Cairn OPTOSPLIT II BYPASS User manual

Manual
OPTOSPLIT II
BYPASS
This guide details initial set up and installation of your OptoSplit II Bypass (BP) image splitter. Each unit is serial numbered,
calibrated and QC’d prior to delivery, therefore minimal setup is required providing you are familiar with the key controls.
For the purpose of this manual, all words in bold blue refer to parts labelled in diagrams throughout this document.
A supplementary Troubleshooting Guide is also available to address frequently asked questions during OptoSplit operation.
Please do not hesitate to contact us if you have any questions or issues during installation or operation
([e-mail]).
1) Mounting to your microscope
It is best practice to firstly ensure you have optimised your microscope light source in the absence of the
OptoSplit, with the camera mounted directly on the c-mount. Many technical queries are raised relating to
uneven illumination, which is often not introduced by the image splitter. Once both your transmitted and
fluorescent light source have been correctly aligned and focussed, the OptoSplit can be mounted on a 1x
c-mount; for deviations from this, please contact us for advice.
1.1) Correct camera orientation
The OptoSplit output port connects to your camera c-mount via a camera mounting thread. The silver
knurled section on the output port can be rotated to secure the camera, whilst also ensuring any cables
connected do not get tangled.
The OptoSplit input port then connects to your microscope c-mount.
email: [email protected].uk [e-mail]
[phone] www.cairn-research.co.uk
For correct alignment of the two emission channels, the orientation of the camera once attached to the
OptoSplit II BP unit is important. Always ensure the top of your camera lies parallel with the top of the
OptoSplit. If fitting onto an upright microscope, this camera orientation remains the same.
BYPASS SPLIT
OPTOSPLIT
cairn-research.co.uk
Bypass
Input port
Camera mounting thread
Output port

email: [email protected].uk [e-mail]
[phone] www.cairn-research.co.uk
1.2) Support legs
Two support legs are supplied for use with an inverted microscope to support the weight of the
unit.
1. Secure the support leg bar to the underside of the OptoSplit using the two screws provided
2. Screw the support legs to the end of the bar
3. The height can be adjusted on each support leg – we recommend a spirit level is used for final
adjustment.
2) Key controls
This section details the key controls for all operating modes – all labels are referred to in
subsequent text in bold blue font.
BYPASS SPLIT
OPTOSPLIT
cairn-research.co.uk
Bypass
Zyla
Support leg bar
Support legs
Trim adjustment and clamp screw
Top face of camera

email: [email protected].uk [e-mail]
[phone] www.cairn-research.co.uk
2.1) OptoSplit panels
B: Left side panel
A: Top Panel
BYPASS SPLIT
OPTOSPLIT
cairn-research.co.uk
Bypass
Bypass / split lever
XY controls for Bypass image
Split control
Rectangular aperture
adjusters
SPLIT
REFLECTED PATH
V
H
Reflected H & V controls
Split control
Door cover
Support leg

email: [email protected].uk [e-mail]
[phone] www.cairn-research.co.uk
C: Right side panel
D: Underside
3) Centralising the Bypass image
The OptoSplit II Bypass unit requires a small adjustment once installed to ensure the Bypass
image is projected onto the centre of the camera sensor. It’s best practise to ensure this is carried
out prior to performing dual channel imaging, but this adjustment can be checked periodically.
Firstly, it’s useful to explore how to define a field of view and become familiar with the adjustable
rectangular aperture.
Support leg bar
Support legs
Trim adjustment and clamp screw
Transmitted V control
Support leg

email: [email protected].uk [e-mail]
[phone] www.cairn-research.co.uk
3.1) Defining field of view
An adjustable rectangular aperture (or diaphragm) precisely defines the region of interest on half
of the camera sensor. On occasions, the OptoSplit is supplied with a Cairn OptoMask diaphragm,
which requires a specific setup procedure – please refer to supplementary documentation when
appropriate.
1. Slide the Bypass / Split Lever to ‘Bypass’
2. Ensure there is no Cairn filter cube installed in the unit by removing the magnetic door cover
labelled REFLECTED PATH. Replace the door cover.
3. Adjust the two rectangular aperture adjusters (one horizontal, the other vertical) to create a
single rectangular region of interest visible on your camera.
4. Ensure the image is square, with no rotation or slant in either direction. Slightly rotate the
camera if required – a rectangular Region of Interest within your imaging software is useful as
a reference for aligning the rectangular aperture.
5. The aperture edges should be in sharp focus, as the rectangular diaphragm is precisely locat-
ed in the microscope focal plane.*
* If the rectangular diaphragm is not in sharp focus on your camera, please refer to the OptoSplit
II BP Troubleshooting Guide in supplementary material to resolve.
Aperture adjusters
Aperture
mechanism
Turn aperture handles to lock and
unlock the aperture adjusters

email: [email protected].uk [e-mail]
[phone] www.cairn-research.co.uk
3.2) Centring the Bypass Image
Once a rectangular region of interest has been defined, this image can then be centred in Bypass
mode.
1. The XY controls for the Bypass image are located on the input port of the OptoSplit.
2. Using the 1.5mm allen key provided, adjust each of the two controls in turn whilst observing
your single image on the camera. One control will result in a horizontal movement, the other
vertical.
3. Centre the Bypass image using an iterative approach with the two controls. A rectangular Re-
gion of Interest within your imaging software is useful as a reference tool to ensure the image
is centred and square.
4) Obtaining a split image
The predominant operating mode of two identical images projected onto each half of your camera
sensor is achieved by installation of a Cairn filter cube.
For initial setup, we recommend using the supplied Calibration Cube (with a 50% / 50% mirror)
and a focussed image of a graticule with transmitted light.
1. Slide the Bypass / Split lever to ‘Split’.
2. Install the calibration cube, by removing the magnetic door cover labelled REFLECTED PATH.
3. Insert the calibration cube, which has a magnetic fixing and will easily locate into place, with
the small handle facing outwards.
4. Replace the door cover.
5. Ensure the rectangular aperture is closed enough in both dimensions to see two distinct
images.
4.1) Installing the Calibration Cube

email: [email protected].uk [e-mail]
[phone] www.cairn-research.co.uk
4.2) Split mode: image alignment
Once the calibration cube is in place, two identical images will be visible on a live camera image.
It’s best practice to setup the unit with the reflected (shorter) wavelength channel on the right and
the transmitted (longer) wavelength channel on the left:
Firstly, identify each channel - the reflected image can be moved vertically and horizontally using
the provided 1.5mm allen key on the H&V controls on the magnetic door cover.
Once you are aware which channel is which, turn your attention to the transmitted image and
position this image on the left-hand side first.
1. Turn the split control clockwise to position the transmitted image horizontally close to the
edge of the camera sensor.
2. Adjust the Transmitted V control (using the 1.5mm allen key provided) to optimise the vertical
position of this (red) image.
Split control clockwise
Transmitted V control
*Please note that the
colours on the
adjacent image are for
illustration purposes only.
On a monochrome
camera, both will be
monochrome.

email: [email protected].uk [e-mail]
[phone] www.cairn-research.co.uk
3. Adjust the Reflected H control to optimise the horizontal position of the reflected (green)
image.
Reflected H control
4. Adjust the Reflected V control to optimise the vertical position of the reflected (green) image.
Reflected V control
Final tweaks of the rectangular aperture and Split control will ensure maximal field of view is
projected onto each half of your camera sensor.
‘Vignetting’ may be present once split mode has been optimised. This is characterised by one
image appearing darker on one edge.
4.3) Split mode: Trim adjustment
1. To remove vignetting, identify the TRIM adjustment on the underside of the unit.
2. A clamp screw holds the adjustment in place, therefore this may need to be turned
anticlockwise to release the slider.
3. Slide the TRIM adjustment in one direction (diagonally) until the vignetting is removed. When
moved too far, vignetting will begin to appear on the opposite image.

Remove the 4 screws and
seperate the two halves. Replace the top half and
resecure the screws.
Place the dichroic, followed by
spring clip in
the recess
Remove the dichroic spring clip
email: [email protected].uk [e-mail]
[phone] www.cairn-research.co.uk
4. Slide the control until vignetting is removed from both images.
5. Re-tighten the clamp screw.
Often OptoSplit II BP units are supplied with populated filter cubes, however (if required), these
can be installed using the guidance notes below.
As UK distributors, we have worked very closely with Chroma to develop a dichroic mirror range
for use in our image splitters with minimal image distortion. As a result, we only recommend the
Chroma Ultra-Flat (UF-2) 2mm thick dichroic mirror range.
The Cairn Filter Cube accepts dichroic mirrors of the following size: 26mm x 38mm x 2mm. Other
dichroic mirrors will fit, but may not be optimal.
We are of course happy to provide filter and dichroic mirror advice for your specific application, so
please get in touch.
5) Filter cube assembly
5.1) Installing dichroic mirrors and emission filters
Please consult filter manufacturers instructions regarding orientation of the filter.
Installing Filters into the Cairn Filter Cube
Installing Dichroic Mirrors into the Cairn Filter Cube
1)
2)
3)
4)
CAIRN
www.cairn-research.co.uk
CAIRN
www.cairn-research.co.uk
CAIRN
www.cairn-research.co.uk
Turn the locking
ring anticlockwise
to remove it. (Using
the supplied lter
removal tool).
Carefully place
the lter inside
the recess.
Replace the locking
ring and rotate it
clockwise to secure.
Filter removal
tool.

email: [email protected].uk [e-mail]
[phone] www.cairn-research.co.uk
5.2) Polarisation (anisotropy) studies
For polarisation experiments, a Cairn filter cube with an integrated polarising beamsplitter cube*
is also available as an additional accessory. In addition, a rotating 25mm polariser is available to
act as a ‘clean-up’ for the reflected S wave.
* Please note the polarising beamsplitter cube is very fragile!
1. Insert the polarising beamsplitter cube by removing the cube door and allowing the magnetic
cube to locate within the OptoSplit.
2. Replace the door cover.
3. Remove the blank cover for the auxiliary drop-in position: reflected path. (Note the blank hold-
ers will have no angled thread indictor and act as a protector for this pocket).
4. Insert the rotating polariser drop-in into the reflected auxiliary position.
5. The half-width blank auxiliary holder supplied will also be required to fill the space once the
rotating polariser is in place.
6. Rotate the polariser until the reflected image (on the right-hand side) achieves maximal intensity,
indicating the correct orientation.
BYPASS SPLIT
OPTOSPLIT
cairn-research.co.uk
Bypass
Door Cover
Auxiliary drop-in position:
reflected path
Auxiliary holder with angled thread
(suitable for emission filters)
Auxiliary drop-in position:
transmitted path
Cairn polarising
beamsplitter cube
Rotating 18mm
polariser drop-in

email: [email protected].uk [e-mail]
[phone] www.cairn-research.co.uk
6) Analysing dual channel data
Additional plugins and modules are available in a variety of software packages for analysis of
dual channel data. For example, the ‘Splitview’ module in MetaMorph (Molecular Devices) or ‘Field
Split’ in iQ (Andor) are useful tools.
We have also written an ImageJ plugin to generate a composite RGB image with the ability to
pixel nudge each image in X and Y.
The .jar file can be downloaded from the ‘Software’ page on our website
(https://www.cairn-research.co.uk/support/software/).
Paste the (unzipped) file into the ‘plugins’ folder in your root MicroManager folder on the C drive.
A MicroManager plugin for use with version 2.0 is also currently in development. Keep an eye on
our website for more details.
Please contact us for any further assistance regarding data analysis ([e-mail])
7) Bypass Mode
The OptoSplit II Bypass is designed with a quick, simple mechanism for full field imaging without
the need to remove the image splitter. A 100% mirror moves into position in ‘Bypass’ mode which
blocks the reflected (shorter) wavelength, resulting in a single, central image projected onto the
camera sensor.
In Bypass mode with the Cairn filter cube in place, the single image observed will be the trans-
mitted (longer) wavelength channel. Depending upon your experimental requirement in Bypass
mode, there are several scenarios for optimal operation.

email: [email protected].uk [e-mail]
[phone] www.cairn-research.co.uk
7.1) Obtaining a full field image of your longest channel split conditions
e.g. A full field mCherry image when splitting GFP / mCherry
1. Leave the Cairn filter cube and existing microscope cube in place
2. Slide the Bypass / Split lever to ‘Bypass’
3. Centre the Bypass image (if required) – refer to section 3.2
4. Open the rectangular aperture. Please note, it’s best practise to only open the aperture
enough to be just outside of your camera field of view to minimise scattered light.
To return to ‘Split’ conditions:
5. Slide the Bypass / Split lever to ‘Split’
6. Close the rectangular aperture to ensure only half the camera chip is illuminated.
7. As the bypass mechanism does not interfere with any split controls, no further adjustment of
the two split images should be necessary.
7.2) Obtaining a full field image of a longer emission wavelength than your split conditions
e.g. A full field Cy5 image when splitting GFP / mCherry
1. Leave the Cairn filter cube in place and change your microscope cube to a position suitable for
Cy5 imaging.
2. The mCherry emission filter will need to be removed as it will impair the Cy5 channel. For
ease of removal, we recommend mounting the mCherry emission filter in the auxiliary drop-in
position: transmitted path, rather than in the Cairn filter cube.
• Locate the auxiliary holder with angled thread indicated by the angled line on the handle.
• Remove the retaining ring (using the tool provided).
• Mount the emission filter with the active face (indicated with an arrow) pointing upwards.
• Replace the retaining ring.
• Replace the auxiliary holder in the transmitted path, maintaining the orientation of the
active face towards the microscope.
3. Slide the Bypass / Split lever to ‘Bypass’.
4. Centre the Bypass image (if required) – refer to section 3.2.
5. Open the rectangular aperture.
To return to ‘Split’ conditions:
6. Slide the Bypass / Split lever to ‘Split’.
7. Replace the mCherry emission filter in the transmitted path.
8. Return your microscope cube to a position suitable for use with the OptoSplit.
9. Close the rectangular aperture to ensure only half the camera chip is illuminated.
10. As the bypass mechanism does not interfere with any split controls, no further adjustment
of the two split images should be necessary.

email: [email protected].uk [e-mail]
[phone] www.cairn-research.co.uk
7.3) Obtaining a full field image of a shorter emission wavelength than your split conditions
OR imaging a variety of dyes utilising the microscope cubes.
e.g. A full field DAPI image when splitting GFP / mCherry
1. Remove the Cairn filter cube, gaining access via the magnetic door cover.
2. Change your microscope cube to a position suitable for DAPI imaging.
3. Slide the Bypass / Split lever to ‘Bypass’
4. Centre the Bypass image (if required) – refer to section 3.2
5. Open the rectangular aperture.
To return to ‘Split’ conditions:
6. Slide the Bypass / Split lever to ‘Split’
7. Replace the Cairn filter cube
8. Return your microscope cube to a position suitable for use with the OptoSplit
9. Close the rectangular aperture to ensure only half the camera chip is illuminated.
10. Although designed to generate minimal displacement when replacing the Cairn filter cube,
final adjustment of split conditions may be necessary – please refer to section 4.2
Please refer to the supplementary Troubleshooting Guide for additional information not de-
scribed in this manual.
Do not hesitate to contact us if you have any questions [e-mail]
445mm
60mm
130mm
135mm