Scientifica MDU Manual instruction

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Multiphoton
Detection Unit (MDU)
Setup and operation manual
Revision 1.0
Manual Part No.: S-MDU-MANUAL

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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)...................................................................................5
1.2.1 Product overview..............................................................................................................................5
1.2.2 Product Walkthrough........................................................................................................................7
2.0 Packing List...............................................................................................................................................8
2.1 Standard items........................................................................................................................................8
2.1.1 Optical block features.....................................................................................................................10
2.1.2 Detector module features ...............................................................................................................12
2.1.3 Control racks..................................................................................................................................14
3.0 Mechanical setup....................................................................................................................................16
3.1 Fitting the above-stage MDU to the SliceScope ....................................................................................16
3.2 Above-stage variant (fixed objective).....................................................................................................18
3.2.1 Checks and preparation .................................................................................................................18
3.2.2 Fitting Objectives............................................................................................................................18
3.2.3 Direct objective attachment ............................................................................................................19
3.3 Above-stage variant manual with manual objective changer (in vivo)....................................................20
3.4 Above-stage variant with Motorised Objective Changer (MOC).............................................................22
3.4.1 Checks and Preparation.................................................................................................................23
3.4.2 Fitting a DIC Prism .........................................................................................................................23
3.4.3 Removing the rear PMT module.....................................................................................................23
3.4.4 Removing the detection module and bracket from the MOC...........................................................24
3.4.5 MOC Variant Reassembly..............................................................................................................26
3.4.6 Re-assembly of the rear PMT module ............................................................................................28
3.4.7 Installation of MOC variant onto the SliceScope.............................................................................28
3.4.8 Objective installation.......................................................................................................................29
3.5 Substage variant ..................................................................................................................................30
3.5.1 Preparation.....................................................................................................................................30
3.5.2 Installation of substage variant onto SliceScope.............................................................................32
3.5.3 Fitting the Condenser to substage variant ......................................................................................34
3.6 Filter Cube Installation...........................................................................................................................35
3.6.1 Filter Cube Population....................................................................................................................35
3.6.2 Fitting the cube to the carrier.........................................................................................................35
3.6.3 Installation in the Optical Block.......................................................................................................36
3.6.4 Extraction of the filter cube and carrier ...........................................................................................37
4.0 Optical System Preparation ...................................................................................................................38
4.1 Transmission Viewing ...........................................................................................................................38
4.2 Transmitted Laser Imaging....................................................................................................................38
4.3 Laser Beam Delivery.............................................................................................................................38
4.4 Control Software and Data Acquisition..................................................................................................39
5.0 Electrical setup .......................................................................................................................................40
5.1 Electrical setup - controller connections ................................................................................................40
5.1.1 Typical electrical configuration for complete system.......................................................................41
6.0 Operation.................................................................................................................................................42
6.1 Powering up the system........................................................................................................................42
6.1.1 Before switching on........................................................................................................................42
6.1.2 Switch-on routine............................................................................................................................42

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6.1.3 Temporary switch-off procedure.....................................................................................................43
6.1.4 Safe switch-off procedure...............................................................................................................43
7.0 Troubleshooting......................................................................................................................................44
8.0 Specifications..........................................................................................................................................51
8.1 Above-Stage Variant with fixed objective and U-AAC condenser..........................................................51
8.2 Above-Stage MOC Variant with U-AAC condenser ...............................................................................52
Notes.......................................................................................................................................................52
8.3 Above-Stage Fixed Objective Variant with Substage variant.................................................................53
Notes.......................................................................................................................................................53
8.4 Above-Stage Variant with MOC and Substage Variant..........................................................................54
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

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1.0 Introduction
1.1 Handling Scientifica equipment –precautions
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
this manual can adversely affect the performance and operation of your device and
may invalidate your warranty.
High Voltage Hazard Warning
The PMTs in the detector modules operate with 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 and the safety interlock defeated.
Please do not open the detectors!
PMT Over-drive caution
The preamplifiers integrated within each detector module have sufficient gain in most cases to saturate
(generate full-scale output signal) well before the PMT delivers its maximum safe output current. The data
acquisition system can be set up to saturate before full-scale signals are attained. 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 defeat
the safety interlocks or to disassemble the main optical block. Make proper use of the “master safety”
switch on the controller and move the laser / visible dichroic mirror to the “safe” (withdrawn) position whenever
working on the microscope with the lights on.
To prevent inadvertent damage to the PMTs, the system incorporates various safety features: the PMTs are
cut off whenever the laser dichroic mirror is withdrawn from the beam, and as a secondary precaution 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 PMTs are cut off whenever the filter cube is removed. Lastly, the controller has a
“master safety” switch which can disable both detectors when the preparation is to be viewed under
conventional illumination or during setting up.
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. 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.

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Operation at very high altitude (above 3000 metres) may also affect the high voltage supplies. Contact
Scientifica for advice if your laboratory is in 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 very 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.
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 electro-physiological 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 (MDU)
This manual is intended for installation of an MDU assuming no Scanhead has been previously installed.
Some steps are irrelevant if there is a Scanhead present. Contact Scientifica if further assistance is needed.
1.2.1 Product overview
Scientifica’s Multiphoton Detection Unit (MDU) is an optoelectronic device, designed to integrate with
Scientifica’s SliceScope microscope frame, for the detection and pre-amplification of florescent signals.
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. This configuration is typically used for in vivo and in vitro studies. There are three
main ways to mount the objectives onto the MDU:
Direct attachment
A sliding manual rail
A Motorised Objective Changer (MOC)
Below stage configuration:
Multiphoton images also can be acquired by detecting the fluorescence scattered through thin samples such
as a tissue slice. A variant of the optical module is available which couples to an immersion-type, high NA

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condenser lens acting as the primary collection optic instead of a conventional microscope objective. 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.
Above and below stage (simultaneous) configuration
In addition to using above or below stage MDUs independently, both can be applied simultaneously to
optimise fluorescent collection. Both above stage and below stage MDUs can be fitted with one or two
detection modules. This means that up to four channels of signal collection can be recorded simultaneously.
It is also possible to gain access to the transmitted laser radiation that has passed through the sample: this
can be used to form an image using a suitable (photo-diode) detector.
Two controllers are required for this four-channel system, giving fully independent control of each channel’s
gain settings.

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1.2.2 Product Walkthrough
Figure A: System Overview
1
Substage MDU
2
Above-stage MDU
3 a+b
Safe imaging switches
4
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-
Immersion lens). An optional manual objective changer is available to
permit the use of low-power objectives for setting up and larger, higher-
power objectives for imaging.
5
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 can be withdrawn from the optical path by a
safe imaging switch on the front of the optical block, leaving clear access to
the objective.
6
IR laser blocking filter (excludes scattered laser radiation at wavelengths
longer than 680 nm from the detectors).
Within U-
MF2 –
includes
8a, 8b &
7
An un-populated filter cube is provided, but it need not be used if only one
detection channel is required.
7
Separator dichroic - enables simultaneous detection in two different
wavebands for experiments using more than one fluorophore. An un-

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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
Component
Comprises:
Above-
stage
variant
(fixed
objective)
One main optics module fitted with either
oOne PMT module (rear position) and One
blanking plate (side position) or
oTwo PMT modules.
One or Two signal cables (3.5 metres RG174
Coax, SMA to BNC with 50 –Ohm terminations).
One Support bracket (factory fitted)
Two M5 X 25 mm bolts for attaching to the
SliceScope
One Kit of Allen keys, including one 4 mm ball-
ended driver for the main attachment bolts.
Two transit caps (upper and lower)
One set of telescopic tubes (two pieces)
One U-MF2 Olympus filter cube (un-populated)
One 1U rack-mount two-channel controller unit
(without rack mounting screws)
One universal mains to 12V DC power supply,
low noise, with 2.5 mm DC barrel plug.
One user manual.
One mixed pollen grain slide (2P test target)
populated filter cube is provided, but it need not be used if only one
detection channel is required.
8a+b
Dye-specific emission filters (purchased separately)
9a+b
Miniature photo-multiplier tubes (PMT) together with an integrated high-
voltage power supply and an active voltage divider circuit which ensures
excellent linearity even for wide dynamic range targets under rapid scan
conditions.
Three PMT variants are offered (manufactured by Hamamatsu, Japan): a
blue/green sensitive R9880U-210 with very high efficiency in the blue to
offset poor transmission in some objective lenses, a red-sensitive R9880U-
01 and an extended-red-sensitivity R9880U-20. For bespoke
configurations (e.g. GaAsP PMTs) please contact Scientifica Ltd.

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Above-
stage
variant
(Manual
Objective
Changer)
One main optics module fitted with objective slide
guide rails and either
oOne PMT module (rear position) and One
blanking plate (side position) or
oTwo PMT modules.
One or Two signal cables (3.5 metres RG174
Coax, SMA to BNC with 50 –Ohm terminations).
Two objective slides
Adaptor rings for objective sliders: M25, M27 &
RMS
One Support bracket (factory fitted)
Two M5 X 25 mm bolts for attaching to the
SliceScope
One kit of Allen keys, including one 4 mm ball-
ended driver for the main attachment bolts.
Two transit caps (upper and lower)
One set of telescopic tubes (two pieces)
One U-MF2 Olympus filter cube (un-populated)
One 1U rack-mount two-channel controller unit
(without rack mounting screws)
One universal mains to 12V DC power supply,
low noise, with 2.5 mm DC barrel plug.
One user manual.
One mixed pollen grain slide (2P test target)
Above-
stage
variant
(MOC)
One Motorised Objective Changer (MOC) fitted
with specific DIC prism holder and shroud.
One specific mounting bracket (pre-assembled)
One main optics module fitted with either
oOne PMT module (rear position) and One
blanking plate (side position) or
oTwo PMT modules.
One or Two signal cables (3.5 metres RG174
Coax, SMA to BNC with 50 –Ohm terminations).
Two M5 X 30 mm bolts for attaching to the
SliceScope
One Kit of Allen keys, including one 4 mm ball-
ended driver for the main attachment bolts.
One transit cap (upper only)
One set of telescopic tubes (two pieces)
One U-MF2 Olympus filter cube (un-populated)
One 1U rack-mount two-channel controller unit
(without rack mounting screws)
One universal mains to 12V DC power supply,
low noise, with 2.5 mm DC barrel plug.
One user manual.
One mixed pollen grain slide (2P test target)
Sub-stage
variant
One sub-stage specific main optics module fitted
with condenser adapter plate and either
oOne PMT module (rear position) and One
blanking plate (side position) or
oTwo PMT modules.
One or Two signal cables (3.5 metres RG174
Coax, SMA to BNC with 50 –Ohm terminations).

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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.
Two transit caps (upper and lower)
One U-MF2 Olympus filter cube (un-populated)
One 1U rack-mount two-channel controller unit
(without rack mounting screws)
One universal mains to 12V DC power supply,
low noise, with 2.5 mm DC barrel plug.
One user manual.
One mixed pollen grain slide (2P test target)
2.1.1 Optical block features
The white disk is a transit cover located in the laser input port which should be removed (un-
screwed) 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.
oNote that Figure B shows an above-stage variant of the optical block; the substage variant has
a slightly different plate surrounding the port on the top surface, and a different transit cap.
The silver lever at the front of the optics block controls the laser / visible dichroic mirror.
oMove 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 optical path is blocked by a mechanical shutter, and the dichroic
mirror is protected from falling dust by the lid of the optical module. In addition, the high-
voltage supply to all detector modules is cut off as a safety precaution.
oMove 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 multiphoton detection. The high-
voltage safety interlock is de-activated, allowing detector operation (as long as the other
interlocks are not active).
Beside the main block a U-MF2 filter cube is shown in Figure D mounted on its carrier. This cube can
accept a standard fluorescence filter set (one dichroic mirror 26 by 38 mm and 1± 0.05mm mm thick,
and up to two 25 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.
There are two detector modules attached to the main block: one is located at the back (with a red-
labelled cable) and at the side (blue labelled cable). The cables attach to the controller unit via 8-pin
mini-DIN connectors. Signals emerge from the gold coaxial SMA connectors and are routed to your
data acquisition system or oscilloscope.