Photostimulation Module

Overview

Label Name Function
A Image display area Displays image based on selected tab (G) and allows to add free-hand stimulation points (point and click)
B 3×3 stimulation grid Use the Grid Parameters (S) to define stimulation grids
C Free-hand stimulation points Example of a free-hand selection of stimulation points
D Green bounding box Defines the accessible area for stimulations
E Grid overlay The grid overlay is controlled directly by the active SciScan window
F Image display tools Select the magnifying glass for digital zooming in or out (press shift) or select the point tool for defining stimulation points in the image
G Channel Select Switches between available channels
H Image information Information about current image resolution, digital zoom status, intensity and location of the selected pixel (hover mouse over image area (A))
I Reset Zoom Resets the digital zoom to fill the available image area
J Status Provides information about the current calibration and stimulation protocol (e.g. invalid values in the Stimulation Protocol (R) need to be corrected before a stimulation can be started – “Stim and Image” (K) and “Stimulate” (L) buttons will be deactivated)
K Stim and Image Starts a Galvo or Resonant image acquisition and a parallel photo stimulation (data will be saved – file path and name need to be set up in the SciScan window)
L Stimulate Starts a single photo stimulation without imaging according to the specified Stimulation Protocol (R)
M General Stimulation Parameters Defines the stimulation parameters for the next stimulation point selection (parameters can be modified for single points in the Stimulation Protocol (R) retrospectively)
N Imaging Path Shutter Indicates state of imaging path shutter, open or closed, state can be manually changed if not locked on the SciScan window
O Stimulation Path Shutter Indicates state of stimulation path shutter, open or closed, state can be manually changed if not locked on the SciScan window
P Calibrate Opens the calibration user-interface for matching the stimulation path with the imaging path (this button blinks red when the current image has not been calibrated – Calibrate to proceed)
Q File Operations Load or save Stimulation Protocol (R)
R Stimulation Protocol Stimulation Protocol example (note, that red cells indicate invalid values)
S Grid Parameters Define stimulation grids (current Stimulation Protocol (R) will be flushed generating a new grid)
T Protocol Parameters Additional timing parameters for the Stimulation Protocol (R)
U Protocol editor Defined stimulation points in Stimulation Protocol (R) can be enabled, disabled, reversed, randomized or deleted
V Wait for external trigger Starting a stimulation by clicking “Stim and Image” (K) or “Stimulate” (L) button waits for an external trigger when ticked (please refer to the HyperScope wiring guide for trigger input specifications)
W Protocol information Provides information about duration of a stimulation group and the total stimulation protocol
X Build Waveform & Estimate Timing Updates the currently defined Stimulation Protocol (R) for the hardware (as default it’s automatically updated with every newly added stimulation point)

Dual Light Path Calibration user-interface

Label Name Function
A Raw Image Display Displays either an Imaging Path image (Galvo or Resonant) or a Stimulation Path image (use Get Ref Image (E) to  pull the current image from the SciScan window)
B Image Display Selector Switches between Imaging Path image and Stimulation Path image display (A) – Image Histogram (C) switches accordingly
C Image Histogram Image Histogram of the active image in (A)
D Calibration Summary Summarises the current calibration status and what type of images are available for calibration
E Get Ref Image Pulls the current image from the SciScan window (Imaging Path GalvoImaging Path Resonant or Stimulation Path Galvo image)
F Calibration File I/O Load, Save or Delete calibration file
G Reset Zoom Resets the digital zoom to fill the available image area
H Image Overlay / Pixel Mapping Switch between the Image Overlay display and the Pixel Mapping graph
I Overlay channel selection Select the display of the Red and/or Blue overlay channel
J Image Overlay Display Overlay of the currently pulled Imaging Path (Blue) and Stimulation Path (Red) image
K Y-Offset (Pixels) Translate the Stimulation Path (Red) image in the Y-axis to match it to the Imaging Path (Blue) image (= Calibration step I)
L Image Overlay Information Information about the overlay image
M X-Offset (Pixels) Translate the Stimulation Path (Red) image in the X-axis to match it to the Imaging Path (Blue) image (= Calibration step II)
N Linear Zoom Scale the Stimulation Path (Red) image in the X- and Y-axis to match it to the Imaging Path (Blue) image (= Calibration step III)
O Estimate Zoom Optional for support match the scaling of both images (can be done in conjunction with Calibration step 3)
P Sinusoidal Phase Correction (radians) Compensation of the nonlinear compression/stretch of the Resonant scanner in the Stimulation Path (Red) image (= Calibration step IV, Resonant only)

Dual light path calibration procedure (RGG+GG, RG+GG and GG+GG system only!)

IMPORTANT Note, that the calibration depends on the current scan parameters and the selected Imaging Path. Another calibration needs to be done when one of these changes.

If a calibration for a specific combination does not exist, the photostimulation module will indicate this as follows:

  • Image display area (A) greyed out and inaccessible for adding stimulation points
  • “Stim and Image” button (K) and “Stimulate” (L) button will be deactivated
  • “Calibrate” (P) button blinks red
  • Status (J) will display the message: “Calibration unavailable for current scanner settings! Click on ‘Calibrate’ to start”

 

However, the calibration files “GalvoCalibration.ini” and “ResoCalibration.ini” in “[SciScan root path]\Parallel Uncaging Module\” contain the latest saved calibrations of different scan configurations, that means once a specific scan configuration has been calibrated, photo stimulation will be available.

 
Step Calibration preparation – Part I: Imaging Path
1) Start-up a SciScan imaging path: Either Imaging Path Resonant or Imaging Path Galvo. This guide will show the calibration for a resonant image, as it comprises one more step.
 
2) optional Optional (recommended for RGG+GG systems): The arrangement of scan mirrors inside the HyperScope Imaging Path physically will force an inverted x-axis, comparing Imaging Path Resonant and Imaging Path Galvo.

To match both Imaging Path Resonant and Imaging Path Galvo, the Galvo image needs to be inverted on the x-axis. To do so, tick x.invert.image in the SciScan AO tab for the Imaging Path Galvo, but NOT for the Stimulation Path Galvo:

 
3) Set up an image (press “FOCUS” or “RECORD” on the SciScan window) with a preferred specimen (Pollen Grains recommended), to calibrate the photo stimulation on the Stimulation Path to the Imaging Path acquisition.
4) Start the Photostimulation Module from the SciScan Menu (top left corner of the SciScan window):
 
5) Open the Dual Light Path Calibration module by clicking on the “Calibrate” button in the Photostimulation Module (top right corner):
 
Note, that the “Calibrate” button is NOT available on the Stimulation Path, because this path does not require any calibration for photo stimulations without parallel imaging!

To access the Dual Light Path Calibration module, switch to either Imaging Path Resonant or Imaging Path Galvo.

6) Adjust the position of the specimen with the XYZ stage.

Position an unambiguous structure with high contrast into the exact centre of the image. Use the Grid Overlay (E) on the SciScan window if needed – 1×1 grid gives a perfectly centred crosshair.

In addition, unambiguous high contrast structures are required in the whole image periphery at the same time. The more the better the calibration. Note, that moving the stage after this step again will lead to an invalid calibration, as exactly the same position must be imaged on the Stimulation Path later.

7) To increase the signal-to-noise ratio of the image, activate the display averaging function on the SciScan window “avg type”.

Set the number of frames to average “avg of” to a reasonable value (e.g. 30 for fast Resonant acquisitions).

The more frames being averaged, the higher the image quality, the better the calibration. To see an effect, acquire the amount of specified frames to average again (“FOCUS”).

8) Double check scan parameters and position of the specimen. Last chance for modifications (repeat the steps above, if required)!
9) Pull the current Imaging Path image from the SciScan window into the Dual Light Path Calibration module by clicking on the “Get Ref Image” button (E).

For live images activate the “Auto Refresh?” function (usually not required). Keep in mind that the last displayed image will be used (“FOCUS” again, if a fresh image is required)!

 
10) Switch to the Stimulation Path Galvo on the Main UI as follows:
 
Step Calibration preparation – Part II: Stimulation Path
1) Match the image resolution to the one used for the Imaging Path image on the SciScan window:
  OR

 

2) Set up an image (press “FOCUS” or “RECORD” on the SciScan window).

Keep in mind – do not move the XYZ stage, as this invalidates the calibration!

To increase the signal-to-noise ratio of the current Stimulation Path image as well, activate the display averaging function again (see Calibration preparation – Part I: Imaging Path step 7).

3) Pull the current Stimulation Path image from the SciScan window into the Dual Light Path Calibration module by clicking on the “Get Ref Image” button (E).

For live images activate the “Auto Refresh?” function (usually not required). Keep in mind that the last displayed image will be used (“FOCUS” again, if a fresh image is required)!

 
Step Dual Light Path Calibration
Note Once an image for the Imaging Path and the Stimulation Path are loaded into the Dual Light Path Calibration module, they can be matched to each other, using two different levels of manipulation for Galvo imaging and an extra one for Resonant imaging:

  1. Image translation in x- and y-axis (step I and II)
  2. Linear image scaling in x- and y-axis (step III)
  3. Nonlinear image scaling in x-axis (Resonant imaging only, step IV)

Those manipulations are applied to the Stimulation Path image in order to map the stimulation positions to the correct pixel positions in the Imaging path.

Note Before the actual calibration (step I to III/IV), make sure the Red and Blue channels are set up properly.

To do so, activate both channels (both ticked in the Overlay channel selection (I), see Dual Light Path Calibration). Adjust the contrast of each channel individually using the Image Histogram (C). The unambiguous structures need to be displayed with a high contrast in both channels, as shown here:

vs.

Deselect the “auto scale” and move both ends of the slider, until the contrast is optimal for each channel. To switch between the Image Histogram (C) for the Red (Stimulation Path) and the Blue (Imaging Path) channel, use the Image Display Selector (B):

 

I Translate the Stimulation Path (Red) image in the Y-axis to match it to the Imaging Path (Blue) image
 
II Translate the Stimulation Path (Red) image in the X-axis to match it to the Imaging Path (Blue) image
 
III Scale the Stimulation Path (Red) image in the X- and Y-axis to match it to the Imaging Path (Blue) image
 
IV Compensate the non-linear compression/stretch of the Resonant scanner in the Stimulation Path (Red) image
 
Note It might be necessary to do several iterations of steps I-IV to achieve a good calibration! Be patient – the better the match, the more precise the calibration! Use the sliders for coarse adjustments and the digital display and the increment/decrement buttons for fine tuning. If need be, change the last digits in the digital display for extra fine tuning.
Result  
Note Don’t forget to save the calibration, otherwise it was for nothing!
 
To save Time

Whilst there is a Stimulation Path image available, one can do the calibration for the other still uncalibrated Imaging Path (RGG+GG systems only). Just get another image from the respective path (in this example this would be the Imaging Path Galvo) and calibrate again following the steps above.

 

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