Multiphoton Detection Unit with Extra Large optics (MDU XL) Setup and operation manual

(1)

Multiphoton

Detection Unit with Extra Large

optics (MDU XL)

(2)

Page 2 of 32

Revision 1.0

(3)

Page 3 of 32

1.0 Introduction

1.1 Handling Scientifica equipment – precautions

This section contains important safety information related to general use of the Scientifica MDU XL. The MDU XL 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 this manual can adversely affect the performance and operation of your device and may invalidate your warranty.

High voltage hazard warning

The photomultiplier-tubes (PMTs) in the detector modules operate with internal voltages of up to -1000 V. Whilst the detector modules are closed, there is no risk to the user from these voltages, but a serious risk to health might occur should the module be opened.

Please do not open the detectors!

PMT over-drive caution

The output from the detector modules is not amplified, requiring an external preamplifier for most applications. Some detector variants may be fitted with an average output current trip circuit that cuts power if dangerous signal levels are encountered. Prolonged operation above, or near the maximum, rated current output will damage the PMTs or give rise to excessive noise.

PMT over-exposure caution

PMTs operate best when kept cool, under power and in the dark! Exposure to bright lights can lead to damage and reduced sensitivity, as well as excessive noise. The system is designed to prevent accidental exposure to bright lights and to cut off the high-voltage should this possibility arise. Do not attempt to disassemble the main optical block. Make proper use of the “enable” button on the controller and move the laser/visible dichroic mirror to the “safe” (withdrawn/left hand) position whenever working on the microscope with the lights on.

To prevent inadvertent damage to the PMTs, the system incorporates various safety features: the optical path is blocked by a mechanical shutter which keeps the detectors dark and maintains their noise at a low level. In addition, the controller has a Safe/Enable button which can disable both detectors when the preparation is to be viewed under conventional illumination or during setting up.

None of these safety measures can replace careful handling of the detectors. The GaAsP PMTs used in the MDU XL have very sensitive photoactive areas. Even when switched off (no high voltage or gain applied to the PMT), indirect room light can be sufficient to permanently damage the GaAsP photocathode, leading to reduced sensitivity (i.e. quantum yield). Take great care when opening the filter cube door, and only do so with the controller switched off and in as much darkness as possible. Close the door immediately after removing the filter block to reduce exposure to any ambient light and only re-open when you are ready to quickly refit the filter block.

Environmental conditions caution

If the system has been stored in a cold place and is suddenly brought into contact with warm air, condensation may form. This will “fog” the optics, but it may also affect the high-voltage supplies in the detector modules.

(5)

Page 5 of 32 A cold system should be allowed to equilibrate to room temperature before operation (which may take a few hours in some cases).

The detection system should not be operated at humidity levels exceeding 80% or if there is any risk of condensation.

Operation at high altitude (above 2000 metres) may also affect the high voltage supplies. Contact Scientifica for advice if your laboratory is at high altitude.

Cleanliness caution

The optical block is delivered with two transit caps fitted to prevent the ingress of dirt and dust. These caps should be fitted if the unit is stored for any length of time. When fitted on a microscope, the laser/visible dichroic mirror should be moved to the “safe” (withdrawn) position whenever the unit is not in use to prevent dust falling onto its surface. Any dust or other contamination will degrade the laser beam quality and will cause scatter, both of which may impact adversely on signal quality. The door through which the filter cube is inserted should be kept shut and the detector modules should be left in place at all times. Use the telescopic tubes around the laser beam path to help prevent dust ingress. Do not open the main optics block.

Eye safety

Multiphoton imaging requires the use of high-power infrared lasers which are inherently hazardous to eyes and invisible. Under conditions of normal use, the multiphoton module will simply permit an infrared laser to pass through and no harmful stray beams will emerge in unexpected directions. To maintain this safe condition, do not disassemble or modify the optical block. You are strongly advised to wear suitable safety goggles at all times when installing, aligning and operating the multiphoton imaging system.

Please also consider local and national laser safety guidelines and legislation. Magnetic fields

The optical block uses small but strong magnets in several locations. These magnets pose no hazard to health but could cause problems to credit cards or other sensitive materials. It is also prudent to route signal wires leading from electrodes to electrophysiological head-stage amplifiers via very short, direct and well-secured routes as far from the underside of the main optics block as possible to avoid microphonic pick-up effects.

1.2 The Scientifica Multiphoton Detection Unit XL (MDU XL)

This manual is intended for installation of an MDU XL on an existing Scientifica multiphoton microscope. Contact Scientifica if further assistance is needed.

1.2.1 Product overview

Scientifica’s Multiphoton Detection Unit (MDU XL) is an optoelectronic device, designed to integrate with Scientifica’s SliceScope microscope frame, for the detection of fluorescent signals. It forms a key component of Scientifica’s modular Multiphoton Imaging System, integrating with all Scientifica scanheads, including the HyperScope, which use a scanning mirror system to raster a near-infrared laser across biological samples. In contrast to the standard Scientifica MDU, the MDU XL is only available in the above stage configuration. To detect fluorescence signals emitted from the objective focus in the sample, the MDU XL collects photons using the attached microscope objective (not included). The extra-large optics of the MDU XL are designed to transmit all fluorescence photons collected by high-NA objectives with a backaperture of up to 20 mm. Such objectives are commonly used for imaging deep in scattering tissue. There are two main ways to mount the objectives onto the MDU:

• Direct attachment

(6)

Page 6 of 32

1.2.2 Product walkthrough

Figure A System overview: The left panel shows the MDU XL mounted on a SliceScope frame. The right panel shows the MDU XL with the top cover removed in the top view outside of any frame or mounting.

1 MDU XL

2 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. Two M32 threaded removeable single position objective holders, as well as thread adapters for M27, M25 and RMS threaded objectives are included with each MDU XL as standard.

3 Pre-fitted and aligned dichroic mirror which transmits laser radiation (wavelength longer than 665 nm) through the optical block but which intercepts and directs visible fluorescence from the sample towards the detectors. This dichroic mirror is withdrawn from the optical path using the lever on the front of the optical block, leaving clear access to the objective, while protecting the PMTs from light exposure by closing the PMT shutter (4).

4 PMT shutter. Moved to block light from reaching the PMTs when moving the dichroic out of the optical path for widefield illumination.

5 Collection lens

6 Scientifica Custom Filter Cube –

The filter cube holds an IR blocking filter (7), the 40x60 mm channel separator dichroic (8), and two 32 mm bandpass filters (9a and b).

7 IR laser blocking filter (excludes backscattered laser radiation at wavelengths longer than 680 nm from the detectors).

8 Separator dichroic - enables simultaneous detection in two different wavebands for experiments using more than one fluorophore.

9a+b Dye-specific emission filters; these spectrally clean up the light transmitted (9a) or reflected (9b) by the dichroic mirror (8).

10a+b Photo multiplier tube (PMT) modules, holding the GaAsP PMTs. Two options are available: standard, protected GaAsP PMTs and gated GaAsP PMTs.

(7)

Page 7 of 32

2.0 Packing list

If the outside of the shipping packaging is damaged, notify your shipping department immediately. The shipping department may wish to notify the carrier at this point.

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.

2.1 Standard items

• 1 MDU XL optics module with either 2x Gated GaAsP PMT detectors or 2x Protected GaAsP PMT detectors.

• 2x 250mm SMA – BNC signal cables • 2x 3m BNC-BNC cables

• 1 support bracket

• 2 M5 X 12 mm bolts for attaching to the SliceScope • 2 transit caps (upper and lower)

• 1 1U rack-mount MDU-8000-20 two-channel controller unit (without rack mounting screws) • Power cable for controller

• 1 user manual.

• 1 mixed pollen grain slide (2P test target)

• Objective thread adapter rings for: RMS, extended length RMS, M25, M27 and M32 extender • 2x brass objective mounts

• XL filter cube with 525/50 32mm emission filter, 620/60 32mm emission filter and 40x60 565LP dichroic for colour separation.

3.0 Features

3.1 Optical block features

The black disk is a transit cover located in the laser input port which should be removed (unscrewed) before use and replaced with a short telescoping tube which encloses the incoming laser beam. A similar transit cover is fitted on the objective port directly below the input port (see Figure B).

The silver lever at the front of the optics block controls the laser/visible dichroic mirror. Move the lever counter-clockwise (to the LEFT) to remove the dichroic mirror from the beam path. This is the position used for direct access to the objective (multiphoton imaging is not possible). In this state the fluorescence detection path is blocked by a mechanical shutter, and the dichroic mirror is protected from falling dust by the lid of the optical module. Move the lever clockwise (to the RIGHT) to place the dichroic mirror in the optical path and to remove the mechanical shutter. This is the position used for detection of fluorescence during multiphoton imaging. Beside the main block, Figure C shows an extra-large optics filter cube mounted on its carrier. This cube accepts dedicated MDU XL fluorescence filter sets (one dichroic mirror 60 x 40 mm and 0.5 ± 0.05mm mm thick, and two 32 mm diameter band-pass filters). The filter block cube assembly is inserted into the optics block and the door (outlined by an orange O-ring seal) is screwed shut using a knurled screw.

(8)

Page 8 of 32 Figure A: Optical block front, underside view with the black transit cap and the sliding objective holder fitted

There are two detector modules attached to the main block: one is located at the back and at the side. The cables from the detector modules attach to the controller unit via 8-pin mini-DIN connectors. The actual PMT signals are accessed from the gold coaxial SMA connectors. These can be connected to your data acquisition system or oscilloscope. Gated GaAsP PMT modules have an additional BNC terminal located next to the SMA signal connector. This port is for applying a 5V TTL signal to activate the fast PMT gate.

Figure C: Scientifica customer filter cube for extra-large optics, shown half inserted into the optical block of the MDU XL

(9)

Page 9 of 32

3.2 Detector module features

Each detector module is an integrated, self-contained unit which emits a non-filtered, non-amplified analogue signal. The optical block is fitted with two identical detectors.

• The detector module incorporates a lens and stray light baffles that ensure optimum collection efficiency whilst spreading the collected light across the sensitive surface of the PMTs. This avoids any problems associated with bright spots or non-uniformity of response during a scan.

• The detectors are either the H10770PA-40 (“Protected”) or H11706P-40 (“gated”) GaAsP PMTs from Hamamatsu.

• The detector’s gain is controlled by a control voltage (0 to 0.9 V) applied by the controller rack unit. The actual high voltage supply is contained within the PMT encasement. For intuitive control, the controller unit displays the corresponding high voltage generated in the PMT. There is no need for any separate or external power supplies.

• The internal high-voltage is applied to the detector using an active voltage divider chain; this ensures that the detector is maintained in a stable operating condition no matter what signals are being drawn from it – the detector response is kept highly linear.

• The signal output is an SMA coaxial connector, designed to drive an input impedance of 50 Ohm. • If the detector module is equipped with gated detectors, a BNC connector is present next to the SMA

connector. This is the gate input connector. It accepts a digital input (low: 0 V, high: above 3 V, up to 5 V), with a high signal closing the PMT module’s gate. The specified maximum gate duration is 10 ms. While longer durations can be possible, the actual maximum gate duration will depend on several parameters (e.g. PMT voltage, duration since last off time). If the actual maximum gate duration is exceeded, the gate will open even if the input signal is still high! The gate only protects the dynodes and internal circuitry of the PMT against overload damage during high light intensities; it does not protect the very sensitive GaAsP photocathode in any way.

(10)

Page 10 of 32

3.3 Control racks

Figure B: Rack-mounting controller, front view

Figure C: Rack-mounting controller, rear view

3.3.1 Rear panel

• The controller is supplied as a 1U height rack-mounted unit (see Figures D and E). The main power switch is a push-button on the far left of the front panel.

• Power (90 – 260 V, 47 – 63 Hz, 1.6 A fuse) is supplied via an IEC connector on the right side of the rear panel.

• PC control (USB Type B socket) This is the preferred method for software control of PMT controller located on the far left of the controller. Using voltage input via the BNC connectors (see below) should only be used for external PMT control, if USB control is not supported by the imaging software.

• Connection points for two PMTs are provided on the rear panel (“PMT A” and “PMT B”). The 8-pin mini-DIN sockets connect the cables from the detector modules. Non-locking connectors are used to prevent damage if the cables are pulled by accident; ensure that both connectors are pushed firmly home.

• The BNC connectors next to the PMT connection points can be used to control the PMT voltage from the imaging software (input range 0 – 10 V). These BNC connectors are connected to the corresponding channels on the imaging data acquisition device, as configured in the imaging software (see the documentation for the software you are using for details). Preference should be given to the USB connector for external PMT control.

• One 6-pin mini-DIN connector (marked “PD”) is provided on the rear panel: this is intended to supply power to a photodiode module for transmitted laser imaging (available as an optional add-on). Do not attempt to connect the main detectors cables to this socket – it is not compatible!

(11)

Page 11 of 32

3.3.2 Front panel

• An LCD display is provided on the front panel. This displays information on the connected PMTs, as well as their status: if the PMTs are enabled, the set high voltage for each PMT is displayed, otherwise the PMT is indicated as “disabled” or “can enable”.

• The two rotary controls are used to adjust the high voltage applied to each PMT detector independently in the range 0 V to about -1000 V (left knob for PMT A, right knob for PMT B). The gain of each detector rises as a power function of the applied voltage.

• A “master safety” button is provided just right of the main power switch. This button toggles the PMTs between “enabled” or “safe” (i.e. disabled).

o In the case of Protected GaAsP detectors, if the overload protection is triggered, the PMTs are automatically disabled. A power cycle may be required to re-enable the PMTs using this safety button. Though it is strongly recommended to remove the source of the overload prior to re-enabling the PMTs.

o The Enable/Disable button can be used to suspend an experiment for a short time and then to return to previously set voltage levels. If, however, the experimental conditions are changed significantly, it may be prudent to turn both rotary controls down to minimum (anti-clockwise) before re-enabling high voltages with the toggle switch. This will help to prevent accidental overload of the detectors.

• On the right side of the front panel the “INT/EXT” switch allows the user to switch between internal (i.e. rotary knob) and external (i.e. imaging software via the input BNC or USB on the back panel) control of the PMT voltage.

(12)

Page 12 of 32

4.0 Mechanical setup

This section details the mechanical, configuration required for the differing mounting variants of the MDU XL.

4.1 Fitting the MDU XL to the SliceScope frame

Figure D: (top VivoScope, bottom SliceScope) Left: Rear view of MDU XL, note the tongue running down the centre of the mounting bracket. Right: Front view of Z-axis plate, note the groove running down the centre of the mounting plate.

(13)

Page 13 of 32 The MDU XL attaches to the upper focusing plate of the SliceScope via a bracket and two M5 X 12 mm hex cap-head bolts. The bracket has a narrow tongue that locates in a slot machined into the focus plate. A 4 mm ball-head hex driver is recommended for attaching the bracket to the SliceScope.

The main optical block is attached to its bracket using one M4 bolt and three M5 bolts which are located underneath the rear detector module. Refer to the procedure in the appendix for the removal of a detector module (9.2 Detector Module removal)

The motorised focus plate on the SliceScope has three pairs of M5 tapped holes for attaching various parts. For mounting the MDU XL, the focus plate should be driven to the top-most position. The optical block is then mounted as high as possible. This ensures the largest possible focusing range for each system, while not risking damage to the system by motor movement.

• If the MDU XL is being installed on an existing system that is already completely set up, remove the black plastic transit cover from the top entry port of the optical block; retain the cover for transport or storage.

• Support the optical block and offer it up to the motorised focus plate so that the tongue in the bracket fits into the central groove in the focus plate.

• Using a ball-ended hex driver, insert one M5 X 12 mm hex cap-head bolt through the slot in the bracket so that it engages in the middle of the three M5 tapped holes on one side of the focus plate (marked “A” in Figure F). Screw the bolt in until it stops but do not tighten it yet.

• Insert the bolt on the other side and screw it in until it stops. Again, do not tighten it.

• Allow the optical block to slide down on the slots in the bracket until the M5 bolt heads are at the top of the slots as shown in Figure G.

• If it was not already removed, unscrew the black plastic transit cover from the top entry port of the optical block; retain the cover for shipping and storage. The threaded hole into which this cap was fitted is designed to accept a short black plastic tube which acts as a light shield together with a second coaxial plastic tube. This tube should be fitted only when the detector module has been fitted to the SliceScope frame. (Ignore step if scanhead already present).

• Locate the inner telescopic light-barrier tube and screw it into the top entry port of the optics block. You may have to introduce the tube through the hole in the SliceScope’s top plate.

• Locate the outer telescope tube (black plastic) and drop it down through the hole in the microscope top plate so that it fits around the inner telescopic tube. (Please note if scanhead is present this tube would also already be present).

o If the fit between the tubes is tight, loosen both M5 fixing bolts slightly, and then gently tilt the optical block from side to side to ease the fit of the tubes. Once the fit is free, tighten both M5 bolts.

(14)

Page 14 of 32 If the experiment height proves inconvenient, the height of the optical block can be adjusted by loosening the M5 bolts through the bracket and sliding the module up and down. It is even possible to fit the bolts through the other sets of tapped holes in the motorised focus plate. Be aware that such changes may require the laser beam delivery optics to require adjustment and may restrict the range of travel.

4.2 Mounting objectives

Please remember: adjustment or removal of any screws or components other than those noted in this manual can adversely affect the performance and operation of your equipment and may invalidate your warranty.

Note that the nominal experiment height without an objective is 339.5 mm above the base of the microscope (with the focus stage position in the highest position, Figure H) unless otherwise stated.

Figure E : MDU XL fitted to the Z-focus plate of a SliceScope: note the position of the bolt heads in the slots

(15)

Page 15 of 32 4.2.1 Checks and preparation

• Check the input dichroic mirror:

o If it is not already there, move the silver actuator lever on the front of the optical block to the left (anti-clockwise). The lever should move freely and pull into its final position. This is the safe (default) position to which the control should be returned whenever imaging is not underway.

• If the dichroic mirror carriage does not move freely, refer to the troubleshooting section.

• Unscrew the black plastic transit cap from the lower entry port of the optics block. Retain this cap for shipping and storage.

• Using the SliceScope motorised controls, adjust the upper Z-focus drive to its central position. The central position is reached when the top of the moving block is level with the top of the static back-plate.

Figure H: The MDU XL installed on a HyperScope with SliceScope frame, indicating the nominal experimental height (i.e. distance between optical table surface and the objective shoulder). The actual experimental height will depend on the objective being used. If a larger distance to the table is required, the microscope can be mounted on riser blocks.

(16)

Page 16 of 32 4.2.2 Mounting objectives

The MDU XL is provided with two M32 threaded objective changers and five different objective adaptors, RMS, extended RMS, M25, M27 and extended M32. Select the correct threaded adaptors for the chosen objective and carefully screw the adaptor into the objective changer and then screw the objective into the adaptor.

Care should be taken during handling of the objective to prevent contact with the lenses which may cause image degradation. Care should also be taken not to cross thread the objective into the adaptor to prevent damage to either component.

Once the objective has been affixed to the objective adaptor (Figure J), the adaptor can be slid into the objective holder, located on the underside of the MDU XL (Figure K). Once slid fully into the objective holder, the objective can be locked into place by rotating the slider to the left (Figure L). The objective should always be put into the locked position for imaging and to prevent accidental damage to the objective and for strict optical alignment.

Figure I: VivoScope frame with MDU XL and a mounted objective. Note that for better visualization, the scanhead is not shown in this figure.

(17)

Page 17 of 32 To remove the objective, simply rotate the slider back to the open position and carefully pull the slider with the objective out of the objective holder.

Figure J: Attaching an objective to the objective slider

Figure K: Sliding objective slider into the mount at the bottom of the MDU XL

(18)

Page 18 of 32

4.3 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). In addition to the standard bandpass emission filters and colour separation dichroic the filter cube also houses the MDU XL’s IR blocking filter affixed to the front face. This filter is designed to block stray IR light from reaching the detectors. This means that even if no emission filter is required for your imaging application the filter cube must be installed. Single-channel operation typically requires the filter cube to be populated with a single 32mm diameter emission filter installed into the rear position of the filter cube. Two-channel operation requires a dichroic mirror to be installed in the cube to enable the second detector to receive some light as well as emission filters for both channels. In the case that additional blocking optics are required (i.e to further block visible light introduced through 1P stimulation, a second 32mm diameter filter can be installed in series with the standard emission filters.

A single XL-Optics filter cube, with filters and dichroic mirror installed is provided with each detection system. The cube can accept standard-sized fluorescence optical filter sets to suit the experimental requirements. The required filters are 32 mm, and thus standard 1” optics cannot be used in the detection unit. Please contact your Scientifica representative for additional filter sets.

The filter cube must be located precisely within the optical block for efficient operation; a suitable carrier is provided which slides into a compartment within the optical block behind an access door on the right-hand side. The base of the carrier incorporates a dovetail slide for positioning.

4.3.1 Filter cube population

It is assumed that the filter cube will be configured to transmit longer wavelengths and to reflect shorter wavelengths via the side-arm.

Please note, to help protect detector sensitivity it is always advised to change the detection unit filter cube in as close to complete darkness as possible and try keep the filter cube door open for as short a time as possible. PMT controller should be turned off during this operation to avoid accidently applying high voltage to the detectors.

(19)

Page 19 of 32 4.3.2 Installation in the optical block

Image M: Extended Optic Filter Cube. Exchanging the filter set consists of each bandpass filter as well as the dichroic being replaced. Removing the screws holding the two main cube parts together (1) provides access to the dichroic. To remove this, the retaining screws (3) are loosened and the mirror spring (4) carefully removed. Remove the old dichroic mirror (5). With the new dichroic in place (assure correct orientation by referring to the manufacturers datasheet), the mirror spring (4) is replaced and the retaining screws (3) re-inserted and tightened. Next, exchange first one and then the other bandpass filter. For this the locking ring (7) is carefully removed and the actual bandpass filter (12) removed and the new bandpass filter inserted (ensure correct orientation by referring to the manufacturers datasheet). Finally, the locking ring is carefully inserted and tightened. Please ensure all optics are handled with care and in lines with standard optics handling procedures (supplied by the manufacturer of the dichroic mirror and bandpass filters).

1 M3 X 8 Cap Head Screw 8 M53 x 1 to M43 x 1 Housing for 50mm Diameter Filter 2 3.0 (m6) x 8 Dowel Pin 9 Blocking Filter 50mm Diameter

3 M3 X 6 Button Head Screw 10 O-ring 1mm CS x 52mm ID Nitrile 70 ShA

4 Mirror Spring 11 M53 Locking Ring for 50mm Diameter Filter

5 Dichroic Mirror 12 32mm Emission Filter ET620/60M-2P

6 32mm Emission Filter – ET525/50M-2P 13 Filter Cube Upper Housing 7 32mm Emission Filter Locking Ring 14 Filter Cube Lower Housing

(20)

Page 20 of 32 • Ensure no power is applied to the PMT controller and room lights are kept to a minimum while the MDU

XL door is open

• Locate the filter compartment door on the right-hand side of the optical block. Unscrew the knurled screw until the door swings open freely. Be careful not to lose the white plastic retaining washer that holds the knurled screw in place.

• Post the filter cube 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.

• Close the door and tighten the knurled screw until the door sits hard against the side of the optical block, compressing the O-ring seal to prevent the entry of stray light.

4.3.3 Extraction of the filter cube and carrier

• Ensure no power is applied to the PMT controller and room lights are kept to a minimum while the MDU XL door is open

• Unscrew the knurled screw on the filter cavity door and open the door fully (be careful not to lose the white plastic retaining washer on the screw).

• The cube can be withdrawn from the optical block by pulling gently and squarely on the cube: using a finger nail on the small spring strip is a convenient method.

• Close the compartment door and secure it afterwards to prevent the ingress of dirt and dust and to keep the detectors protected from stray light.

(21)

Page 21 of 32 Caution:

• It is important that the door is closed tightly: this prevents light leakage, which can increase stray light contamination of your measurements, as well as potentially damage the PMTs.

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. Ground straps are best made from short, wide sections of copper braid (like the sheath of large-diameter coaxial cable).

• Route the screened control cables from each detector module around the microscope in a convenient manner and bring them to the rear of the controller.

o Remember that the detection module must be free to move in the vertical direction when the microscope is focused. Ensure that the cables are sufficiently slack to permit this motion and that they will not interfere with any other equipment in so doing.

• Connect each control cable into one of the 8-pin mini-DIN sockets marked “PMT A” or “PMT B”. The order is not critical. Ensure that both plugs are pushed fully home.

o It may be convenient to add colour-coded sleeves or labels to each cable to identify the two detectors individually.

• Connect a shielded signal cable to the SMA socket on each detector module, ensuring that the SMA plug is fully hand-tight. Route the cables to the data acquisition system in such a way that they will not foul any other equipment whilst moving. Again, it may be useful to place identification labels on the cables.

• If using gated PMTs, connect the blanking output from the imaging system (or the stimulation device) to the gate input BNCs on the back of both PMT modules, using a BNC T-connector to split the signal. • Connect the controller to the mains power using the cable suited for your plug type.

6.0 Operation

6.1 Before switching on

We suggest that you adopt this safety routine when operating the system for the first time, or following any re-configuration:

• Check that all connections are secure.

• It is important that your system should be able to monitor the output of the detector modules to prevent inadvertent over-driving.

o Is the data acquisition system (hardware and software) working correctly?

o Is the oscilloscope (if used) connected and set up correctly (including the triggering)? • Check that the main power switch on the front panel is “OFF” (i.e. not depressed).

(22)

Page 22 of 32 • Check that the filter cube compartment door is properly closed – is the knurled screw tight and is there

no gap between the door and the optical block body?

• Check that the primary dichroic is removed from the light path (lever on MDU is to the left)

• Turn off any microscope illumination source. This is especially important if the illumination contains any visible light at wavelengths shorter than 680 nm because this can enter the detection system directly via the objective lens and over-load the PMTs.

• Turn off any un-necessary lights, and/or cover the equipment.

• Make sure the PMTs remain ‘disabled’ on the controller until you are ready to acquire data.

6.2 Switch-on routine

• Turn both high-voltage controls fully anti-clockwise to minimum and initially switch to internal control using the INT/EXT toggle.

• Turn on main power of the controller using the push-button switch at the left of the case. The LCD display will now be illuminated, showing information on the connected PMTs, and should read “Disabled”. • On the optical block, move the laser / visible dichroic mirror into the beam by rotating the front-mounted

control lever right until it stops. The optical collection system is now opened to receive light.

• When you are ready, press the Safe/Enable button to enable the PMTs. The detector gains can now be adjusted using the two rotary controls.

• Adjusting either rotary control clockwise will increase the high voltage and thereby the gain of the corresponding detector. Adjust the controls gradually. The set high voltage of each channel will be displayed in the central display.

• To test each detector: With the laser off, adjust the high voltage controls gradually while observing the increasing signals; ideally on an oscilloscope.

o As the applied voltage is increased, the signal baseline may shift slightly negative, but it should not move by more than a few mV in a properly set-up microscope. The oscilloscope must be set for DC input coupling to observe this.

o There should be no sign of any 100 Hz or 120 Hz ripple in the signal (if observed, this would indicate the presence of stray room light operated by the local AC supply).

o As the gain is increased, the signal should begin to show more and more short negative spikes; these are individual photons being detected by the PMTs and also dark noise. The baseline should still be near zero: if not, there may be a light leak in the system.

• Return the gain controls to minimum before turning on the laser for imaging; this will prevent accidental overload of the system.

SAFETY NOTE

Whilst the system is in operation the detectors can be damaged if overloaded. Do not turn on room lights or remove any light shield over the experiment whilst the detectors are on (even if the gain is set to minimum). Visible light reflected off the sample area will reach the detectors with great efficiency even if there are no other light leaks in your system! Remember that the maximum output signal will be reached when the PMTS are delivering 50% of their maximum safe output current.

6.3 Temporary switch-off procedure

If you need to make a quick change to the experiment that will not change (increase) the signal levels significantly, simply disable the detectors using the Safe/Enable button, without adjusting the gain controls. This will cut off the supply to both PMTs rendering them safe from overload. When you are ready to resume the

(23)

Page 23 of 32 experiment with the same sensitivity settings, press the Safe/Enable button again to re-enable the detectors and continue, keeping a check on the signal levels. If you need to work on the experiment with the lights on for a longer period, use the safe switch-off procedure below.

Even with the detectors in “Safe” mode or the controller turned off, the photocathode will still be damaged by exposure to light. To prevent this, turn the lever on the front of the MDU XL to the left to shutter the unit.

6.4 Safe switch-off procedure

To shut the system down for a longer period, or to carry out extended adjustments on the experiment with the lights on:

• Turn both gain controls anti-clockwise to minimum; the meters should both indicate zero volts.

• Disable the detectors by pressing the Safe/Enable button and turn the unit off using the power switch. • Withdraw the input dichroic mirror from the optical path by moving the silver lever on the front of the

optical block to the left (anti-clockwise). This has the effect of blocking the optical collection system with a solid shutter, preventing any light reaching the PMTs even when they are off. This ensures that the PMT noise levels remain as low as possible.

(24)

Page 24 of 32

7.0 Troubleshooting

The following procedures can be used to diagnose and fix some common problems. They assume some familiarity with instrumentation and electrics. If you prefer to not carry out these procedures yourself, or if you need further advice, please contact Scientifica directly.

Adjustment or removal of any screws or components other than those noted in this manual can adversely affect the performance and operation of your device and may invalidate your warranty.

Dichroic mirror actuator jams.

• Probable cause: foreign body in front part of the optics block.

o Remove the detection module from the SliceScope and remove any objectives / adapters and telescopic tubes. Look through the laser entry and the objective ports to see if a foreign object is obstructing the dichroic mirror/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.

No activity on the controller (display fails to boot).

• Try moving the power plug at the rear of the controller in case there has been stress on the connector causing a bad contact.

• Try a different mains power cable in case internal fuse has tripped • Check fuse within PMT controller mains connection

• As a final check, try disconnecting the two detector cables (and any photodiode cable) one at a time. If the controller starts to operate, then the fault lies in one of the detectors. Contact Scientifica for help. No high-voltage reading on either detector regardless of “enable” switch position or voltage controls.

• Probable cause: connectivity issue.

o You are strongly advised to set both sensitivity/high voltage controls to no more than 20% of their

travel and to keep the light level relatively low during these checks. This will be sufficient to show

correct operation without damaging the detectors.

o Remove each detector cable from the rear of the controller and inspect the plugs for bent or missing pins: there should be 8 pins visible in each mini-DIN plug. Re-connect the plugs into the controller and ensure that each one is pushed fully home; there should be no significant resistance when the plugs are inserted into their sockets.

o Check both the detector cables for damage.

No apparent signal coming from a detector (but control voltage apparently operational). • Probable cause: data acquisition system issue

(25)

Page 25 of 32 o Disconnect the detection system from the data acquisition unit and carry out an independent test of the data acquisition system (according to the manufacturer’s instructions) to ensure that it is responding correctly. If all is well, proceed with the tests below.

• Probable cause: connectivity and measurement issues.

o The signal connections may be intermittent. Try moving the cables, especially close to the end connections or near any location where the cables may have been sharply bent. If this restores the signal, then the cable(s) should be replaced. If you are using other cables and BNC T-pieces to monitor the signals, then try replacing these as well.

o With the system turned off, remove the signal cable from the problem detector and inspect it for damage. Using a multi-meter set for resistance or continuity measurement check the resistance between the outer shell of the SMA connector and the outer shell of the BNC connector – the reading should be less than 0.5 Ohms. Make a similar measurement between the central contacts of the SMA and BNC connectors – again the result should be less than 0.5 Ohms. Finally, check that there is NO connection (infinite resistance) between the central pin and the outer body of each connector. If

any of these requirements is not met, replace the cable.

o If the cable passes inspection, re-connect it to the detector module and ensure that the SMA connector is screwed fully home (hand-tight). Connect the far end of the cable to an oscilloscope input directly.

o Set the oscilloscope as follows: the input must be DC coupled, with a sensitivity of 0.1 V per division, and any probe setting should be 1:1 gain. Set the triggering mode to AUTO and the trigger source to the input channel to which the cable has been connected.

o Turn on the controller, turn both high-voltage controls to minimum and “enable” the detectors. The oscilloscope should be showing a very small noise level (a few mV peak-to-peak). Gradually increase the highvoltage control corresponding to the detector under test until a voltage of about 200V to -250V is attained. At this point shining a dim torch towards the sample area or turning on dim room lights should produce a visible change in the background signal level of the oscilloscope. If room lights are on, then the oscilloscope should show a significant 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 V) • Probable cause: stray light or detector noise.

o If the images appear to have a high background level or if they are very noisy even if the detector(s) are operating at low to medium voltage, then the system may be suffering from stray light ingress. No stray light can enter the optics block other than via the objective port or the laser port.

o Check that any substage transmission-type illumination source is turned off during imaging even if it is an infrared source (some of which may emit some visible light).

o Stray light is most likely to be coming from visible light which has been scattered off the sample and into the objective lens. Check that the experimental arrangement provides fully dark conditions at the sample during imaging.

o It is possible for visible light to enter the optics block from the top (i.e. from the direction of the scanner). Again, check that there is no stray source of light in that part of the equipment (look for a red emission indicator light on the laser, for example, and cover it).

(26)

Page 26 of 32 Excessive noise during imaging at high detector gain (voltages between -800V and -900V)

• Probable cause: detector noise and dark current or recent detector overload.

o At high gain, some degree of dark current is to be expected: it will often take the form of randomly-timed short impulses clearly visible on the oscilloscope.

o If the noise level is excessive, or significantly higher than usual, and stray light ingress can be ruled out with certainty, then it is possible that one of the detectors has accidentally been exposed to an overload or to an overly bright light when operating at high gain.

o Recovery from such exposure can take several hours so our recommendation is to leave the detector switched off in the dark for 12 hours before testing again if accidental exposure to bright light is suspected.

o 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.

• Smearing or “trails” behind narrow, bright objects along scan lines, narrow features appear too wide (horizontally). Problem mainly evident in high speed scans.

o Probable cause: excessive scan speed for the preamplifier bandwidth.

• Ensure that the sampling rate of the data acquisition system is set to at least twice (preferably three times) the specified bandwidth of the preamplifier.

• Try reducing the scanning speed (either decrease the scanned field or increase the line time, or both); if this reduces or removes the problem then it is probable that the preamplifier cannot cope with the scan speed (it has insufficient bandwidth). If this is a problem for your experiments, contact Scientifica regarding alternative preamplifier bandwidths.

• 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.

• Excess noise in digitized signals (especially compared to the oscilloscope trace)

o Probable cause: under-sampling by the data acquisition system.

• It is important that the sampling rate be set more than twice (preferably three times) as fast as the rated bandwidth of the detectors and preamplifiers; for example, a detector / preamp with a 500 kHz bandwidth should be sampled at a rate of more than 1 MHz, and preferably 1.5 MHz regardless of the scan speed in use. This precaution ensures that the noise floor is as low as possible, and that high-frequency noise is not “aliased” (or mixed) down to lower frequencies where most of the signal energy resides. Sampling at half the recommended rate may increase the apparent noise floor in the digitised data by 40% or more. If you know that the scan will be run more slowly, and that the signal will not occupy the whole preamplifier bandwidth, then apply a suitable FIR digital low-pass filter to the line-scan data coming from the digitiser and then decimate the data stream to achieve the appropriate sampling density. It is never advisable to reduce the sampling rate! If this processing requires too much storage or computational resource, consider using a lower bandwidth detector preamplifier.

(27)

Page 27 of 32 • Low or no multiphoton signal, or unusually large laser power required to obtain any signal.

o Possible cause: insufficient laser spot intensity at the sample (multiphoton fluorescence intensity varies as a power of the applied laser intensity).

• Check that the appropriate or usual laser power is being delivered through the objective and that the laser is functioning within specification (i.e. correct wavelength, stably pulsing, specified power level). Check any beam attenuation system that may be installed – is it functioning and adjusted correctly?

• Is the laser beam filling the rear aperture of the objective lens? If the beam is too narrow, then the objective will form a wider focus than it might otherwise, with lower peak intensity, and multiphoton fluorescence may be reduced. This can occur if you change objectives for a type that has an increased NA or a reduced magnification at the same NA.

• Is there some aberration in the beam delivery optics that is blurring the laser spot at the sample?

o Possible cause: auxiliary filtering issues

• 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.

• Try imaging without the filter cube in the main optics block. The detector at the rear of the detection system should give a strong signal. If it does, verify that the filters in the filter cube match the dichroic in the cube as well as the fluorophore being used.

• Keep in mind that GaAsP detectors are only sensitive between 400 and 700 nm. Fluorophores emitting outside of this range will yield very dim signal at best.

o Possible cause: use of a narrow-field objective for imaging in a highly-scattering sample. • It may be that the multiphoton fluorescence is being generated as expected but is then

being scattered laterally by the sample until it escapes the field of view of the objective. Try using a smaller scan area or changing to a wider-field objective. It is also possible to confirm the system is working as intended by imaging a test sample, such as the pollen grain slide included with the MDU XL.

• Loss of signal with depth

o This is to be expected for two reasons: firstly, the laser intensity contributing to the multiphoton fluorescence is reduced at depth as the sample tissue distorts and scatters the incoming beam. Secondly, the visible fluorescence is strongly scattered by most tissues and does not reach the collecting objective so efficiently.

(28)

Page 28 of 32

8.0 Specifications

8.1 MDU XL with fixed objective and U-AAC condenser

Figure O: MDU XL with U-AAC condenser

Notes: The vertical position of the optical block can be adjusted using the bracket and M5 fixing bolts to suit the objective. If the optical block is lowered too far, the objective will move out of conjugation. The substage condenser and illumination optics block can be removed for in vivo work. If even more space is needed, Scientifica recommend the use of a VivoScope frame in place of the SliceScope frame.

(29)

Page 29 of 32

9.0 Appendix:

9.1 Removing and Replacing a Detector Module

Removal of a detector may be necessary for maintenance, repair, or to allow 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 are fragile: Take care not to drop or otherwise shock them whilst they are removed from the optical block. Take precautions to ensure that they are kept clean. Also ensure you briefly ground yourself prior to removing the modules, to prevent damage to the contained electronics by static discharge. Remember that high voltages are present inside each module: do not work on detector modules with the power / control cables connected and do not open them.

9.2 Detector module removal

• Switch off the controller unit and disconnect both power / control cables from the 8-way mini-DIN sockets on the rear panel. Disconnect the signal cables as well.

• Remove the MDU XL from the microscope following the procedure appropriate for the variant you have. • Each detector module is attached to the main optical block with four M3x12 hex cap-head screws (Figure P). Support the detector module to be removed with one hand and undo the retaining screws with the appropriate hexagon key. The detector module should be withdrawn from the optical block in a straight motion with no twisting.

• Put the PMT module aside covered by a clean and dust-free tissue or inside a clean plastic bag. Be careful not to lose the O-ring from the front of the collection lens housing.

(30)

Page 30 of 32

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. If any dirt is evident, remove it with a soft brush but avoid blowing it into the optical block. • Inspect the front of the PMT module before assembling it (use only dim red light for this inspection to

protect the GaAsP photocathode):

o The lens should be clean, free of dust and finger-prints.

o The O-ring should be present in its groove (it may be red or black in colour), and it should be smooth and clean. This O-ring acts as a stray-light shield.

• Lift the module and insert the four M3x12 screws into the holes.

Gently offer the PMT module up to the optical block so that the lens and holder enters the hole in the optical block and keep the module as level as possible. When the module is fully home against the optical block, gently tighten the four M3X12 screws.

1 M3 X 12 Cap Head Screw

2 PMT

3 MDU XL – Main Body

4 (Figure Q) PMT Protective Sticker 5 (Figure Q) PMT Protective Cover

(31)

Page 31 of 32

10.0 Warranty, technical queries and

returns

The standard warranty for all Scientifica designed and manufactured goods are two-years. However, 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.

The warranty will not apply if the instrument has been damaged by accident, misuse, or as a result of modification by persons other than Scientifica Ltd personnel.

Before returning an instrument, please obtain a returns number from your local representative or Scientifica Ltd in the UK.

Please refer to our terms and conditions of sales for information on our legal obligations. However, we pride ourselves on going beyond the call of duty and seek to remedy where we can and in a timely manner.

For any warranty queries please contact your local distributor or Scientifica Ltd directly.

Tel: +44 (0) 1825 749933

E-mail: [email protected]

(32)

Page 32 of 32

11.0 About Scientifica

Scientifica specialises in the manufacture and distribution of innovative, superior equipment for electrophysiology researchers, based on principles of careful design and solid engineering.

By responding to the unique demands placed on the equipment of electrophysiologists and thinking laterally around these challenges, we've developed an impressive product range used in laboratories throughout the world, including many top universities, research centres and pharmaceutical companies.

Scientifica Limited is a private limited company registered in England and Wales, registration number 3286415. VAT number: 684 3558 00.

Registered office address: 52c Borough High Street, London, SE1 1XN, UK.

1A Kingfisher Court, Brambleside, Bellbrook Industrial Estate, Uckfield, TN22 1QQ,

United Kingdom

Tel +44 (0) 1825 749 933 Fax +44 (0) 1825 749 934 [email protected]