EJP-00266-2008 – revision
SUPPLEMENTAL MATERIAL
1. Evaluation of pulsed-laser performance
Laser performance can be evaluated using a power meter to measure output power (e.g. ThorLabs S-110 with S212-A detector or Newport Handheld Power Meter 840-C with series 818 detectors) and a spectrometer (e.g. Avantes fiber optics spectrometer USB S2000/PC2000) to determine the spectral bandwidth of the pulses. The latter is an important diagnostic because the spectral bandwidth is inversely related to pulse width (tp); the shorter the pulse width, the broader the bandwidth (BW) such that with
, (S1)
where lc is the center wavelength and c is the speed of light. The inverse relationship between pulse width and bandwidth arises because the Fourier synthesis of shorter pulses requires a larger number of different-frequency sinusoids than the synthesis of longer pulses.
2. Mathematical analysis of the optical arrangement of the stimulus projection
The so-called "ABCD" matrix method (Siegman, A.E., 1986, Lasers, University Science Books, Sausalito, CA) exploits the fact that in the paraxial approximation (rays close to the optical axis) a 2 by 2 matrix can be used to calculate how position and angle of any light ray change as it propagates through an optical system.
To model the overall paraxial beam path we used the following matrix product
, (S2)
with free space propagation over a distance d in a material with index n represented by
, (S3)
a lens of focal length represented by
, (S4)
and the focal lengths of objective, tube lens, scan lens, projection eye piece, and focus-adjustment lens represented by, , ,,, and, respectively. The distances between optical elements are given by etc. is the focus distance, with being the focus shift. is the displacement of Leye from the position for that . In order for there to be an actual image of the LCoS at the distance we need since where a ray that starts at the LCoS comes out at a plane located at should only depend on the point but not the angle of origin. We further use , ,,, and , due to the construction of the microscope, and , since tube and scan lens together need to form a telescope. This leaves as parameters still to be determined , , . If we want the stimulus to be focused onto the 2P image plane () the image of the LCD must be at infinity after the eyepiece, i.e. for the matrix
(S5)
the lower right element has to be zero () since for the image to be at infinity the exit angle of a ray must not depend on the angle it leaves the LCoS. A further condition is given by the required demagnification of the LCoS chip, which displays 800x600 pixels with a pitch of 12.0 µm horizontally and 12.3 mm vertically, resulting in an active area of 9.6 mm (H) x 7.4 mm (V). If we set dfal-LCD = 73.6 mm the image if the LCD is (de)magnified by a factor of 0.122. Both magnification and the parfocal condition are independent of the distance between the eyepiece and the scan lens () if stimulus and laser focus are parfocal. If the stimulus focus is shifted from the laser focus the magnification does change (from 0.122 for the parfocal case to 0.167 and 0.210 for a stimulus planes 100 and 200 mm below the laser focus, respectively, which requires the focus-adjustment lens to be moved by 4.50 and 6.95 mm towards the LCoS chip). These demagnification factors are sufficient to ensure that for the LCD chip size of 10x8 mm an area of more than ~1mm in diameter can be stimulated.
3. Alignment of the visual stimulator
Our procedure for aligning (in xyz directions) and scaling the stimulus uses the accessory optics (see main article section 2.2.5) and is carried out with a water-filled recording chamber placed onto the stage of the microscope. We assume that the laser-scanned field of view and the objective-side CCD share a common center. To define a reference position that can be viewed with both cameras (cf. 2.2.5) a glass electrode tip is brought down to the bottom of the recording chamber and positioned (using the objective-side camera; see main article section 2.2.5 and Fig. 5C) such that its tip coincides with that common center. It is crucial that the electrode is not moved for the remainder of the alignment procedure.
In the next step condenser-side camera (the IR LED of the trans-illuminator is moved aside and switched off, see 2.2.5) and LCoS illumination LED(s) are switched on and light from the stimulator is allowed to reach the chamber by sliding M7 (DMvStim) into the beam path while removing M8 (DMcam; see Fig. 3B and 2.2.5). The condenser-side camera is then aligned with the objective axis by moving the condenser in x, y, and z until the tip of the electrode is centered and focused on the condenser-side camera. Depending on the setting of the focus adjustment lens Lfal (see 2.2.2) the stimulus is not necessarily in focus. In fact, its focal plane should be below the bottom of the chamber. To calibrate the stimulus focus location the objective is moved upwards by the desired z offset between stimulus and laser foci (typically around 100 µm) and the position of Lfal is adjusted along the optical axis until the test stimulus (a cross hair at the stimulator center as well as rectangles of defined sizes is in focus; Fig. 5D).
Next the cross hair is shifted (using the visual stimulation program QDS) until it is aligned with the electrode tip in software. Only a minor (< 20% of the field width) shift should be needed. Larger shifts indicate a coarse misalignment of the stimulus arm. (In this case it should be verified that the stimulus beam exiting the eyepiece hits M7 at a 22.5° angle, is parallel to the optic table and enters the center of the scan lens.) The last step is to calibrate the stimulus scaling. This is done by displacing the objective by a known distance (using the position readout of the manipulator moving the objective). The resulting shift of the stimulus image in the condenser-side camera provides the stimulus scaling.
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