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 [email protected]).

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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II.Package Contents

Optosplit Image Splitter

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Optosplit Body

Filter Cube(s) ____________

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2 slot covers

Shutter to occlude either optical path

Auxillary filter/lens holder

Coupling

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Rectangular diaphragm with female c mount input

Output focussing adjuster with male c mount output

Filters

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Dichroic filter ____________

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Short emitter

Long emitter

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Dichroic filter ____________

Short emitter

Long emitter

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Dichroic filter ____________

Short emitter ____________

Long emitter ____________

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Dichroic filter ____________

Short emitter ____________

Long emitter ____________

Package Contents

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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 3 : 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 3.2 as realignment is not trivial.

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1.

2.

3.

Separating the two Images

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.

Figure 9 : Image separation

Adjust vertical position of short wavelength image

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.

Figure 10 : Vertical alignment

Ready to record

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.

Figure 11 : Correct alignment

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:

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Rotating mirror cradle to give precise symmetrical control of the degree of separation whilst maintaining identical path lengths.

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A half-size fully silvered mirror at the output port for recombining the images.

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Fine adjustment controls for camera focus and vertical alignment.

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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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5 Technical Support

e-mail : [email protected]

Web : http://www.cairn-research..co.uk/

Address : Cairn Research Ltd

Graveney Road

Faversham

Kent

ME13 8UP

Telephone : +44 (0) 1795 590 140

Fax : +44 (0) 1795 594 510

Technical Support

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