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
BW
c 2
ct p
,
(S1)
where c 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
M tot  S  w, nwater   L  f obj   S  d obj tl ,1  L  ftl   S  dtl  sl ,1  L  f sl  
S  d sl ep ,1  L  f ep   S  d ep  fal   fal ,1  L  f fal   S  d fal  LCD   fal ,1
,
(S2)
with free space propagation over a distance d in a material with index n represented by
1 d 
n,
S  d , n  
0 1 


a lens of focal length f represented by
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(S3)
EJP-00266-2008 – revision
 1
L f   
 1/ f
0
,
1
(S4)
and the focal lengths of objective, tube lens, scan lens, projection eye piece, and focusadjustment lens represented by, f obj , f tl , f sl , f ep , and f fal , respectively. The distances
between optical elements are given by d obtl etc. w is the focus distance, with w  f obj
being the focus shift.  fal is the displacement of Leye from the position for that w  f obj  0 .
In order for there to be an actual image of the LCoS at the distance w we need
Mtot [1, 2]  0 since where a ray that starts at the LCoS comes out at a plane located at w
should only depend on the point but not the angle of origin. We further use f obj  9mm ,
ftl  200mm , f sl  50mm , f ep  39mm , f fal  50mm and d ob _ tl  95mm , due to the
construction of the microscope, and d tl _ sl  f tl  f sl , since tube and scan lens together
need to form a telescope. This leaves as parameters still to be determined d sl ep , d ep  fal ,
d fal  LCD . If we want the stimulus to be focused onto the 2P image plane ( w  0 ) the image
of the LCD must be at infinity after the eyepiece, i.e. for the matrix
M ep  L  f ep  S  d ep  fal  L  f fal  S  d fal  LCD 
(S5)
the lower right element has to be zero ( M ep [2, 2]  0 ) 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 m 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 ( d sl ep ) 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 m 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 ~1 mm in diameter can be stimulated.
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EJP-00266-2008 – revision
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 laserscanned 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 objectiveside 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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