NOTE: You will get a message ‘Video Savant will now reset’ followed by ‘Cannot Register LUT Graph Class’ and you will be asked to verify the new dimensions. This is all normal—just click OK for each.

2.) Mounting your sample and getting samples oriented:

A.) If necessary, drop a very small drop of water onto the sample and mount your sample in the appropriate sample carrier. (The 20x objective is a default and is water immersion—other lenses are available and see Max to use these).

B.) With the eyepiece slider pulled out (output to eyes), turn on the transmitted light source for brightfield viewing. You can now use the focus knobs on the scope and the joystick for the stage to locate your sample.

C.) Turn off the transmitted light and click ‘Open Shutter’ on the application page. You should now be able to focus through the eyepiece, using 2-photon illumination on the sample on which you focussed in B.).

3.) Using the PMTs.

Switching to PMT Collection:

In the confocal application, ‘close’ the shutter. Make sure the transmitted light source is ‘off’ and then move the slider so that emission goes to the PMTs. Failure to turn off the transmitted light source first can saturate the PMTs and you may have to wait minute/hours for them to calm down.

Click the ‘Live’ Button. The shutter will open and images will start being collected at video rate. You should be able to view the image on the Video Savant screen. This ‘Live’ mode is useful for setting z-series and multiple stage positions (below). You can boost intensity by boosting PMT voltages (stay within the –400-->-1000 Volt range) or by integrating more frames.

The Stream Filter and Displays:

Behind the scenes, the video card is taking the input from each PMT and using a ‘stream filter’ in VS to convert the raster scan of the sample into images. This involves both interleaving the image as well as trimming data from the edge of the scan pattern where scan behavior is non-linear.

The details of this stream filter can be seen by clicking ‘Stream Filter’ within the main Video Savant control panel. This bit of code is the brainchild of Mike Sanderson.

Then click on the ‘Stream Filter’ on the left hand panel and click ‘Properties’. You should see the following dialog box.

One reason this scope is particularly quick is that data is collected on both the forward and reverse paths of the scan mirrors. However, as the galvos heat up on this scope and sometimes on a day to day basis, they may move slightly faster or slower than predicted by the synchronization signals that control them. A value of around 0 was initially set for the ‘Odd Offset 1’ (in this screenshot, channel was set to offset much more. This is highly atypical--usually these values are identical for all channels) but values can change slightly from day to day. You’ll know this needs to be changed since every other line will look a bit wobbly and some objects will look like dual objects.

A variety of channels can be displayed as a live upright file containing each color stacked vertically, a tiled set of up to four images, nd/or a small image containing a color composite. As opposed to the image in VS, this will always represent the output of the stream filtering process.

Within the stream filter, you can choose which inputs (0-3) are displayed as which colors on a color-composite (using the dialogue) but only 3 colors max - use 0,1, 2 - R,G, B. The 4th would have to remain B/W in the alternate display modes.In addition, you can choose (see ‘setting up timelapses’ below) how many 1/30th second acquisition frames will be integrated to make the final image. This is ultimately the same number as the stream filter ‘average’ and does not need to be entered here unless you want to see the effects live (the acquire button places the value in the ‘Timelapse’ tab into this register—if you don’t hit acquire, you have to enter alternate averaging numbers in the box in the ‘stream filter’ dialog box.

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