Multiphoton Detection Unit (MDU)

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1 Experts in Electrophysiology Microscope Equipment Multiphoton Detection Unit (MDU) For integration with the Scientifica SliceScope Pro motorised microscopy system

2 Multiphoton Detection Unit Two-photon excitation microscopy is a fluorescence imaging technique, applying a pulsed infrared laser for the imaging of living tissue.

2 2 Multiphoton Detection Unit Two-photon excitation microscopy is a fluorescence imaging technique, applying a pulsed infrared laser for the imaging of living tissue. The ~80MHz pulsing of the laser ensures that IR photons reach the focal plane in quick succession, exciting the fluorescent dyes. Using infrared light minimizes scattering in the tissue and two-photon absorption reduces the out-of-focus excitation for strongly suppressed background signal. These combine to achieve a vastly increased penetration depth for these microscopes. Multiphoton Detection Unit - demonstrating the ease of filter cube exchange High Quality Data Scientifica has discussed the need of two-photon imaging data with many researchers and we understand high quality data is required for publication in prestigious journals. We have therefore developed a PMT detection module which is optically pre-aligned and efficient for maximized photon collection and improved image quality. Compact and Flexible The compact design also makes the PMT detection module perfect for electrophysiologists who require the placement of multiple manipulators around the sample. Scientifica understand the need for flexibility in a two-photon system and have therefore designed the module to be compatible with a wide variety of objectives. Open-source The Multiphoton Detection Unit, together with our SliceScope microscopy system, is the perfect open source two photon solution for researchers looking to design their own scanhead. It utilises widely available freeware including ScanImage and HelioScan, dramatically reducing the cost of a two-photon microscope, making the technique accessible to more laboratories.

3 The Multiphoton Detection Unit in combination with a SliceScope Pro 3000 & two MicroStar Micromanipulators 3

4 Product Case Study As part of our collaborative development process, the Multiphoton Detection unit was applied in a prominent London laboratory to patch and image rodent neuronal cells.

4 4 Product Case Study As part of our collaborative development process, the Multiphoton Detection unit was applied in a prominent London laboratory to patch and image rodent neuronal cells. The results demonstrate the high-resolution, low-noise images accessible through this innovation. Figure 1: Detection of calcium changes in spines and boutons from layer V pyramidal cells in slices from mouse visual cortex using Scientifica s MultiPhoton Detection Unit. The patch pipette was filled with 40 um Alexa594, to enable identification of structures, and 200 um Fluo5-F, used to monitor changes in calcium. ScanImage (v3.7) was used to set up a line scan through the spine and bouton in A and B respectively (indicated with red arrows). All images taken are 512x512 pixels, zoom levels of 1x to 8x were used with a line speed of 1ms/line. A current injection into the cell, resulting in five action potentials at 30Hz, induced the calcium changes detected by Fluo5-F. Both graphs, A and B, show the calcium dynamics (white trace) overlayed with the five action potentials (red trace). Image courtesy of Sjostrom laboratory University College London

Optics and PMTs (Photomultiplier Tubes) 5 Electronics Our unique electronic design, with built-in Scientifica manufactured preamplifiers and high voltage power supplies, eradicates potential noise

There are no excessive highfrequency noise components to degrade the signal and there is sufficient bandwidth to accommodate rapid image scanning (0.5 ms per line).

The oversized optics allow for efficient photon collection from small and large back aperture objectives and the oilimmersion condenser from Olympus.

5 Optics and PMTs (Photomultiplier Tubes) 5 Electronics Our unique electronic design, with built-in Scientifica manufactured preamplifiers and high voltage power supplies, eradicates potential noise which improves the signal-tonoise ratio of the recorded images. Each PMT delivers a pre-filtered output via a coaxial connector that can be fed into a data acquisition system. There are no excessive highfrequency noise components to degrade the signal and there is sufficient bandwidth to accommodate rapid image scanning (0.5 ms per line). Optimised Optics The Scientifica Multiphoton Detection Unit features pre-aligned optics which makes it easy for the user to integrate with the SliceScope microscope. The oversized optics allow for efficient photon collection from small and large back aperture objectives and the oilimmersion condenser from Olympus. This gives researchers the ability to collect as many photons as possible from a large field of view. It is fitted with a 680nm IR-blocking filter to prevent the IR laser light from reaching the PMTs. The user may insert conventional fluorescence filter sets, housed in an Olympus filter cube, to optimize signals and differentiate between different fluorophores. A diachroic mirror can be withdrawn from the beam to permit access for conventional fluorescence imaging and then replaced without disturbing the image.the above stage variant fixes to the Z-focus drive on the SliceScope and, together with the objective, moves in the Z-axis ensuring a fixed distance between the objective back aperture and the PMTs. For this reason, no matter how deep the imaging plane is, all photons will be focused on the PMTs. User Selected PMTs We use the industry renowned Hamamatsu PMTs in all our modules. By giving users the choice of PMTs more sensitive in the blue/green (including GFP and EGFP), red or extended-red (ideal for Alexa594) wavelengths, the Multiphoton Detection Unit can be customized to suit different dyes used for two-photon microscopy.

6 Features In-Vitro & In-Vivo Options A key feature of the Scientifica Multiphoton Detection Unit is that it can be applied above or below the sample.

In-Vitro Application When imaging in-vitro, the objective and condenser can be used to collected as many ballistic (unscattered) and scattered photons from the sample as possible.

The Multiphoton Detection Unit can also be used with Scientifica s Motorised Objective Changer for the hands-free movement between two RMS threaded objectives.

6 6 Features In-Vitro & In-Vivo Options A key feature of the Scientifica Multiphoton Detection Unit is that it can be applied above or below the sample. Alternatively, a module can be applied in each of these two positions simulataneously, for optimum signal collection in up to four channels. In-Vitro Application When imaging in-vitro, the objective and condenser can be used to collected as many ballistic (unscattered) and scattered photons from the sample as possible. Scientifica Multiphoton Detection Units can be used with a range of objectives and an oil immersion condenser to collect photons which can be summed together to provide a more intense signal. The Multiphoton Detection Unit can also be used with Scientifica s Motorised Objective Changer for the hands-free movement between two RMS threaded objectives. In-Vivo Application Multiphoton Detection Units together with the in-vivo SliceScope configuration are the perfect solution for in-vivo two-photon imaging. Large, low magnification, High NA objectives (e.g. Olympus 20X) can be screwed directly into the underside of the Multiphoton Detection Unit or used with the in-vivo objective slider if objectives need to be changed frequently.

7 7 Multiphoton Unit Lightpaths The diagram below details the light path of the applied excitation laser and the PMT collection of the resultant scattered and unscattered photons. It features a setup with both above-sample and below-sample modules applied.

8 Features Compatible Objectives The above-stage configuration can be fitted with adapter rings which allow the user to use objectives particularly sought after for two-photon microscopy.

8 8 Features Compatible Objectives The above-stage configuration can be fitted with adapter rings which allow the user to use objectives particularly sought after for two-photon microscopy. These include the following objectives: Objective Working Distance RMS threaded Objectives Olympus 40x 3mm (LUMPLFLN40XW) Olympus 60x 2mm (LUMPLFN60XW) M25 threaded Objectives Olympus 20x 2mm (XLUMPLFLN20XW) NA Size of back aperture 7mm 7mm 20mm M27 threaded Objectives Zeiss Objectives 1.9mm mm M32 threaded Objectives Nikon 16x (LWD16X) 3mm Nikon 25x (CFI75 2mm Apochromat 25XW) mm 18mm

9 1. 2. 1. Motorised Objective Changer Controlled from switches on our control hardware & software, the MOC

Advantages Ultra smooth motorised movement allows hands-free objective exchange Retains Par Focality and Par

Sliding changer Ideal for In-Vivo multiphoton imaging, when manual exchange of objectives is prefered.

9 Motorised Objective Changer Controlled from switches on our control hardware & software, the MOC has two main advantages over manual changers. 3. Advantages Ultra smooth motorised movement allows hands-free objective exchange Retains Par Focality and Par Centrality, so you switch between either lens and keep your sample perfectly in focus 2. Sliding changer Ideal for In-Vivo multiphoton imaging, when manual exchange of objectives is prefered. Each objective can be mounted on a separate mounting carriage. 3. Screw in option For single objective multiphoton imaging. Adaptors are available to suit a wide range of objectives.

10 10 Control and Safety All Scientifica products are designed to be as safe, versatile and easy to use as possible. This is to allow our customers peace of mind when purchasing any of our electrophysiology equipment. The careful design of the Multiphoton Detection Unit extends to the user control interface, ensuring simple and safe operation. Module Controller The Multiphoton Detection Unit is supplied with a two-channel 1U rack controller for each PMT. The two digital readouts displays the high-voltage (up to 1000V) applied to each PMT. As the gain can therefore be independently controlled for each detector, signal levels can be optimised for different fluorophores. The controller is also fitted with a Safe / Enable switch for turning off the highvoltage applied to the PMTs providing an additional level of PMT safety. Safety Features We understand the cost implications of setting up a two-photon microscope and therefore have included safety features to the module and controller to limit possible user-related damage to the PMTs. The high voltage power supply to the PMTs will be cut-off when: The user moves the primary dichroic out of the light path. Removes either Multiphoton Detection Units from optical block. Opens the cover to the filter cube to change emission filters. The user uses the disable switch on the controller.

11 SliceScope Integration SliceScope Pro 3000 & 6000 Depending on your sample type and preference on sample and manipulator mounting, we d recommend that multiphoton technology is best applied using

SliceScope Pro 3000 With the X and Y translation of the SliceScope restricted by the lazer inputs required for Multiphoton imaging, the SliceScope Pro 3000 features a motorised top plate to enable

11 11 SliceScope Integration SliceScope Pro 3000 & 6000 Depending on your sample type and preference on sample and manipulator mounting, we d recommend that multiphoton technology is best applied using the following SliceScope Pro systems. SliceScope Pro 3000 With the X and Y translation of the SliceScope restricted by the lazer inputs required for Multiphoton imaging, the SliceScope Pro 3000 features a motorised top plate to enable smooth movement of the sample and micromanipulators. With 25 mm of super smooth travel in X and Y at a resolution of 20 nm, the ultralow noise electronics mean that you can continue to record sensitive signal even during sample adjustment. The smooth remote control movement achieves the excellent stability required to retain established patches. The large top plate enables the mounting of a full range of equipment, fixed via our unique sliding carriages or via 1/4-20 tapped hole pattern. SliceScope Pro 6000 The SliceScope Pro 6000 features a low level base plate on which to build an electrophysiology rig. This base plate can be translated in X and Y axis and the sample height can be easily adjusted using a super-stable post and platform system. A key feature of the motorised base plate is that the in-vitro sample chamber featured above can be exchanged for a tapped hole in-vivo mounting plate. As the substage optics on the SliceScope can also be removed, the setup becomes an optimal setup for in-vivo studies, enabling the placement of life support equipment and other accessories in very close proximity. This versatility makes the SliceScope Pro 6000 a cost-effective option for those likely to investigate both in-vitro and in-vivo preparations.

12 12 Technical Specifications General Laser/Visible Dichroic Mirror Laser Blocking Filter Objective max exit aperture Angular Collection Objective compatibility Spectral Filtration Detection Number of Channels Detector types Electrical filtration Detector System Bandwidth Preamplifier gain Signal Range Output connection Power requirements Power Supply Note: Specifications SEMROCK FF665-DiO2, factory-fitted SEMROCK FF01-680/SP, factory fitted 24 mm diameter +/- 5 degrees maximum from 20 mm objective, +/- 3.5 degrees from 24 mm objective Water-dipping M32X0.75, M27X0.75, M25 X0.75 and RMS threaded types (direct or via adapters) One Olympus U-MF2 filter cube supplied (un-populated) compatible with standard fluorescence filter sets (25 mm filters and 24 X 36mm dichroic mirrors 1 mm thick. One or Two PMT-based detector modules containing integrated high-voltage supply, active voltage divider, preamplifier and low-pass filter / buffer. Hamamatsu R9880U-210, R9880U-01, R9880U-20 or GaAsP (detector in preparation, expected Q3 2011). Second-order constant-delay (linear phase) low pass filter integrated into detector module. Nominal delay 495 ns. 500 khz corner frequency (See note. Other v 200,000 (see note) 0 to 2 V into 50 Ohms (output may vary from -3.5V to +3.5V). SMA (3.5 metre signal leads supplied) +12V DC, 0.5 Amps (2.5 mm DC barrel connector, centre pin positive) Low noise universal mains DC-in output Preamplifier gains and bandwidths may vary from the nominal values if you have ordered a customised detector module.

13 Ordering Information 13 The units can be specified using the following code format: MDU-XXXX-YY-ZZ XXXX 2000 use with objective for photon collection from above the sample 2500 use with oil condenser (U-AAC) for photon collection from below the sample (in-vitro only) YY / ZZ 20 blue/green sensitive PMT (on side port when used in combination with red-sensitive PMT) 40 red- sensitive PMT (on rear port when used in combination with blue/green-sensitive PMT) Get in touch Our dedicated product specialist team and Multiphoton experts can help you with any further requirements or questions you might have regarding the Multiphoton Detection Unit. We can also help you create an optimum setup for your research requirements Warranty All Scientifica instruments are sold with a two year warranty to give you complete peace of mind. This covers all defects in manufacturing and materials, providing the system is registered with us within 30 days of delivery. An extended warranty can be purchased if desired after this two year period. For more information or to order within the UK and Eire, please contact Scientifica directly. Outside these areas, please contact your local distributor or visit our website.

14 14 Schematics Multiphoton Detection Unit, above and substage ± ± All schematics feature the Motorised Objective Changer (see page 9)

15 15 Scientifica 386 SliceScope with Multiphoton Detection Unit (above objective only) ± ± Scientifica SliceScope with Multiphoton Detection Unit (Above view)

16 Revision 3 Tel: +44(0) Fax: +44(0) info@scientifica.uk.com Web: SCIENTIFICA LTD Kingfisher Court Brambleside Bellbrook Industrial Estate Uckfield East Sussex TN22 1QQ UK Your nearest distributor

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