Cairn OptoSplit III User manual

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Important Information
Please Read Before Installing and
Operating Your OptoSplit III
For maximum reliability we recommend using the equipment within
certain guidelines. If in any doubt, then please feel free to contact our
technical support department who are always willing to help. (e-mail
[email protected]o.uk)
Note: Please do not adjust any control
before reading this manual.
The OptoSplit is supplied pre-aligned and centred, please refer to this
manual before making any adjustments.
When changing the Cairn Filter Cube, care should be taken not to
touch any optical surfaces. If any lenses, lters, or mirrors are marked
then they should be wiped with a clean lens tissue.

Contents
Introduction
Installation guide
Installing Filters into the Cairn Filter Cube
Installing Dichroic Mirrors into the Cairn Filter Cube
Inserting the Cairn Filter Cube into the Optopsplit III
Auxilliary Component Mounts
Installing the OptoSplit III in the Lightpath
Operation of the OptoSplit III
Adjusting and Locking the Aperture
Adjusting the Position of the Images
Focussing the Camera on the Aperture
Dual and Single Image Modes of Use
Locating the OptoSplit Images on the Camera
Centering Images on the Camera
Image Optimisation (Trim Adjustment)
Technical Summary
Technical support
1
2
2.1
2.2
2.3
2.4
2.5
3
3.1
3.2
3.3
3.4
3.5
3.6
3.7
4
5
1
2
2
3
3
5
5
6
7
7
10
11
12
15
16
17
18

Page 1
1 Introduction
In uorescence imaging applications it is often useful to acquire simul-
taneous images at multiple emission wavelengths. Traditionally such
applications have been restricted by the speed of an electronic lter
changer, or by the cost and complexity of adding extra detection hard-
ware. In many instances the region of interest (ROI) does not require
the full resolution of the camera, so the ideal solution would be to
simultaneously image at dierent wavelengths on the same camera
chip. In conjunction with a research microscope and a suitable CCD
camera the Cairn OptoSplit III allows the researcher to do exactly this.
The Cairn OptoSplit III is usually supplied with a rectangular aperture to
dene a ROI and includes controls to allow up to three images to be
positioned accurately and conveniently within the camera frame.
Images can be acquired using any imaging software and processed
either manually o-line or using an appropriate analysis tool such as
the Splitview module in Universal Imaging’s Meta series software or
Field Split in Andor Bioimaging’s iQ.
The instrument is usually congured to attach to the c-mount output
port of a research microscope, with a c-mount CCD camera tted to its
output. The design allows for connection to a variety of alternative
devices, so please consult with us if you intend using it in any other
conguration.
Installation of the OptoSplit III in the light path is straightforward and
does not require any special tools. Should you wish to make use of the
full input image without splitting (non-split mode), then exchanging
the triplecube for the bypass cube, and a simple adjustment of the
input diaphragm will permit this. We do however recommend removal
of the OptoSplit from the light path when the splitting mode is not
required to ensure the maximum light throughput to your detector.

Page 2
2 Installation Guide
2 .1 Installing Filters into the Cairn Filter Cube
Before using the OptoSplit III image splitter you will rst need to install
the appropriate set of lters and mirror for your application into the
Cairn Filter Cube. Instructions for these procedures are in sections 2.1
and 2.2. Always take extreme care when changing or adjusting the
lter and mirror sets to avoid damage or soiling. If you purchased your
OptoSplit III with a full lter set pre-installed, then you may wish to go
to section 2.4 as the following may not be pertenant.
The Cairn Filter Cube has spaces for
three 25mm lters. These are held in
place by locking rings which can be
removed using the tool provided. To
t a lter, remove the locking ring
and place the lter orientated so
that the arrow points into the Cairn
Filter Cube and towards the light
path. Then simply replace and
tighten the locking ring.
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Turn the locking
ring anticlockwise
to remove it.
Carefully place
the lter inside
the recess.
Replace the locking
ring and rotate it
clockwise to secure.
The arrow should be orientated
into the lter cube and towards
the light path.
Long Dichroic
(1st Position)
Short Dichroic
(2nd Position)
Short Wave FilterMiddle Wave Filter
Long
Wave
Filter
(View from bottom
of cube)

Page 3
In most applications, the dichroic beamsplitter(s) will have longpass
characteristics, so the longer wavelength of the lters should be
located in the straight through lter position, and the shorter
wavelength lters in the second/third position.
2 .2 Installing Dichroic Mirrors into the Cairn Filter Cube
To t your dichroic mirrors into the Cairn Filter Cube remove each of the
eight screws that secure the three sections together. You will then be
able to gently pull the cube apart. There is one steel locating rod in
each part of the cube to ensure the correct alignment when it is
reassembled. Once the sections of the cube are seperated remove the
dichroic holder and place the dichroic mirror(s) (active side down) into
the rectangular recess(es). Then simply replace the holder and
reassemble the cube and tighten the screws.
2.3 Inserting the Cairn Filter Cube into the OptoSplit III
The Cairn lter cube is designed to be easily accessible in order to
facilitate quick and easy changes of lter sets. Access to the lter cube
mount is gained by removing the access panel using the two handles
to pull gently and rmly. The lter cube mount will now be visible. The
mount attached to the rear of the Cairn Filter Cube is designed to mate
with the bracket on the internal wall of the OptoSplit III, so that the
small handle on top of the cube will be facing out. Once the Cairn Filter
Cube is mounted successfully, replace the access panel using the two
handles. (See over for diagram.)
Remove the 8 screws and
seperate the three sections. Remove the dichroic holders and
carefully place the mirror(s) in
the recess(es) active side down.
Re-assemble the cube
and replace the screws.

Page 4
Remove the access panel using the
two handles.
Mounting
bracket
Replace the access panel using the
two handles.
Mount the lter cube onto the brackect on the interior of the OptoSplit II.

Page 5
2.5 Installing the OptoSplit III in the Light Path
Before installing the OptoSplit III unit it is important to rst set up the
microscope, camera, and software to give a clear image of an object of
less than half the size the camera frame. Ideally this should be a real
sample with the appropriate optical properties for the installed lter
set. Failing this a standard bright eld image can be used, but this may
lead to arbitrary intensity dierences between the spectrally resolved
images.
First the CCD camera should be mounted on the
microscope C-mount output and the port adjusted
to give the sharpest possible image. Once a clear
image can be seen the camera should be switched
o and removed from the microscope.
The OptoSplit III should then be tted on to the
microscope with the diaphragm
orientated toward the output of
the microscope. The camera can
then be xed onto the output
port of the OptoSplit III, with the
top orientated so that the top of
the camera lies parallel with the
top of the OptoSplit III. If you are
tting onto a vertical mount then
the tops should still be orientated
in the same direction.*
2.4 Auxilliary Component
Mounts
To control the relative intensity of
the two pathways we recommend
using neutral density ltering in the
brighter pathway using the auxilliary
component mounts. This is required
only when there is a signicant
dierence in brightness between
the images. Auxilliary component mounts.
Top of camera
V1
V3 H3
V2 H2

Page 6
* A 1mm nylon spacer is also included with each
unit. This may be required on the camera output
mount, particularly when mounting a shutter in
addition to the OptoSplit.
Having tted the camera to the output port of the
OptoSplit III it should be possible to adjust the
image to see a sharp picture of the aperture edges
with the sample in focus. The image should line up
with the edges of the aperture and should not have
any rotation or slant in either direction.
3Operation of the OptoSplit III
The OptoSplit III uses a single control for adjusting image separation
and allows for dierent sizes of samples to be used. There are
additional controls for rening the ROI, and centering the image.
The image should
not have any slant.
Image.
Image.
TRIM
V1
V3 H3
V2 H2
Split adjuster
V2/H2 adjusters
V3/H3 adjusters
V1 adjuster
Aux. component
mounts
Clamp Screw
Trim Controller
Split adjuster

Page 7
3.1 Adjusting and Locking the Aperture
The OptoSplit III is supplied with an adjustable rectangular aperture
that allows the user to determine the ROI both vertically and
horizontally. Once the ROI has been dened, the aperture can be
locked in place using the aperture adjusters. (See diagram below.)
In order to adjust the ROI, adjust each of the two levers on the input
port of the OptoSplit III until the desired area is dened on the camera.
If you wish to lock the aperture in place, then tighten the levers by
rotating them clockwise, and to loosen them rotate anti-clockwise.
3.2 Adjusting the Position of the Images
For successful image separation it is necessary to rst dene the eld of view,
and ensure the three optical pathways are overlaid.
It is possible to separate the images by rotating the Split control in the
clockwise sense, however image distortion will be evident when overlaying
the two halves of the image.
The imaging procedure that follows assumes a Triple Filter Cube is installed
and the full image is being monitored.
To begin, set a convenient sized aperture using the input aperture levers so
the edges of the aperture are just visible within the eld of view vertically, and
restrict the eld to just under 33% of the horizontal dimension.
It is also important to note that here the reected image (that with the
shortest wavelength), is depicted as being to the right of the screen. You will
be able to identify this image easily after seperating all three as it will move
with adjustments to the V1 control, but not adjustments to the V2 control.
Aperture adjusters
Aperture
mechanism
Turn the aperture handles
clockwise to lock and unlock
the aperture adjusters

Page 8
V1
V3 H3
V2 H2
Split adjuster
V1 adjuster
V3 adjuster V2 adjuster
H3 adjuster H2 adjuster
Here we see the three superimposed
images. Although your image may be
monochromatic, colours have been used
here to dene the three images.
Turning the Split control anti-clockwise
will seperate the reected, and transmit-
ted images symmetrically on the
horizontal axis, whilst the Centre image
will not deviate.
Note: To prevent calibration of the
OptoSplit III o-axis, the Reected (or
short wavelength) image has no
independent horizontal controls, so it is
important that it is adjusted into
position rst, and the Centre, and
Transmitted images are calibrated to it.
Transmitted
image
Centre
image
Reected
image

Page 9
Turning the V1 controller will adjust the
vertical position of the Reected, and the
Centre images, the remaining image is
the Transmitted image which should be
on the left side, if it is on the right, then
adjust the Split control until the outside
images are exchanged
Adjustments to the V2 controller will
move the Centre image along the
vertical axis, and allow for adjustments
to be made independently of the
Reected image.
Adjustments to the H3 controller will
move the Transmitted image along the
horizontal axis.
Adjustments to the H2 controller will
move the Centre image along the
horizontal axis.
When the spectrally resolved images are
side by side on the camera chip you are
ready to record. When carrying out
experiments the aperture should be set
to mask the region of interest tightly so
that the images are located as closely as
possible on the camera chip.

Page 10
3.3 Focussing the Camera on the Aperture
If the OptoSplit III aperture is not in sharp focus, then adjust the ne
focus on the camera as follows:
1. Set a convenient sized aperture using the input
aperture levers so the edges of the aperture are within
the eld of view. Block one light path by tting the
shutter plate provided into the one of the Aux mount
positions.
2. Slacken the two hex screws that retain the output
rotating ring to allow the camera to turn in the
C-mount.
3. Slightly loosen the single hex screw, which locks the
focussing ring.
4. Rotate the focussing ring between the camera and
the OptoSplit III whilst holding both in position and
until the aperture comes sharply into focus.
5. The camera rotating ring should be locked o with
the camera chip orientated precisely in line with the
OptoSplit III, as shown in the gure in the previous
section.
V1
V3 H3
V2 H2
Hex screws
Split control
Focussing ring
Output
rotating
ring
Aperture levers

Page 11
6. The remaining hex screws should then be
re-tightened & the shutter plate removed.
7. If the microscope image is in focus down the eye-
pieces, but blurred when viewed through the
OptoSplit III with the aperture is in focus, then the
microscope C-mount is not parfocal with the eye-
pieces. The side port should be adjusted as described
by the microscope manufacturer if parfocality is
required (recommended), or the microscope focus
control can be used to independently bring the image
into sharp focus on the OptoSplit III eld aperture.
3.4 Dual and Single Image Modes of Use
The OptoSplit III can be setup in single channel or two channel
(OptoSplit) modes, by simply exchanging the lter cube & adjusting
the eld aperture. Single image or “bypass” mode is the simplest of
these, only requiring the eld diaphragm to be fully opened to enable
operation in full frame mode. When using the OptoSplit III with the
regular OptoSplit II cubes for dual image splitting, the setup is similar
to that described above for three channel operation, but the second
reected image is not present. To fully align an OptoSplit II cube in the
OptoSplit III, the adjustment sequence is given below:
1. Set a convenient sized aperture using the input
aperture levers so the edges of the aperture are just
visible within the eld of view vertically, and restrict
the eld to just under 50% of the horizontal
dimension.
2. Identify the image that is reected by the cube
dichroic mirror by adjustment of the V1 vertical
control.
3. Rotate the separation control counter-clockwise until
the reected image outer edge is located just inside
the edge of the sensor.

Page 12
4. Adjust the vertical position of the reected image
using V1 to centre the image. The vertical eld aper-
ture size can be adjusted at this point to optimise the
sensor use.
5. Adjust the vertical height of the transmitted image
using V3 to centre.
6. Adjust the horizontal position of the transmitted
image using H3 so the outer edge of the image is just
inside the sensor edge. If the images overlap, or there
is a large gap between them, then adjust the horizon-
tal eld aperture size to optimise the sensor use, then
readjustment of the split control will allow the outer
edges of the images to be repositioned.
3.5 Locating the OptoSplit images on the camera
If the OptoSplit III controls have been badly misaligned, the images can
show signicant aberrations, or in extreme cases only a single image
may be observable for any setting of the ‘Split’ control. In these cases
the following procedure will enable the device setup to be recong-
ured.
Locating the Reected Image
1. Fit the system calibration cube & shutter the transmit-
ted and centre illumination pathways using the shutter
Direction
of light
Reected image
Centre image
Transmitted image
Split control

Page 13
4. Adjust the vertical position of the reected image
using V1 to centre the image. The vertical eld aper-
ture size can be adjusted at this point to optimise the
sensor use.
5. Adjust the vertical height of the transmitted image
using V3 to centre.
6. Adjust the horizontal position of the transmitted
image using H3 so the outer edge of the image is just
inside the sensor edge. If the images overlap, or there
is a large gap between them, then adjust the horizon-
tal eld aperture size to optimise the sensor use, then
readjustment of the split control will allow the outer
edges of the images to be repositioned.
3.5 Locating the OptoSplit images on the camera
If the OptoSplit III controls have been badly misaligned, the images can
show signicant aberrations, or in extreme cases only a single image
may be observable for any setting of the ‘Split’ control. In these cases
the following procedure will enable the device setup to be recong-
ured.
Locating the Reected Image
1. Fit the system calibration cube & shutter the transmit-
ted and centre illumination pathways using the shutter
plates provided.
2. Dene a small area using the OptoSplit input aperture
diaphragm and ensure the input to the OptoSplit is
illuminated.
3. Attempt to centre the reected image pathway
horizontally using the ‘Split’ control to scan the
position range.
4. If no aperture edges can be located for any position
of the ‘Split’control, enlarge the input aperture and
repeat step 3.
5. Once the image of the input aperture has been
located and centred horizontally, the vertical align-
ment of the aperture can be set using the V1 control.
6. Adjust the input aperture size so that it just underlls
the camera eld of view vertically, and just under one
third of the camera eld horizontally.
Locating the Transmitted Image
1. Move the shutter from the transmitted to the
reected illumination pathway, the calibration cube
should remain in place.
2. With the reected image located, it is likely the
transmitted image will be seen somewhere in the eld
of view. If this is the case, small adjustments of the H3
and V3 controls should be used to adjust this image to
mirror the location of the reected image on the
camera chip.
3. If no image is seen in the eld, we will again scan the
image horizontally. For the transmitted image this is
achieved using the H3 control.
4. If after scanning the horizontal axis no image can be
found, again open the input aperture slightly & repeat
step 3.
5. Once the edges of the aperture have been found,
reduce the input aperture as for step 6 above, and
centre the image in the camera eld using H3 and V3

Page 14
Locating the Centre Image
1. Move the shutter from the centre channel to the
transmitted illumination pathway, the calibration
cube should remain in place.
2. With the reected & transmitted images located, it is
likely the centre image will be seen somewhere in the
eld of view. If this is the case, small adjustments of
the H2 and V2 controls should be used to adjust this
image to mirror the location of the reected image on
the camera chip.
3. If no image is seen in the eld, we will again scan the
image horizontally. For the centre channel image this
is achieved using the H2 control.
4. If after scanning the horizontal axis no image can be
found, again open the input aperture slightly & repeat
step 3.
5. Once the edges of the aperture have been found,
reduce the input aperture as previously, and centre
the image using H2 and V2.
Aligning the Split Images
1. Remove the shutters from all pathways, keeping the
calibration cube in place. The images should be
approximately centred and overlaid at this point, so
the image splitting will occur centred on the system
optical axis.
2. Separate the images by rotating the “split” control
counterclockwise until there are sited adjacent to
each other and separated by a narrow dark band.
3. Fine adjustment of the images should be carried out
as described in section 3.2
The image splitter is now correctly aligned and ready
for use

Page 15
3.6 Image Optimisation
(Trim Adjustment)
Vignetting is when the image
appears darker towards one
edge. This eect can be easily
corrected by adjusting the trim
control. To adjust the trim
control, loosen the clamp screw
on the underside of the OptoSplit
III by a quarter of a turn in order to
free the slider which controls the
trim control. Gently move the
slider until the vignetting is eliminated. If the slider is moved too far
then the vignetting will become apparent on the other image and will
need to be adjusted in the opposite direction until both images are the
same intensity. It is important to remember to re-tighten the clamp
screw once the adjustment is complete.
If the OptoSplit III has been congured into single channel (non-split)
mode and Trim adjustment is required to compensate for vignetting
being observed, it is likely the slider will need to be at the extreme of its
range of movement to either end.
Vignetting is a darkening of the image
towards one edge.
Clamp Screw Trim Controller
TRIM