Intrinsic & epi-fluorescence imaging using the port camera

Some colleagues have asked about the possibility of integrating imaging acquisition from the port camera to do intrinsic and/or epi-fluorescence imaging ahead of targeting a region of interest with two-photon experiments in Scanbox.

We have now added this option to Scanbox.

To use this  option you will need to wire the I2C port on the faceplate of Scanbox.  As nobody seems to be making use of I2c sensors we changed the functionality of these pins.

You will first need a camera with an output TTL signal that provides a rising edge on each frame. Most advanced cameras provide such an option.  That signal needs to be connected to pin #2 (second from the left).  We also need a synchronization signal from our visual stimulus, so we know on which frames it is presented.  A rising edge on pin #3 will be timestamped by the system with the frame number when it occurred.

That’s all you need in terms of hardware connections.

Now, you will find an extra line in the scanbox_config.m file that allows you to select the format you want to use for the port camera.  Some cameras have an 8-bit depth format that is convenient and sufficient if all you are using the camera for is to navigate around the sample. For imaging, you will probably want to use formats with pixel depths ranging from 12 to 16.  You can use Matlab’s imaqtool to see what formats are available for your camera.  Set the pathcamera_format variable to a string reflecting your selection.  For example:


sbconfig.pathcamera_format = 'Mono14';

In Scanbox, you will see a new panel which, at the moment, is sparsely populated with only 3 buttons, but should be enough to get things going.  Two buttons behave in the same way as for the eye and ball cameras: they allow you to define a ROI that will be saved.  The third button “Grab” allows you to manually start and stop the acquisition.  You can only use this button when the port camera is active. Otherwise, you will get a message complaining about it. When the port camera is active, sending a command to start and stop sampling will engage this button as well.  So the same experimental scripts you are now using for two-photon imaging can be used for imaging with the camera port without any change at all.

The data are stored in two separate files, one that contains the images themselves and the other containing the TTL data from the visual stimulation.  To read them, there are two separate functions: sbx_get_ttlevents() and sbx_read_camport_frame().

The first function takes the file name as input and simply returns a list of frame numbers during which a rising edge was present in the “stimulation” input on the I2C connector above.

So, for example:


>> ttl = sbx_get_ttlevents('xx0_111_222')'

ttl =

    200  240  280  320  360  400

This means the onset of six stimuli occurred during those frames.

The second function takes the file name and a vector of frame numbers.  It returns a volume of data where the third dimension corresponds to the selected frames.

So, for example:


>> data = sbx_read_camport_frame('xx0_111_222',ttl(1)-10:ttl(1)+10);

Will read the volume from ten frames before the onset of stimulus #1 to 10 frames after.

As things are now you have to control illumination externally.  We will work to integrate illumination and histograms of ROI values soon.

Sampling on a Surface

Not long ago I mentioned Scanbox’s new ability to sample on a surface. Now you can access this new feature in the GUI by navigating to the Surface Sampling panel.

Surface sampling allows you to link lines of the resonant scan to depths determined by the optotune setting.  In other words, it allows you to sample on a surface along the galvo axis (the vertical axis in Scanbox).  Of course, limits are imposed by the range of the optotune and its dynamical response.

Below is an example of how the process works.  Here, I am imaging a slide of pollen grains that is tilted along the vertical axis.

Because the slide is tilted, different settings of the optotune bring the grains in different lines into focus, as shown at the beginning of the video.

To compensate for the tilt we can establish a link between lines in the scan and depth. To do this, change the optotune setting while focusing, then hit the Link button, and then click on the grains that are in focus.  In this example, I repeat this a handful (3) of times.

Now, when the Enable button is clicked, Scanbox interpolates a value of the optotune for each line given the established links and uploads the resulting values to the Scanbox firmware.  When we image the slide with the link active we see all the grains in focus. In other words, Scanbox is now sampling on a slanted surface and compensating for the tilt of the slide.

This is a useful feature to use when compensating for the curvature of a structure that is being imaged or tilting the imaging plane without physically tilting the objective.

Try it and let me know if you run into any problems.

The use of this feature requires an update of Scanbox and the firmware to version 4.0.

 

Tiling with Knobby

Knobby scheduler allows the user to move the microscope by specifying a list of relative changes in position at given frame numbers.  One quick way to fill up the table is provided by a set of text fields in the knobby panel.  This allows one to perform (x,y,z) tiling with a given range and step size.

tiling

The first row of entries correspond to the range (in um) to be covered in the x, y and z axes respectively.  The second row specifies the step size in each case.  The last entry at the bottom tells Knobby how many frames to sample at each location.

In the example above, Knobby will perform a z-stack with 40um range and 20 um steps (that is, a total of 3 locations), on an (x,y) grid of 3×3 with 200um horizontal and vertical range and steps of 100um.

As you input the desired values the table will update automatically.

Once ready, click the “Arm” checkbox (and the “Return” checkbox if you want knobby to go back to its initial position at the end) and start your acquisition.

Here is the resulting scan for the example above:

Processing Volumetric Data

After collecting volumetric data with the Optotune you can now process your data as follows.

First, use sbxsplit() to generate separate data files for each “slice” of the optotune.

For example, if I have a data file gn7_008_001.sbx collected some data with an optotune waveform having a period of 5 the command

>> sbxsplit('gn7_008_001')

will generate a set of 5 files named gn7_008_001_ot_NNN, where NNN.sbx will range from 000 to 004, corresponding to each separate optical plane.

Second, the resulting files can then be aligned and segmented by treating each plane individually.  This will generate the corresponding *.signals and *.segment files.

Finally, you can call:

>> sbxmerge('gn7_008_001')

This will generate gn7_008_001_merged.signals and gn7_008_001_merged.segment.

The signals matrix will have as many rows as frames were present during acquisition while interpolating the missing samples for each plane (that is, when the optotune was sampling from other planes).

The interpolated signals are then deconvolved as usual to generate an estimate of spiking in the spks matrix.  From here on you can process the data as if it came from a typical experiment where only one plane was sampled.

The mask variable in the segmented file will have a size of [ny nx plane], where [ny, nx] is the size of each frame and plane is the period of the optotune waveform.  Each cell has a unique ID value corresponding to its column in the signals matrix.

sbxmerge.001

Note that each setting of the optotune waveform is treated independently, even though thay may potentially represent the same plane (as it may happen using sinusoidal or triangular z-scanning).

Spatial Calibration for Multiple Objectives

Multiple users of the scope may be running projects that require different types of objectives.  How to keep a spatial calibration for each and switch between them when necessary?

Scanbox now includes an “objective” configuration variable — a cell array of strings each with the name of a different objective.  Right now I have:


% objective list
sbconfig.objectives = {'Nikon 16x','Nikon 25x'};

Now, within the Knobby panel you will see a pull down list containing the objective names:

objectives

Select the desired objective before performing a calibration.  Once the calibration is finished it will be saved.  If a calibration is not present for a given objective the calibration button will read “No Calibration” and the mouse control will be disabled. If you change the objective, simply select the corresponding entry in the pull down list to apply the new calibration (no need to restart Scanbox).

When collecting data the info structure will include the objective name and calibration data:


info =

struct with fields:

resfreq: 7930
postTriggerSamples: 5000
recordsPerBuffer: 512
bytesPerBuffer: 10240000
channels: 2
ballmotion: []
abort_bit: 0
scanbox_version: 2
scanmode: 1
config: [1×1 struct]
sz: [512 796]
otwave: []
otwave_um: []
otparam: []
otwavestyle: 1
volscan: 0
power_depth_link: 0
opto2pow: []
area_line: 1
calibration: [1×13 struct]
objective: 'Nikon 25x'
messages: {}
usernotes: ''

Visualizing Individual Slices during Volumetic Imaging

During volumetric imaging, Scanbox displays all images as they are acquired. This can be inconvenient if we are trying to visually assess the activity of neurons within any one optical slice.  One solution is to display the images separately in a montage by means of a Scanbox plug-in.  However, it would be better to have such an option integrated into Scanbox.  A new feature offers this possibility.

Turning on a  “Slice” checkbox within the Optotune panel will make a pull-down menu within the display appear.  This menu allows selection of the optical slice you want to visualize. Unchecking the slice checkbox allows Scanbox to go back to its normal operation of showing all images in the incoming stream.

Here is a brief demo showing this feature.  Enjoy…

 

 

 

Automatic Control of Laser Power

A new checkbox within the Laser panel (labeled AGC) allows you to turn an automatic control of laser power on and off.

When AGC is on, Scanbox checks the distribution of pixel values on the image every T seconds, and increases or decreases the laser power by a certain factor if the fraction of pixels above a threshold is outside the desired range.

The values of these parameters are found in a new section of the sbconfig.m file:

% Laser AGC
sbconfig.agc_period = 1;            % adjust power every T seconds
sbconfig.agc_factor = [0.93 1.08];  % factor to change laser power down or up if outside prctile bounds
sbconfig.agc_prctile = [1e-5 1e-3]; % bounds on percent pixels saturated wanted
sbconfig.agc_threshold = 250;       % threshold above which is considered saturated (255 is max value)

 

Below is a video showing AGC in action.

At the beginning of the video,  I focus on pollen grains using low power. When the AGC is turned on, it brings the power up. Then, if I increase the PMT gain, the laser power is decreased in response.  If the laser power is changed manually, AGC will re-adjust it to bring the pixel distribution within the desired limits.

 

AGC of laser power is useful when running a z-stack with a range that is larger than 100um or so.  In that case, engaging AGC will make Scanbox adjust laser power as a function of depth. Another situation where AGC may be useful is while running very long experiments/sessions where water may evaporate slowly leading to a reduction of the signal. In that case, turning AGC would compensate and could render the data usable.

 

Automatic Optotune Calibration

We previously explained how to calibrate the optotune manually.  With the introduction of Knobby 2, we are able to make this process automatic.  You will now find a ‘Calibration’ button in the Optotune panel.  To use it, do the following:

  1. Set the optotune slider to its lowest value (slide all the way down)
  2. Bring some pollen grains into focus
  3. Stop focusing
  4. Make sure the data directory has a directory named xx0
  5. Click the ‘Calibrate’ button
  6. Sit back and relax.  Wait for the process to complete.

Knobby will run some z-stack acquisitions for different values of the optotune current setting.  The volumetric data will be used to calculate the shift in z at various values of the current. A panel will display the progress in processing the images (it takes about 2 min). Scanbox will then plot the raw data and a fit by a quadratic polynomial, which may look something like this:

opto_calib

Scanbox will write a calibration file which will take effect next time you start Scanbox.

After restarting Scanbox, you can check the calibration as follows:

  1. Set the optotune slider to its lowest value (slide all the way down)
  2. Set Knobby to super-fine mode
  3. Focus on some pollen grains
  4. Zero Knobby (XYZ)
  5. Move out-of-focus by moving the optotune slider up to some value
  6. Now bring sample back into focus using the z-axis knob
  7. Compare the reading of the z-axis in Knobby’s screen with the depth noted in the optotune panel. These two numbers should match very closely.

 

Click-and-Center with Knobby Mouse Control

If you have been making use of Knobby’s spatial calibration button, you can now move on to interacting with Knobby remotely.

The new Knobby scheduler panel looks a bit expanded.  The speed (coarse/fine/super-fine), the mode (normal/rotate), and zero (XYZ/XYZA) buttons should be self explanatory. They do exactly the same as if you were to be using them on Knobby.  The action you select on the panel will be reflected on Knobby’s screen as well.

knobbymouse

The range, steps and frames entries provide a quick way to edit the scheduler’s table if you intend to do a simple z-stack.  Range is the extent of the distance in z you want to span, steps is the the size of the step (in um), and frames is the number of frames you want to scan at each position.

Finally, there is new checkbox labeled “Mouse control”. When this is active and you are scanning you can easily move around the sample by clicking the cell you want to bring to the center of the screen, as shown by the video example above.  Once you click on a cell, Knobby does the rest. We call this feature click-and-center. This works at all magnifications (even if you change it on the fly). At the same time, scrolling the mouse wheel allows you to focus up and down.  The speed of the movement will be controlled by the selected speed. You may want to start at the slowest speed (Super-fine).

Spatial Calibration and return to origin

We added two new features to the knobby scheduler panel, as shown below by the arrows.

knobby2nf

One (left arrow) allows knobby to perform an automatic spatial calibration of the system, measuring the (x,y) size of pixels at all magnifications.  To perform this calculation do the following:

  1. Focus on some pollen grains.  Make sure one of the pollen grains is well centered on the screen at the highest magnification (x8).
  2. Make you there is an empty folder named xx0 in the data directory you selected.
  3. Hit the calibration button (pointed by the left arrow).
  4. The system will ask you if you want to proceed.
  5. Scanbox will then loop over all magnifications, collecting data for 8 sec and moving the sample by a known distance.
  6. When it finishes, Scanbox will display the optimal value of a resonant gain magnification variable that will make your pixels square.  Note that value.

Now, when you restart Scanbox the system will read the new spatial calibration in the calibration button.  The format is [xsize ysize] [xfov yfov].  The left pair shows the width and height of a pixel (in micrometers), and the right pair shows the width and height of the field-of-view at the current magnification setting (also in micrometers).  In the example above, the (x,y) size is [0.72 0.70] um, meaning the aspect ratio is close to one.

If the aspect ratio is far from one, and you prefer square pixels, do the following:

  1. Change the value of the “gain_resonant_mult” variable in the configuration file to the one suggested by the Scanbox spatial calibration, restart Scanbox.
  2. Re-run the spatial calibration.  Thi will create a new calibration file.
  3. Restart Scanbox to read the newly created calibration file.  Now the aspect ratio should be close to one.

The second feature is one is a checkbox labeled Return. When you run a knobby schedule you have click this box to make knobby return to the initial position it started from. So, for example, upon completion of a z-stack the system will go back to where it started. Note: this feature uses the Store/Recall C function, so if you have anything stored there it will be erased.