Andor Technology Optosplit II User manual

Optosplit II
Image Splitter
User Guide v3.1

Important - Please read before installation
I. Important - Please read before installation
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 (e-mail tech@cairn-
research.co.uk .
The following points should be considered when using the Optosplit for the first time:
1. The Optosplit is supplied pre-aligned and centred, please refer to this manual before making any
adjustments.
Do not adjust smaller knurled screws or recessed hex screw on lid before reading this manual!
2. When changing the filter cube care should be taken not to touch any optical surfaces. If any lenses,
filters, or mirrors are marked then they should be wiped with a lens tissue.
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Package Contents
II.Package Contents
Optosplit Image Splitter
Optosplit Body
Filter Cube(s ____________
2 slot covers
Shutter to occlude either optical path
Auxillary filter/lens holder
Coupling
Rectangular diaphragm with female c mount input
Output focussing adjuster with male c mount output
Filters
Dichroic filter ____________
Short emitter ____________
Long emitter ____________
Dichroic filter ____________
Short emitter ____________
Long emitter ____________
Dichroic filter ____________
Short emitter ____________
Long emitter ____________
Dichroic filter ____________
Short emitter ____________
Long emitter ____________
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III.Table of Contents
I. Important - Please read before installation.......................................................................................... i
II. Package Contents................................................................................................................................... iii
III. Table of Contents................................................................................................................................... v
1 Introduction............................................................................................................................................. 1
2 Installation guide..................................................................................................................................... 3
2.1 Configuring the filter and dichroic mirror holder................................................................ 4
2.2 Installing the Optosplit in the light path................................................................................ 5
2.3 Focussing the camera. ............................................................................................................ 6
3 Operation of the Optosplit................................................................................................................... 7
3.1 Adjusting the position of the images...................................................................................... 7
3.2 Configuring a new filter set...................................................................................................... 9
4 Technical summary............................................................................................................................... 11
5 Technical Support................................................................................................................................. 13

Table of Contents
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Introduction
1 Introduction
In fluorescence imaging applications it is often useful to acquire simultaneous images at two
different emission wavelengths. Traditionally such applications have been restricted by the speed of an
electronic filter changer, or by the cost and complexity of adding a second camera to a system. In many
instances the region of interest does not require the full resolution of the camera so the ideal solution
would be to simultaneously image at two different wavelengths on the same camera chip. In conjunction
with a research microscope and a suitable CCD camera the Cairn Optosplit allows the researcher to do
exactly this.
The Optosplit is usually supplied with a rectangular aperture to define an ROI and includes controls
to allow the two images to be positioned accurately and conveniently within the camera frame. Images
can be acquired using any imaging software and processed either manually off-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.
As supplied, the instrument is configured to attach to the c-mount output port of a research
microscope, with a c-mount CCD camera fitted 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 configuration.
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Installation guide
2 Installation guide
The Optosplit comes supplied with a rectangular aperture, as shown below. This allows you to make the
best use of the camera chip area, but there is the alternative of a circular iris available.
Figure 1 : Optosplit with adjustable rectangular diaphragm
Before installing the Optosplit you will first need to install an appropriate filter cube into the filter
holder. If your Optosplit has been purchased with a full filter set pre-installed then you can skip to
section 2.2 for instructions on fitting it in the optical pathway. Instructions for installing the filter cube are
given in section 2.1.
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2.1 Installing the filter cube
The first stage in configuring the Optosplit is to install the filter set. If you wish to change the
configuration after installation then the instructions here should be followed, but it is not necessary to
remove the Optosplit from the light path. The filter cube position can be accessed by removal of the side
access panel, which is held in place by a set of magnets. The access panel is removed by pulling the two
handles labelled A, shown in figure 2.
Figure 2 : Accessing the filter set
The Optosplit filter cubes accepts 25mm filters and 38x26mm dichroic mirrors. If the cube has not
been supplied with your filter set prefitted, these should be orientated as indicated by the manufacturer.
For our standard filters this is with the arrow pointing towards the sample (i.e. Towards the microscope .
The filters are held in place using the 25mm loking rings supplied, and the dichroics accessed by removing
the four screws holding the two halves of the cube together. The filter cube is fitted to the runner
indicated in figure 3 below.
Figure : Fixing the filters in position
Note: When fixed correctly in the cube the filters sit at a small angle to the optical axis to
prevent reflected light from reaching the detector.
In most applications the dichroic beamsplitter will have long-pass characteristics, so the longer
wavelength of the two filters should be located in the straight through filter position, and the shorter
wavelength filter in the second position. (please refer to the technical summary for details of the light
path . Removal of the dichroic cube is the reverse of the fitting, with the cube sliding out of the mount
when the handle on the cube is pulled towards you.

Installation guide
Note: To control the relative intensity of the two pathways we recommend using neutral density
filtering in the brighter pathway using the auxilliary component mounts (M in fig 2)This
is neccesary only when one image is disproportinately brighter than the other.
Figure 4 : Filter cube in place
2.2 Installing the Optosplit in the light path
Before installing the Optosplit system it is important to first set up the microscope, camera and
software to give a clear image of an object of less than half the size of the camera frame. Ideally this
should be a real sample with appropriate optical properties for the installed filter set. Failing this a
standard brightfield image could be used, but this may lead to arbitrary intensity differences 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 image. Once a clear image can be seen the camera should be switched off
and removed from the microscope. The Optosplit should be fitted onto the microscope with the
aperture orientated towards the C mount port. The CCD camera can then be fitted onto the exit port of
the Optosplit. The exit port of the Optosplit has a rotating ring (green arrow in fig.6 to allow the camera
to be fitted in the same orientation as the Optosplit. This is important to allow the Optosplit to operate
predictably. The correct orientation is for the top of the Optosplit to lie parallel with the top of the
camera, as shown below. If your Optosplit is fitted with a rectangular diaphragm then the image of the
aperture should be upright with no slant.
Figure 5 : Alignment of Optosplit with camera
Having fitted the camera onto the Optosplit it should be possible to see a sharp image of the
aperture on the computer monitor, with the sample in sharp focus within the aperture. During factory set
up the Optosplit is configured with the two images approximately superimposed, so when it is first
installed a single central image should be observed.
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2.3 Focussing the camera on the Optosplit aperture.
Figure 6 : Adjustments to focus the image on the camera
If the Optosplit aperture is not in sharp focus on the camera, then adjust the fine focus on the
camera using the following procedure. Refer to fig.6 above for screw locations:
1. Set a convenient sized aperture using the input aperture levers (less than half the size of the camera
frame , so the edges of the aperture are within the field of view.
2. Slacken the two hex screws that retain the output rotating ring (green arrow to allow the camera to
turn in the C-mount.
3. Separate the two images slightly using the knurled screw facing towards the camera (blue arrow .
4. Slightly loosen the single hex screw, which locks the focussing ring (red arrow .
5. Rotate the focussing ring between the camera and the Optosplit whilst holding the Optosplit and
camera in position until the aperture comes sharply into focus.
6. The camera rotating ring should be locked off with the camera chip orientated precisely in line with
the Optosplit, as shown in the figure in the previous section.
7. The remaining hex screws should then be re-tightened.

Operation of the Optosplit
3 Operation of the Optosplit
The Optosplit is an intuitive device, with a single control for adjusting image separation (blue arrow in fig
7 to allow different sized samples to be used. There are additional controls for defining the field of view,
and image centering which depend on the type of aperture fitted to the Optosplit. The figure below
shows the Optosplit fitted with the rectangular aperture, and its size is adjusted using the pair of levers
indicated by the yellow arrows. If an iris diaphragm is supplied, this is adjusted by rotation of a single lever
in the same location.
Figure 7 : Control positions for aligning the two images
3.1 Adjusting the position of the images.
Once the camera is set up it is useful to familiarise yourself with the operation of the alignment
adjustments. Please note that the diagrams below indicate the images for a system using an appropriately
aligned and centred rectangular diaphragm, if your system has an iris diaphragm then the images will be
shaped accordingly. An image similar to that shown below will be observed when the Optosplit is first
used, with the two images superimposed.
Figure 8 : Superimposed images on aligned system
When the camera is mounted correctly (as described in section 2 above the knurled screw facing
the camera will control the horizontal image separation (blue arrow in fig.7 above , and together with the
aperture levers these are the only controls that will be routinely adjusted during experiments. The
remaining controls on the top-plate all adjust the alignment and centring of the two images and should
not be adjusted unless the filter set has become mis-aligned.
Note: Do not adjust any top plate controls until you are familiar with the basic operation of the
Optosplit and you have referred to section .2 as realignment is not trivial.
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1. Separating the t o Images
Figure 9 : Image separation
Turning the horizontal image separation
adjustment counterclockwise will split the two
images (fig.7 blue arrow .
Note: Only small adjustments are necessary to
separate the images.
2. Adjust vertical position of short avelength image
Figure 10 : Vertical alignment
Should the left and right images be at
different vertical heights, this can be corrected by
adjusting the position of the short wavelength image.
Turning the vertical alignment adjustment (Pre 1
will alter the vertical position of the shorter
wavelength image. Again only very small adjustments
are necessary.
3. Ready to record
Figure 11 : Correct alignment
Once the spectrally resolved images are
located side by side on the camera chip you are
ready to record. When carrying out experiments the
aperture should be set to mask the ROI tightly so
that the two images can be located as closely as
possible on the camera chip.

Operation of the Optosplit
3.2 Centering the Optosplit images on the camera
When the Optosplit is initially installed, or if the filter block goes out of alignment, the Optosplit
images can show significant aberrations, or in extreme cases only a single image may be observable for
any setting of the separation control. In these cases the following procedure will enable the filter set to be
reconfigured. Control locations are shown in figure 7.
1. Define a small area using the Optosplit aperture and centre the shorter wavelength image
horizontally using the separation control (blue arrow, fig.7 .
Note: In a correctly configured Optosplit both images will be superimposed with
the short wavelength image centred.
2. Centre the same image vertically using the vertical offset adjustment on the top-plate of the
Optosplit (Pre 1 .
3. The short wavelength image is now correctly positioned.
4. To centre the long wavelength pathway, we superimpose the longer wavelength image on the
short wavelength image we have just centred. This is done using the knurled screw Pre 3 and hex
screw Pre 4.
5. First we centre the image horizontally by small adjustments of Pre 3. Once the image is exactly
superimposed on the long wavelength image in the centre of the field of view, we can adjust the
vetical position of this image.
Note: If the images are superimposed off-centre then image aberrations can
result, so ensure the short wavelength image is correctly centred before
positioning the long wavelength image.
6. Adjust vertical position using the recessed hex screw, Pre 4.
7. Recheck image horizontal alignment & repeat steps 5 & 6 if required.
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Technical summary
4 Technical summary
The Optosplit uses a conventional dichroic mirror to separate the light into two different spectral
bands. This selection of wavelength is usually augmented by the use of bandpass filters (as shown in the
schematic below.
The design is loosely based on technology described by Kinosita et al. (J.Cell Biol.(1991 115, 67-73 ,
but includes several proprietary features, most notably:
•Rotating mirror cradle to give precise symmetrical control of the degree of separation whilst
maintaining identical path lengths.
•A half-size fully silvered mirror at the output port for recombining the images.
•Fine adjustment controls for camera focus and vertical alignment.
•Optional ROI definition using adjustable rectangular aperture or iris diaphragm.
The system is supplied with high grade AR coated achromatic doublet lenses and dielectrically
coated mirrors for maximum throughput.
Dimensions (H x W x D , in mm, excluding couplings 55 x 110 x 110
Dimensions (H x W x D , in mm, including couplings 55 x 240 x 110
Approximate weight (kg 1.26
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