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Summary of Contents for Scientifica MDU
- Page 1 Multiphoton Detection Unit (MDU) Setup and operation manual Revision 1.0 Manual Part No.: S-MDU-MANUAL Page 1 of 58...
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Page 2: Table Of Contents
Contents Note: This manual describes the features, functions and operation of the Multiphoton Detection Unit (MDU) and associated devices. Before use, carefully read this manual, directions for all accessories, all precautionary information and specifications 1.0 Introduction ............................... 4 1.1 Handling Scientifica equipment – precautions ..................4 1.2 The Scientifica Multiphoton Detection Unit (MDU) ................... - Page 3 9. Appendix: ..............................54 9.1 Removing and Replacing a Detector Module..................54 9.2 Detector Module removal ........................55 9.3 Detector Module Replacement ......................56 10.0 Warranty, Technical Queries and Returns ..................57 11.0 About Scientifica ........................... 58 Page 3 of 58...
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Page 4: Introduction
This section contains important safety information related to general use of the Scientifica MDU. The MDU is a piece of scientific equipment and as such requires care when handling. Adjustment or removal of any screws or components other than those noted in... -
Page 5: The Scientifica Multiphoton Detection Unit (Mdu)
It forms a key component of Scientifica’s modular Multiphoton Imaging system, integrating with the Scientifica Scanhead, which uses a galvanometer- based mirror system to raster a laser across a biological sample. There are three main configurations of the MDU available, the operation of which is covered in this manual. Above stage configuration: To detect fluorescence signals reflected back from the sample, the above stage MDU collects photons using suitable, high NA objectives. - Page 6 In this configuration, the MDU is suspended on an X, Y stage which is in turn attached to the condenser focusing mechanism: this permits the microscope to be adjusted for Köhler illumination in the usual way. The system allows conventional illumination of the sample by either visible or infra-red light when two-photon imaging is not in progress.
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Page 7: Product Walkthrough
Safe imaging switches Objective fittings • A wide range of objectives can be fitted directly to the MDU block, including M32 X 0.75, M27 X 0.75, M25 X 0.75 and RMS thread. Alternatively, a motorised Objective Changer can be connected for use with RMS-thread objectives (such as an X4 and an X40 NA 0.8 Water-... -
Page 8: Packing List
If the shipping carton is not damaged, carefully remove and identify all of the components as listed below. If any of items are missing, contact Scientifica Ltd or your local distributor. Please retain the packaging for storage or future transportation of the system. - Page 9 Above- One main optics module fitted with objective slide stage guide rails and either o One PMT module (rear position) and One variant (Manual blanking plate (side position) or o Two PMT modules. Objective Changer) One or Two signal cables (3.5 metres RG174 Coax, SMA to BNC with 50 –Ohm terminations).
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Page 10: Optical Block Features
One substage support bracket (factory fitted) incorporating X,Y adjustment controls. Two M5 X 20 mm bolts for attaching to the SliceScope One kit of Allen keys, including one 4 mm ball- ended driver for the main attachment bolts. ... - Page 11 o If the filter-cube door is not screwed shut completely, a safety interlock is activated which disables the high voltage supply to both detectors. Figure B: Optical block front, underside view with transit cap fitted. The underside view (Figure B) shows the threaded (M32 X 0.75) objective port, fitted with a transit cap. ...
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Page 12: Detector Module Features
Users may order a manually-operated sliding objective changer option which permits rapid interchange between (for example) a low-power objective (for setting up) and a large, higher power, High-NA wide field objective (for imaging). This option is intended for use with objectives which are too large to fit into the motorised objective changer. - Page 13 o Making bi-directional scanning more reliable by providing a consistent scan-speed-independent signal processing delay. The analogue signal range is 0 to 2 V nominally into a 50 Ohm terminated cable. The output connector is SMA (a SMA to BNC lead and 50 Ohm termination resistor are provided). Page 13 of 58...
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Page 14: Control Racks
2.1.3 Control racks Figure D: Rack-mounting controller, front view Figure E: Rack-mounting controller, rear view The controller is supplied as a 1U height rack-mounted unit. The main power switch is a push-button on the far left of the front panel. ... - Page 15 voltages applied to each tube will depend upon the position of the corresponding rotary controls, and the applied voltages will be displayed on the meters. o The master safety switch can be used to suspend an experiment for a short time and then to return to previously set voltage levels.
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Page 16: Mechanical Setup
MDU. 3.1 Fitting the above-stage MDU to the SliceScope Figure F: Left: rear view of MDU above-stage variant with tongue indicated. Right: front view of Z-axis plate with groove indicated. The above-stage variant attaches to the upper focusing plate of the SliceScope via a bracket and two M5 X 25 mm hex cap-head bolts. - Page 17 Figure G : Above-stage MDU fitted to the Z-focus plate: note the position of the bolt heads in the slots. Unscrew the white plastic transit cover from the top entry port of the optical block; retain the cover for shipping and storage.
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Page 18: Above-Stage Variant (Fixed Objective)
If the dichroic mirror carriage does not move freely, refer to the troubleshooting section. If the dichroic mirror appears cracked or is apparently absent, contact Scientifica for assistance. If the dichroic mirror is dusty, refer to the maintenance section for cleaning procedures. -
Page 19: Direct Objective Attachment
If direct fitting is undesirable, or you wish to use two different objectives on a regular basis, then two forms of objective-changer are available – one manual (see below) and one motorised (see separate section 3.4 Above-stage variant with Motorised Objective Changer (MOC) covering the MOC variant). 3.2.3 Direct objective attachment The under-side of the optical block presents a hole with an M32 X 0.75 metric thread which is directly compatible with some large microscope objectives: simply screw the objective into this hole until the shoulder... -
Page 20: Above-Stage Variant Manual With Manual Objective Changer (In Vivo)
3.3 Above-stage variant manual with manual objective changer (in vivo) Figure H: In vivo variant with manual objective changer Users might wish to change objectives in a more rapid and convenient manner (perhaps using a lower-power objective during setting up, and a higher power objective for imaging). A manual objective-changing mechanism is available for this purpose. - Page 21 Manual Objective Changer Notes The interchangeable objective holders are nominally 6 mm thick; this means that the objective lens will be 6 mm below its usual mounting plane, and the experiment height should be modified from the nominal 219 mm to 213 mm (with +/- 12.5 mm range). ...
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Page 22: Above-Stage Variant With Motorised Objective Changer (Moc)
3.4 Above-stage variant with Motorised Objective Changer (MOC) Figure J: Above-stage variant with motorised objective changer (MOC). The MOC (Motorised Objective Changer) variant is designed to permit users to change rapidly and automatically between a low-power objective lens (such as X4 or X10) used for setting up a sample and electrodes and a higher power imaging objective (such as 40X, NA 0.8 or 60X, NA 1.0 from Olympus). -
Page 23: Checks And Preparation
3.4.1 Checks and Preparation Check that you have all the required parts: o A MOC assembly including pre-fitted DIC prism holder and cover / light shield. o A MOC-compatible bracket o The Optical block with two detectors fitted o Two M5 X 30 Hex cap-head bolts and one M4 X 25 hex cap-head bolt. ... -
Page 24: Removing The Detection Module And Bracket From The Moc
3.4.4 Removing the detection module and bracket from the MOC Figure K: The above-sample / MOC variant with the rear detector removed. Three M5 bolts and one M4 bolt (through the rear of the bracket) hold the detector to the MOC. ... - Page 25 DIC prism installation Figure L: MOC shown with separate DIC prism and holder Figure M: DIC prism and holder in place within MOC Locate the four M3 bolts that retain the DIC prism cover, and unscrew them to reveal the DIC prism holder beneath.
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Page 26: Moc Variant Reassembly
Figure N: DIC prism installed onto the MOC with cover in place. 3.4.5 MOC Variant Reassembly Figure O : The MOC (with DIC prism holder) and the bracket prior to re-assembly. Place the MOC on a flat surface, and sit the mounting bracket over the rear of the MOC so that the three 5mm holes in the bracket line up with the three M5 tapped holes in the MOC. - Page 27 Figure P: Optical block assembled to the bracket and MOC using three M5 bolts and one M4 bolt (located in the rear face of the mounting bracket). Fit three M5 X 16 hex cap-head bolts through the clearance holes in the optical block and the bracket and screw them loosely into the MOC top plate until they just stop rotating.
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Page 28: Re-Assembly Of The Rear Pmt Module
3.4.6 Re-assembly of the rear PMT module Refer to the procedure in the appendix 9.3 Detector Module Replacement for replacing the rear PMT substage. 3.4.7 Installation of MOC variant onto the SliceScope Figure Q: Side view of the optical block, bracket and MOC showing the side slot (under the rear detector module) which accepts the M5 X 30 fixing bolts. -
Page 29: Objective Installation
Figure R: Installing the MOC-variant to the SliceScope Insert the second bolt and drive it home, but do not tighten it fully. Locate the inner telescopic light-barrier tube and screw it into the top entry port of the optics block. You will have to introduce the tube through the hole in the SliceScope’s top plate. -
Page 30: Substage Variant
3.5 Substage variant Figure S: Substage variant The substage variant of the multiphoton detection system uses a high-NA oil-immersion condenser as a light collector instead of a microscope objective. This condenser can also be used for illuminating the sample for conventional imaging, and the system is provided with manual X, Y adjustments and a motorised focus adjustment (on the SliceScope). - Page 31 o Hexagon drivers including ball-ended 4 mm driver for M5 bolts. Using the SliceScope’s motorised controls, position the condenser focusing plate to its central position. Check that the substage lens at the base of the SliceScope is fitted with its plastic transit cover before starting the installation procedure.
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Page 32: Installation Of Substage Variant Onto Slicescope
3.5.2 Installation of substage variant onto SliceScope Figure U: The lower condenser focus plate showing the correct mounting holes (labelled “A”) for the substage multiphoton detection module. The condenser-focusing Z-axis plate on the lower part of the SliceScope Column has three pairs of M5 tapped holes as for the upper z-axis plate. - Page 33 Figure V: Mounting the substage variant onto the condenser focus plate of a SliceScope. Insert one M5 X 20 hex cap-head bolt into the slot on one side of the mounting bracket, hold it in place with a 4mm ball-ended hex driver, and offer the whole assembly up flush to the lower Z-axis focus plate so that the bolt engages with the central M5 tapped hole.
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Page 34: Fitting The Condenser To Substage Variant
3.5.3 Fitting the Condenser to substage variant Slide Figure W: Substage detection module fitted onto the SliceScope with the condenser in its mount. Dotted arrow indicating screw to secure condenser in place. Using a small hex driver, un-screw the three soft-tip retaining screws until the plastic tips are flush with the internal bore of the adapter plate. -
Page 35: Filter Cube Installation
3.6 Filter Cube Installation The filter cube is used to enable simultaneous two-channel detection (by means of a dichroic mirror / beam- splitter) and to define the spectral responses of each channel (by means of appropriate band-pass interference filters). Use of the filter cube is not mandatory: for single-channel operation no cube is required. Two- channel operation however requires at least a dichroic mirror to be installed in the cube to enable the second detector to receive some light. -
Page 36: Installation In The Optical Block
The cube should be retained firmly on the carrier by the spring-loading. It should not be loose or able to slide off under its own weight. If the cube is found to be loose, do not insert it into the optical block: instead contact Scientifica for help. -
Page 37: Extraction Of The Filter Cube And Carrier
Check that the filter cube has been pushed fully onto the carrier as described above. Post the filter cube carrier squarely into the opening so that the dovetail on the under-side of the carrier engages with the groove in the floor of the optical block. Push the assembly home gently and smoothly until the rear surface is flush with the side of the casing. -
Page 38: Optical System Preparation
4.0 Optical System Preparation In order to perform multiphoton imaging with the detection modules, it is necessary to prepare laser beam delivery and scanner optics. 4.1 Transmission Viewing For best image quality, we recommend using Köhler illumination. The intermediate diffuser within the SliceScope’s substage optics should be removed from the beam (the silver lever at the lower left side of the SliceScope should be moved to the horizontal position). -
Page 39: Control Software And Data Acquisition
A means for monitoring the near IR laser power being delivered to the sample (for example a power meter and a glass beam-splitter). 4.4 Control Software and Data Acquisition The Scanhead is compatible with HelioScan and ScanImage Please consult relevant manuals. HelioScan User Guide and information available for download here. -
Page 40: Electrical Setup
5.0 Electrical setup 5.1 Electrical setup - controller connections Mount the controller in a suitable 19-inch rack. It may be necessary to connect a ground strap from the lug at the rear of the controller to the optical table or to another suitable grounding point depending on the local screening arrangements. -
Page 41: Typical Electrical Configuration For Complete System
5.1.1 Typical electrical configuration for complete system Figure AA: Typical electrical configuration for complete system Page 41 of 58... -
Page 42: Operation
Turn on main power at the controller using the push-button switch at the left of the case. o Both meters and the Scientifica logo should illuminate. o Both meters will read a small voltage, usually positive. The value has no significance. -
Page 43: Temporary Switch-Off Procedure
Adjust the high voltage controls gradually whilst observing the signals – an oscilloscope is probably the most convenient means, but the data acquisition system can be used also – but with the laser still off. o As the applied voltage is increased, the signal baseline may shift slightly positive, but it should not move by more than a few mV in a properly set-up microscope. -
Page 44: Troubleshooting
/ shutter mechanism. Do not open optical block but look through laser entry/light port to identify the jam item. If easily accessible try and remove the object carefully with tweezers. If fearful of touching mirrors call Scientifica for help. Do not disassemble the main optical block. - Page 45 Check to see that correct function is restored. If you have no success, the fault may lie elsewhere: contact Scientifica for assistance. Page 45 of 58...
- Page 46 100Hz or 120 Hz oscillating signal on top of an offset. If the expected response is not seen then turn the system off and contact Scientifica for assistance. Excessive noise and offset during imaging at low detector gain (voltages between 0V and -700 ...
- Page 47 If, after a period in the dark, the detector noise has not reduced to previous levels, the detector may need to be replaced. Contact Scientifica for advice. Variable noise and offset down an image.
- Page 48 If this is a problem for your experiments contact Scientifica regarding customised preamplifier bandwidths. o If your calculations suggest that the scan speed and resolution in use should not be causing a problem, it is possible that the pre-amplifier filter is faulty. Contact Scientifica for assistance. ...
- Page 49 Possible cause: auxiliary filtration issues or detector mis-placement o It is possible that the fluorescence emission is being blocked by the filter placed in the removable filter cube, or that the detectors are placed in the wrong positions to suit the filters installed.
- Page 50 too close to the edge of the objective’s field of view, then fluorescence will be lost and the images appears shaded at the corners. The effect is usually worse as the imaging depth is increased. o Try reducing the width and height of the scan. o If a wide field is important for your experiments, consider using a wider-field objective lens.
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Page 51: Specifications
8.0 Specifications 8.1 Above-Stage Variant with fixed objective and U-AAC condenser Figure BB: Above-stage variant with fixed objective and U-AAC condenser Notes: The figures shows a single “large” (75 mm parfocal distance) objective screwed directly into the optical block, but other combinations are possible, including a short (e.g. 45 mm parfocal) objective coupled to a 30 mm extension adapter (provided), or even connected directly to the optical block. -
Page 52: Above-Stage Moc Variant With U-Aac Condenser
8.2 Above-Stage MOC Variant with U-AAC condenser Figure CC: Above-stage MOC variant with U- AAC condenser Notes The Motorised Objective Changer (MOC) will only accept RMS-thread objectives. It is typically populated with a low-power air objective (e.g. X4) and a higher-power water- immersion objective (e.g. -
Page 53: Above-Stage Fixed Objective Variant With Substage Variant
8.3 Above-Stage Fixed Objective Variant with Substage variant Figure DD Notes This is a four-channel system which is useful for work with thicker tissue slices. Each detection module has its own controller. The lower detection module utilizes an oil-immersion condenser for light collection, and is mounted on an adjustable suspension so that the illumination and collecting can be adjusted and centred in the conventional manner. -
Page 54: Above-Stage Variant With Moc And Substage Variant
8.4 Above-Stage Variant with MOC and Substage Variant Figure EE 9. Appendix: 9.1 Removing and Replacing a Detector Module Removal of a detector may be necessary for maintenance, or repair, or for access to the mounting bracket at the rear of the optical block. The following procedure explains how to remove and replace a detector. The detector modules should be regarded as “fragile”: Take care not to drop or otherwise shock them whilst they are removed from the optical block. -
Page 55: Detector Module Removal
9.2 Detector Module removal Detector Modules are retained by two long M3 bolts, one in the corner of the module and one through the centre. Switch off the controller unit and disconnect both power / control cables from the 8-way mini-DIN sockets on the rear panel. -
Page 56: Detector Module Replacement
9.3 Detector Module Replacement Inspect the optical block where the detector module is to be attached: look to see that there is no dirt or damage around the three small contact pins located above the large hole in the optics block. If any dirt is evident, remove it with a soft brush but avoid blowing it into the optical block. -
Page 57: Warranty, Technical Queries And Returns
Scientifica’s Multiphoton Imaging system, includes components from other companies, which offer a twelve- month warranty. For an extended warranty on the full system (including external companies) please contact your Scientifica representative. All warranties cover defects in manufacturing and materials. In this unlikely event, Scientifica will manage the repair and replacement of all components. -
Page 58: About Scientifica
Scientifica Limited is a private limited company registered in England and Wales, registration number 3286415. VAT number: 684 3558 00.
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