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Parallel fluorescence spectroscopy tools for micro and nano-analytical applications down to single biomolecules

Final Report Summary - PARAFLUO (Parallel fluorescence spectroscopy tools for micro and nano-analytical applications down to single biomolecules)

Executive summary:

In fluorescence-lifetime imaging microscopy (FLIM), a map of the emission lifetime in a cell is obtained. PARAFLUO aims to develop an innovative instrumentation system that enhances and extends the FLIM effectiveness, enabling to obtain simultaneous spectrally resolved data (sFLIM) for the various components of the emission. There is widespread consensus that sFLIM will support a better understanding of the biological processes involved, which may be paramount for the (patho)physiology of tissues and organisms and provide better insight in key medical issues, as origin and growth of tumors. This instrumentation will be useful also in other applications, e.g. multi-spectral profiling of objects by laser detection and ranging.

Project Context and Objectives:

All biological processes rely on the complex interactions of biomolecules such as proteins, DNA, RNA, lipids and sugars. These interactions usually take place in large molecular assemblies. Typical examples are the "molecular machines" involved in signal transduction, transcription and translation of genomic information, protein degradation, or intracellular transport processes. A better understanding of these processes is paramount for a better understanding of the (patho)physiology of tissues and organisms and gives a base for gaining a better insight in key medical issues, such as the origin and growth mechanisms of tumors. Analytical techniques that provide information on these interactions are nowadays currently in use not only at the cutting edge of biomolecule research, but also in laboratories of medical and biological ambients. Fluorescence microscopy and spectroscopy is widely employed. FRET (Forster resonance energy transfer in acceptor/donor pair) is employed for measuring intermolecular distances on a nanometer scale, thus obtaining information about the tagged proteins and their environment. Established FRET techniques rely on measurements of changes either of the spectral intensity or of the lifetime of the fluorescence.

The rising quest of analytical instrumentation suitable for this field provided a driving force for industrial developments. Such developments have been mainly achieved by dedicated SMEs that originate from research ambients. European small and medium-sized entreprises (SME)s have been very successful in this field and have gained an acknowledged leadership at world-level, but the competition is hard and requires being continuously at the forefront of the technological advancement. A challenging request presently arises from the market and calls the attention of the SMEs. FRET measurements of spectral intensity and of lifetime taken individually do not give a complete information about the involved fluorophore characteristics. There is a wide consensus among experimenters that for a better understanding of the biological process involved at cellular level it is necessary to acquire at every cell region under investigation simultaneously the spectral and temporal fluorescence data. This requires developing optoelectronic instrumentation capable of carrying out spectrally resolved fluorescence-lifetime imaging microscopy (sFLIM).

Project Results:

The results of the PARAFLUO project are provided here below by Workpackage. For the figures mentioned in the text, please refer to the attached document.

WP1 Management

Planning and scheduling (P1-MPD)
This task provides accurate planning of different actions during the project's lifetime. Activities that have been performed are:
1) collecting the inputs and needs for changes / checking the evolution of the work and assuming consequent decisions according to budget, workplan and objectives.
On the occasion of the kick off meeting, the Coordinator collected information on the status of the WPs and the following inputs to update the workplan arose:
- as for WP2, based on the analysis of advantages and risks, it was proposed that some exploratory devices with diameter of 80micron and pitch of 250 micron will be included in the fabrication runs, in order to explore the prospect of further progress;
- as for WP4, taking into account the spectroscopy application envisaged, it was proposed to design the array with the first 4 lenslets optimized for 500nm wavelength and the other 4 lenslets optimized for 600nm wavelength.

On the occasion of the PARAFLUO project meeting on February 10th, the following inputs to update the workplan arose:
- as for WP2 and WP3, since remarkable problems have been met for obtaining the silicon wafers with quality adequate to the purpose of the project, a new supplier of wafers was found, who guarantees wafer quality. However, since the delivery of these wafers was estimated to take two more months, it was decided to proceed and launch the first fabrication run by employing a batch of epitaxiated silicon wafers available at POLIMI as residual from previous activities.

On the occasion of the PARAFLUO project meeting on October 5th 2010, the Coordinator collected information on the status of the WPs and the following inputs to the workplan arose:
- as for WP6, it was agreed to perform some first tests of the lenslet design by using a CCD camera in place of the SPAD array. The tests should be performed in order to test the complete optical setup including the microlenses, but without having yet available the SPAD array. The aim was to correct possible uncertainties in the optical calculations;
- the work for the development of the Si-Ge ASICs is taking a time longer than the planned one, because in the approach taken for reducing the risk and cost more iterations of the fabrication are necessary;
- also the development of the micro-lens array of the 8x1 detector would benefit from an extension of the activities. In fact, a remarkable improvement of the light collection has been verified, but optical design and simulation predict even higher values that could be pursued with further experimental work.

Moreover, during the meeting held on February 25th 2011 in Bolzano (see details below), the beneficiary P5 CNR communicated that the silicon foundry in Bologna suffered important technical failures concerning the ion implanter and the oven-flux controller. Fabrication steps of essential importance and confidential nature were affected and for technical and confidentiality reasons it was not advisable to resort to other foundries. Repair and reconditioning of the apparatus took a time that had been originally planned for important technological trials, aiming to improve the performance of the 8x1 detector. Particularly important were tests of fabrication with different substrates, planned for ascertaining the conditions for obtaining best results in production.

For the reasons mentioned above, the Consortium asked an extension of the contract duration of three months, shifting the term from 31 May 2011 to 31 August 2011, thus allowing to complete the work and better attain the goals of the PARAFLUO Project. For this reason, an amendment has been submitted to the EC in March 2011. In this context, the Coordinator has been supported by P9 CFc in the completion of the formal procedures (revision of Annex I and Grant Agreement Preparation Forms).

2) communications between WP Leaders and/or WP participants and arranging meetings where necessary
During the project period, several meetings among WP Leaders took place:

June 15-18, 2009: Meeting held during the conference Laser World of Photonics 2009, Munich. Participants: POLIMI, MPD, PQ and HWU. Objectives: preliminary discussion about the start-up of the project activity
June 25, 2009: Meeting held at PQ site. Participants: PQ, FHB. Objectives: start up meeting of WP5.
July 16, 2009: Meeting held at CNR site in Bologna. Participants: POLIMI and CNR. Objectives:
a) discuss the circuitry suitable to be integrated with the SPAD detector, to be used to buffer the timing signal inside the multichannel module;
b) planning of the characterization activity to be performed in order to be able to model the MOSFET devices;
c) define how to proceed for the joint design of the mask layout;
d) planning the first 6 months of activity in WP2 and WP3.

August 24, 2009: Meeting held at FHB site. Participants: PQ, FHB. Objectives: establishing design guidelines, interface cells to the HydraHarp system.
September 9, 2009: Meeting held at the premises of the manufacturer of the TDC chip. Participants: PQ, FHB. Objectives: clarify libraries and design kits, generating some test structures.
September 23, 2009: Meeting held at CNR site in Bologna. Participants: POLIMI and CNR. Objectives:
a) Discussion about the results obtained in the MOSFET parameter extraction using the model BSIM3; main parameters to be used and their effect on the model;
b) Alignment of the Polysilicon layer inside MOSFET devices: electrical problems and mask tolerances;
c) Electrical measurements methods developed at POLIMI about SPAD quenched with different circuits realized using discrete elements: performances, delay in the response, timing.

December 2, 2009: Conference call. Participants: MPD, POLIMI, PQ and FHB. Objectives: Short intermediate report of all teams, with special attention to the interfacing between 8x1 SPAD module and TCSPC apparatus.
December 11, 2009: Meeting held at PQ site. Participants: PQ, FHB. Objectives: fixing for January tape-out, test structure for PLL and time reading channels.
February 10, 2010: Telephone conference. Participants: All project teams. Objectives: Short intermediate report of all teams and consultations for the preparation of the report of the first year.
February 18, 2010: Meeting held at CNR site in Bologna. Participants: POLIMI and CNR. Objectives:
a) presentation and discussion of the new process flow;
b) planning of electrical characterization of the single SPAD, of the 8x1 SPAD arrays, of MOSFET devices and of various MOSFET circuits in the wafers of the new run;
c) planning of activity for months 9th -12th for WP2 and WP3.

February 24, 2010: Meeting held at POLIMI site in Milano. Participants: POLIMI and MPD. Objectives:
a) setting design guidelines for auxiliary electronic blocks and SPAD array package;
b) planning of activity for months 9th -12th (WP2, task 2.3).

April 15, 2010: Web conference. Participants: HWU, HPL, PQ. Objectives: Discussion of the optical system for collecting light on the 8x1 SPAD detector in the measurement setup.

May 17, 2010: Meeting held at POLIMI site in Milano. Participants: POLIMI and CNR. Objectives:
a) analysis of the first results of the electrical characterization of the integrated silicon devices and circuits;
b) planning of activity for months 13th -18th for WP2 and WP3.

May 24, 2010: Meeting held at POLIMI site in Milano. Participants: POLIMI and MPD. Objectives:
a) evaluate design (task 2.3) and prepare tests;
b) planning of activity for months 13th -22th for WP2.

May 28, 2010: Meeting held at PQ site. Participants: PQ, FHB. Objectives: evaluate design and prepare tests of the fast integrated circuitry in SiGe.

July 26th, 2010: Telephone conference. Participants: MPD, CFc, POLIMI. Objectives: to revise the final version of the periodic report to be submitted by the Coordinator to the EC.
August 17th, 2010: Meeting held at PQ site in Berlin. Participants: PQ, FHB. Objectives:
a) test measurements of 1-channel TDC;
b) discussion of the measurement results.

September 27th, 2010: Telephone conference. Participants: All project teams. Objectives:
a) to discuss the project financial status after the end of the first periodic report;
b) to discuss the transaction and the invoicing procedures required between RTD Performers and small and medium-sized entreprises (SME) Participants.

October 5th, 2010: Meeting held at POLIMI site in Milano. Participants: all partners. Objectives: the meeting was intended for discussing the status of the PARAFLUO Project activities at the end of year 1, taking into account achievements, problems, corrective actions and planning of next activities for concluding successfully the project.

December 10th, 2010: Telephone conference. Participants: All project teams. Objectives: discuss SPAD and TDC progress.

December 21st , 2010: Meeting held at POLIMI site in Milano. Participants: MPD, POLIMI. Objectives:
a) status of task 2.4;
b) review of the sub-systems assembly/interface strategy;
c) planning of activity for months 20th -24th for WP2.

February 15th , 2011: Meeting held at FHB site in Brandenburg. Participants: PQ, FHB. Objectives:
a) evaluate the 3-channel TDC chip and prepare further tests;
b) planning and execution of EMV measurements.

February 25th , 2011: Meeting held at MPD site in Bolzano. Participants: MPD, POLIMI and CNR. Objectives:
a) Status of the PARAFLUO project;
b) Discussion about the requested project extension;
c) production of arrays: current status and future;
d) new MPD activity in collaboration with PoliMI in the designing the iAQC.

March 28th , 2011: Web conference. Participants: POLIMI, PQ. Objectives:
a) performance of prototype of 8x1 detector;
b) bonding of microlens array on CCD test system.

April 1st , 2011: Meeting held at POLIMI site in Milano. Participants: POLIMI and CNR. Objectives:
a) discussion of the problem encountered during the new process flow;
b) results of the electrical characterization on the single SPAD;
c) planning of electrical characterization of SPAD arrays.

May 16th , 2011: Meeting held at POLIMI site in Milano. Participants: MPD, POLIMI. Objectives:
a) review of the PARAFLUO module performance;
b) discussion on potential improvements in the module's engineering.

May 23rd, 2011: Telephone conference. Participants: MPD, POLIMI, CFc. Objectives: to discuss the PARAFLUO dissemination and exploitation strategies and results.

May 25th , 2011: Meeting held at PQ site in Berlin. Participants: PQ, FHB. Objectives:
a) discussion about latest measurement results;
b) causes analyses of crosstalk effects between on-chip channels;
c) decision to the version with separated TDC chips for each channel.

June 16th , 2011; Meeting at UGOE site in Göttingen. Participants: UGOE, PQ. Objectives: final realization of dispersing system.
July 14th, 2011: Meeting held at PQ site in Berlin. Participants: PQ, FHB. Objectives:
a) communication between the TDC chip and the Virtex6 FPGA platform;
b) test assemblies for specification of TDC chips and verification of its input signals.

August 24th , 2011: Meeting held at PQ site in Berlin. Participants: PQ, FHB. Objectives:
a) delivery of project results and the latest TDC testchip;
b) discussion about measurement results and solving bonding problems with the TDC chip.

Progress and cost reporting (P1-MPD - P9- CFc)
This task establishes a reporting structure to the European Commission and for internal communication within the project. Activities performed include:
1) providing reporting templates to all participants
P9 CFc, under the supervision of the Coordinator, created ad-hoc templates for the collection of the necessary data for the reporting activities. In details, the following templates have been designed and provided to partners:
- table for the collection of costs sustained by RTD Performers;
- table for the collection of costs sustained by SME Participants;
- table for the collection of costs sustained by Other enterprises;
- table for the collection of data related to the dissemination activities performed;
- table for the collection of information related to the administrative officers in charge;
- table with deliverable status;
- template for the drafting of the deliverables.

2) maintaining a document repository
The private area of the website has been structured by P1 MPD and P9 CFc in order to be a repository of the necessary documents for the management of the project, such as the Grant Agreement, the Consortium Agreement, the minutes of the meetings held and the presentations shown during meetings, the deliverables that are submitted as well as the common templates (i.e. deliverable templates).

3) creating and submitting the periodic report.
P9 CFc assisted the Coordinator in the collection of data from the partners and prepared a first draft of reporting documents. As for the description of the technical activities and results, P9 CFc forwarded the first draft to the Coordinator for revision and improvement.

As for the financial part of the report, a first check about the eligibility of costs and the consistency of costs and person-months with the related activities has been performed. The global situation of the whole project has been evaluated and possible unclear situations have been clarified directly with the partners involved. The latter activities have been carried on together with the Coordinator through teleconferences.

On the basis of the activities listed above, the final draft of the report has been created and P9 CFc contributed to a formal and layout revision. All partners completed their forms C and submitted to the Coordinator. In this context, P9 CFc assisted all partners in the procedures for registering their ECAS accounts and in facilitating the resolution of problems through the dedicated helpdesk. Before the submission of the report to the European Commission through the online systems available (QUEST and FORCE), the Coordinator approved the Report from both the technical and the financial point of view.

In the second period, the Final Report has been also prepared and submitted.

Monitoring, control and quality management (All partners)
This task ensures monitoring and control of the established plans. Activities performed include:
1) work progress control
During the technical meetings held, the progress of each WP has been monitored and discussed. Moreover, during the second period, the Coordinator, assisted by P9 CFc, monitored the attainment of planned milestones and the completion of project deliverables. In this context, P9 CFc assisted the Coordinator in the revision of the document layout.
2) cost control
During the kick off meeting, the Coordinator with the support of P9 CFc related about WP1 "Management" informing all partners about the reporting obligations and the reporting schedule. It was proposed that all partners provide in January 2010 the preliminary financial figures in order to be able to accurately monitor the financial situation before the formal reporting of the first year in June 2010. Early in February 2010 the internal status of project resources has been ascertained and collected. The final aim was to make possible a faster collection of data in the formal reporting stage at the end of the first year.

During the second period, on September 27th 2010, with authorisation of the Coordinator and together with the Coordinator, a skype conference has been organised by P9 CFc with the administrative officers of the various partners to discuss with them the project financial status after the end of the first periodic report and, in particular, the transaction and the invoicing procedures required between RTD Performers and SME Participants. The situations discussed during the skype conference were then followed by direct contacts with the partners.

These activities were performed in order to:
i) identify at early stage the possible risk situations and prepare recovery plans where needed;
ii) lead the partners to become familiar with the reporting requirements, giving them the possibility to ask specific clarifications.

During the PARAFLUO meeting held on October 5th 2010, the Coordinator with the support of P9 CFc updated all partners about the reporting obligations and the reporting schedule. They also reminded the necessity of paying careful attention to the administrative/financial issues discussed during the skype conference held.

Logistics (P1 MPD - P9 CFc)
The following project meetings have been held:

1) kick off meeting, July 9th, 2009.
The meeting was mainly devoted to discuss the organization of the activities and the key technical aspects of the project.

The following persons attended the meeting on behalf of the partners:
Sergio Cova (MPD)
Massimo Ghioni and Ivan Rech (POLIMI)
Rainer Erdmann, Benedikt Kramer and Michael Wahl (PQ)
Gerald Kell (FHB)
Gerald Buller (HWU)
Carla Finocchiaro and Serena Cogoni (CFc)
whereas the following persons and partners were represented as here reported:
Joerg Enderlein (UGOE) represented by Rainer Erdmann
Piera Maccagnani (CNR) represented by Ivan Rech
Caspar Clark (HPL) represented by Gerald Buller

After welcoming all participants, the Coordinator recalled that the project should be completed within two years from the official starting date of June 1st, 2009. The term is therefore May 31st, 2011 and the time available for the amount of work to be done is relatively short. The Coordinator recalled the main lines of the work and the Workpackages reported in Annex 1 of the EC Grant Agreement. He proposed to discuss the present situation in general and to review the prospect of activity in the next months with particular attention to WP2, WP3, WP4 and WP5.

2) PARAFLUO project meeting, February 10th, 2010.

The final agenda for reporting and discussion was:
- Work on the complete 8x1 detector: chip, packaging and module box (WP2 and WP3);
- Work on the lenslet array for the 8x1 detector (WP4);
- Work on the microscope optomechanical system (WP6);
- Work on the fast integrated circuitry in SiGe technology (WP5);
- Status of the subcontracts among SMEs and RTD performers (WP1);
- Instructions and hints for the project reports and for financial monitoring (WP1 and WP7);

The following persons attended the meeting on behalf of the partners:
Sergio Cova (MPD)
Andrea Giudice (MPD)
Massimo Ghioni and Ivan Rech (POLIMI)
Piera Maccagnani (CNR)
Rainer Erdmann and Benedikt Krämer (PQ)
Joerg Enderlein and Ingo Gregor (UGOE)
Gerald Kell and Martin Ahlborg (FHB)
Gerald Buller and Mo Taghizadeh (HWU)
Kamila Gurdala and Serena Cogoni (CFc)

3) PARAFLUO project meeting, October 5th 2010 in Milan (Italy).
The following persons attended the meeting on behalf of the partners:
Sergio Cova (MPD)
Andrea Giudice (MPD)
Roberto Biasi (MPD)
Massimo Ghioni (POLIMI)
Ivan Rech (POLIMI)
Angelo Gulinatti (POLIMI)
Piera Maccagnani (CNR)
Francesco Moscatelli (CNR)
Rainer Erdmann (PQ)
Benedikt Kramer (PQ)
Ingo Gregor (UGOE)
Gerald Kell (FHB)
Gerald Buller (HPL)
Mo Taghizadeh (HWU)
Andrew Waddie (HWU)
Aongus McCarthy (HWU)
Carla Finocchiaro (CFc)
The meeting was intended to discuss the status of the PARAFLUO project activities at the end of Year 1, taking into account achievements, problems, corrective actions and planning of next activities for concluding successfully the project.

In the frame of this task, P9 CFc supported P1 MPD in the collection of availabilities from the partners for the meeting date, in the definition of the meeting agenda as well as in the revision of the meeting minutes.

Legal and Administrative issues (P1 MDP - P9 CFc)
Along the project duration, P1 MPD and P9 CFc monitored that the partners were acting according to the Consortium Agreement, defined during the negotiation phase as well as according to the contracts defined among RTD Performers and SME Participants in relation to administrative issues.

WP2: Electronics and detector design for a 8x1 SPAD array. Development of a compact multichannel module.

Design of the linear SPAD array (POLIMI-CNR)
A linear SPAD array was designed according to the specifications set by all partners. The SPAD elements have 3 different diameters for the active area: 30 μm, 50 μm and 80 μm.

Besides the SPAD detector, each pixel includes a monolithically integrated pick-up circuit that has been specifically designed to reduce the capacitive load on the sensing node and, in turn, the couplings between adjacent channels. Another objective is to strongly reduce any low-pass filtering of the current flowing through the device, in order to improve the timing jitter.

The avalanche current flows through a sensing resistor (Rpoly) and the voltage drop across it is used as input to a typical nMOS inverter used to buffer the timing signal. The inverter includes two nMOS transistors that are laid out in a concentric fashion; M1 is located inside in order to optimize the area occupation. The aspect ratio of M1 is 10 times smaller than that of M2 in order to ensure an adequate low logic level.

In order to design and simulate the pick-up electronics, the basic behavior of MOS devices fabricated by using the mixed SPAD-nMOS technological process was fully characterized and modeled using the SPICE model BSIM3 v3.2.2. The model developed for the MOSFET devices was used to simulate the behavior of ratioed logic nMOS circuit, with two circular nMOS transistors. The agreement between simulation and experimental data is quite good.

Preliminary measurements performed on test structures including a SPAD device and a monolithically integrated pick-up circuit showed that it is possible to achieve a remarkable timing jitter of about 30ps FWHM with an equivalent threshold of 100 mV, much higher than that typically used with external pick-up circuit (10 mV).

Design and fabrication of integrated Active Quenching Circuits for SPAD array operation (POLIMI)
A key component of the multichannel detection module is the Active Quenching Circuit (AQC) that is needed to properly operate each SPAD element. In order to build a compact system, a fully integrated AQC (iAQC) has been developed as application specific integrated circuit (ASIC), by exploiting a high-voltage 0.35 μm CMOS technology available from the Europractice program. The circuit design was focused on the goals of minimizing power dissipation and having a minimum time delay between avalanche ignition and quenching and between quenching and reset (dead time). The minimum desired dead time is about 50 ns, which corresponds to a maximum saturated counting rate of about 20 Mcps.

Assembly and characterization of the multichannel module (POLIMI, MPD)
A compact module has been fabricated in order to easily fit the 8x1 photon counting detector into a microscopy system for single molecule spectroscopy. The complete system is mainly constituted by:
i)detection head including the three fabricated chips (SPAD with integrated electronics, iAQC and comparator arrays) and a TEC cooler,
ii) power and output boards and,
iii) closed-circuit liquid cooling system.

WP3 Technology for fabrication of a monolithic 8x1 SPAD array detector

Process flow definition and fabrication of linear SPAD arrays (CNR, POLIMI)
A deep investigation on the effect of the most critical steps in the fabrication process was performed in order to reduce the intrinsic noise of the SPAD detector. In particular:
- we identified some technological processes that had been used in the past as possible source of contaminations and we replaced each of them with another one that provided improved characteristics in terms of contaminations;
- we tested the effect of two different gettering processes;
- we changed the standard wafer cleaning employed at the end of each photolithographic process.

On-wafer characterization of the fabricated SPAD arrays (CNR)
The main goal of this task is a complete characterization of the new mixed SPAD-nMOS process employed for manufacturing the multichannel modules. On-wafer characterization was performed with the aim at reaching three main objectives:
1. analysis of the performances obtained for the manufactured MOSFET devices, extraction of the most important parameters and comparison with the expected values;
2. analysis of the performances obtained for the inverter circuits used to buffer the timing signal inside the multichannel module;
3. analysis of the performances obtained for SPAD devices and verification that the full SPAD-nMOS process does not affect the detector performance.

Process flow refinement and fabrication of a second SPAD array generation (CNR)
The characterization performed on single SPAD devices (first batch) highlighted that the DCR level is deeply influenced by the substrate quality. Therefore, with the aim of reducing the DCR level, new silicon substrates and new epilayers have been employed in the fabrication of a second batch of SPAD arrays. Furthermore, in order to reduce the optical crosstalk inside the SPAD array chip, highly doped substrates have been employed. The second fabrication run was carried out in the second year of the project with 6 different substrates and a complete electrical characterization of the single SPAD devices and of the 8x1 SPAD array fabricated on the new wafers was performed.

WP4 Microoptics for focusing and filtering onto the 8x1 array detector

Design and fabrication of microlens array (HWU-HPL-POLIMI)
A diffractive microlens array was used to increase the fill factor of the 1 x 8 SPAD detector array. The detectors elements had a 50 μm diameter circular active area and were on a 250 μm pitch. The microlens array consisted of a 1 x 8 array of diffractive microlenses, with a focal length of 1.040 mm, fabricated on a fused silica substrate. The array of diffractive microlenses is composed of two different microlens designs. The first, optimised for an illumination wavelength of 500nm, is designed to cover the wavelength range from 450nm and 550nm while the second, optimised for an illumination wavelength of 600nm covers from 550nm to 650nm.

Integration of microlens array and SPAD array (HWU-HPL-POLIMI)
A number of different approaches to the lens integration were first experimented using a range of photolithographic and micro-structuring hardware. The initial approach, using a Karl Suss MJB3 mask aligner, gave promising results with a translational placement accuracy of better than 5 micrometres although some degree of rotational misalignment was observed. We then started using a Karl Suss FC6 flip chip bonder which is better suited to the alignment process as it possesses a dual microscope system which allows the simultaneous observation of both the microlens surface and the SPAD chip. We have performed initial alignment tests using dummy substrates and have achieved alignment accuracies greater than those from the initial tests with no significant rotational misalignment.

WP5 ASIC-based based multichannel integrated time correlated single photon counting (TCSPC) system

Matching the ECL library for Multi Channel TCSPC applications (FHB)

The major results obtained in this respect are here listed.
- New or improved ECL cells, successfully designed, simulated and added to the existing library.
A five-stage timing oscillator vco_fivsuty was devised as the key element that generates the primary timing information for all other cells. A shift register SR32 was implemented to adjust every stage of this oscillator from a standard serial CMOS interface. A 4-bit asynchronous counter ctha5_4bit was developed for the low resolution timing share.
- An improved FSM (finite state machine) for fast readout of the ECL counter.
The result is the macro cell FSM_div6g, which as finite state machine generates a 3-bit Johnson code. The maximum clock rate for this state machine is matched to the vco_fivsuty cell and is higher than 6GHz (simulated and measured on a test structure).
- Simulation of the readout counter based on VHDL model.
Results were positively evaluated by the ModelSim Simulator. Problems were met due to metastable state transmissions and were solved by implementing a redundant flip-flop structure into the synchronization and by adjusting the logic of the time readout channels.
- Minimization of the PLL jitter effects and improvement of the long term stability of the PLL.
The test structures contained a phase comparator phdiff-_comp and a short-time integrator lowpass as new elements. These cells were designed to close the PLL loop. Measurements of this loop have shown a residual time-jitter of about 3ps rms. The contribution to the long term stability due to our circuit structures is below 3ps.

Detail of activities
As for the design of the PLL stabilized clock generator, the master timing information with the control unit is generated by a PLL stabilized clock generator.

In order to keep the power consumption at moderate to level, the speed of the circuitry was not pushed up to the maximum limit. If the time resolution requirement is set to about 20ps, the power consumption of an 8-channel chip may be restricted to approx. 1.5 watts. Provided that the PLL is locked on, the effective timing resolution will be determined by the external crystal frequency.

Matching the channel design suitable for multi-channel applications (FHB)
The major results obtained in this respect can be summarized as follows.
- Creation of a minimum of new ECL cells for the SPAD interface. A standard comparator cell was created in month 6 of the project; it was designed for low-voltage inputs below 100mV and ECL-matched outputs. In the following, as agreed with the project partners, the cell has been modified for a standard NIM input. The new modified cell was created in month 10. The timing behaviour is satisfactory for our application: the propagation delay is better than 25ps and the timing differences are below 3ps.
- Definition of the chip architecture and simulation of different structural variants were done in month 9. The stabilized clock generator and the time read-out channel were feed together. The modifications that we have done make possible a connection of up to eight time read channels to one PLL stabilized clock generator.
- Evaluation of the SG25H3 technology. It turned out that at the present stage not all the cells required for our design can be implemented in this technology. For future TDC applications, the SG25H3 technology will be undoubtedly a good choice, since it can provide the best performance to cost ratio. However, since not all the ECL standard cells are available today and many of them are even not yet specified, the development of a TDC in the SG25H3 technology cannot be attained within the time span of the PARAFLUO project. The SG25H3 technology should be considered for TDC projects over a longer time span.
- Evaluation of the SG13 technology. It turned out that this technology is not yet ready for precise timing applications. At the moment, some ECL standard cells and design kit adequate to evaluate test structures for the TDC application are not yet available. Therefore, this technology was discarded by the PARAFLUO project.
- The SG25H1 technology from the IHP turned out to be the only one suitable and available within the time span of the PARAFLUO project is. Therefore, in order to fulfil all the specifications set in the PARAFLUO project, it was decided to carry on the design with the SG25H1 technology. It is worth stressing, however, that this technology is the most expensive one for the time being.
- The full lot of schematics of the multi channel TCSPC chip were thus carried to the SG25H1 technology in order to prepare the next working steps. This operation, based on the results of the former steps, implied a lot of formal work although it did not require to develop new ideas.

Details of activities
The timing information of the PLL-stabilized clock generator will be latched whenever an input event is detected.

The LOW, MID and HIGH bits are coming from the PLL clock unit and will be latched after an event that must be indicated at the input. It is very important to fix the exact timing point by capturing the MID and HIGH bits in the right sequence. Three different readout registers were designed.

Full design of first 8-channel TDC chip (FHB)
The main result obtained in this respect was the layout generation of the TDC chip. The steps successfully completed by the end of April 2010 were:
- Design steps, DRC, ERC, extraction and analogue simulations
- Verification, generation of GDS data files
- Design review
- Finalized chip design and deliver to the chip factory

Mount and test the initial design and redesign (FHB)
At the beginning of the PARAFLUO project it was planned to realize the TDC as an integrated 8-channel chip. Later, simulations have shown that the expected power dissipation is too large for application of the SGB25V technology. Therefore, all the needed cells were transferred to the SG25H1 technology. This more expensive technology halves the heat dissipation at the same speed and chip area. To evaluate this technology, the first realization of a TDC chip was planned and realized as a 3-channel version. This permitted all the possibilities to evaluate the SG25H1 technology for a multichannel TDC in one chip. The manufacturing of the chip was realized with EUROPRACTICE.

Mount and test the final version, documentation (FHB)
Because of the crosstalk between different channels, the existing design of an 8-channel TDC chip was not given to the manufacturer. Instead of this, the 8-fold single chip version was given preference. Every one-channel TDC chip contains the PLL stabilized clock generator and one readout channel. The synchronization is provided by a common crystal clock source. The distribution of this common clock may be realized by a careful design of all wiring traces to the channel modules. Under these conditions, a jitter between the common crystal clocks of the PLL blocks in any TDC chip will not exceed 5ps. By choice of the SGB25V technology, the power consumption of every TDC chip is slightly below 1W. Therefore, this more inexpensive technology was chosen.

FPGA design for TDC data processing (FHB-PQ)
Firstly, the communication between TDC chip and FPGA were tested.

After successful testing of the communication between TDC chip and FPGA, stable choice of a master clock frequency was possible. After the developments concerning the interface between the chip-based TDC modules and the Virtex5 FPGA, the integration of this configuration into the HydraHarp system was necessary. In order to accommodate the higher data throughput of a collection of 8 channel TDC modules, the HydraHarp bus system has been expanded and the event data sorter has been parallelized. Basic functionality was verified by means of simulations and test generator data.

Software design adaption and overall design (PQ)
The software of the HydraHarp platform was expanded to accommodate eight-channel data handling in each module. This included the redesign of the module firmware as well as modifications of the low level data structures in the software library of the HydraHarp system. The device driver was overhauled to work with the latest versions of Windows including the 64-bit versions. The graphical user interface of the HydraHarp software required no significant modifications since it was readily designed to handle up to 32 channels. Only some real-time data analysis features were improved for speed. Due to delays in physically completing the 8-channel module, testing of the overall system was started late. Within the project extension it was possible to test basic functionality but full system testing could not be performed in time.

WP6 Integration of the 8x1 array detector and TCSPC multichannel electronics into a microscopy system

Incorporation of the spectrally dispersing element, the microlens array, and detector array into the existing confocal scanning microscope MT200 (UGOE-PQ)

The design and implementation of the spectrograph dispersing element into the confocal scanning microscope MT200 was achieved in the first period of the project. For that purpose, special mechanical elements have been custom-built in our workshop for holding the spectrograph. It was important to find a mechanical solution which makes possible an easy alignment without sacrificing stability. Thus, we used available CAD software to design the mechanical holder and to optimize the design with respect to mechanical stability of the whole imaging system. Because the 8-channel SPAD detector module was not yet available, we temporally installed a single-photon sensitive back-illuminated EM-CCD camera (Andor iXon) as detector, so that we were able to record spectrally resolved fluorescence images in our confocal set-up with single-molecule sensitivity. This helped also to experimentally check the mechanical stability of the set-up and of the alignment of all the opto-mechanical parts.

Spot size and position
In order to determine the position and size of the focal spots that are created by the microlens array we illuminated the CCD homogeneously with spectrally filtered light. The light was emitted from a non-coherent light bulb and collimated to form an adequately planar wave-front.

Optical cross-talk
From the measurement we can also obtain the amount of light that is guided to the neighboring focus and compare it to the signal of the primary focus.

The measurements of the optical cross-talk show that a significant amount of light is guided onto the next pixel in the row. Whereas the cross-talk to the previous pixel is less than 5%, it reaches values above 30% in the forward direction. We believe that this is a consequence of the missing correction lens in front of the microlens array. This leads to an inclined illumination of the structure and shifts the position of the spot. To correct for this the beam has to be moved farther than necessary and thus hits partially the next microlens structure.

An important figure of merit is the absolute transmission of the whole dispersing system. By comparing the total signal in the focused spots with the total signal in an area of the chip that has the same size than the microlens structure we were able to obtain the total transmission of the structure. As expected, it varies with increasing difference between the design wavelength and the actually used wavelength of light. In the usable wavelength range, the total efficiency is around 60% to 80%. With a reduced optical cross-talk we assume that one can consistently reach values larger than 75%.

Development of dedicated software for fast and efficient data evaluation (UGOE-PQ)
This task is a key one for WP6 and probably for the whole project, because the applicability of spectrally resolved fluorescence lifetime measurements strongly depends on the ability to reduce the amount of raw data and to process them within an acceptable time. The data as it is acquired by the microscope system will be composed, in its final version, of eight spectral channels with fluorescence decay curves of greater than 102 time channels per curve, each channel containing 2 byte of information. This leaves us with a data amount of ca. 20 kB per image pixel, which results, for a 1000 by 1000 pixel image, in a total amount of 2 GB data per image.

The treatment of spectrally resolved FLIM data requires several steps of pre-evaluation for efficiently dealing with the huge amount of raw data. In a first step, we compute all TCSPC histograms for each pixel and each spectral channel. If the data about the instrumental response function IRF is not available, we estimate the IRF by an appropriate model. The next step corrects the data for possible time-shifts of the IRF in each spectral channel. In general all the data are then shifted in time so that the center of mass of the IRF in each channel lies at 1.5 ns. If no explicit values are given, we estimate dark-count rate and afterpulsing probability of the detector by the signal observed in the time-bins before the onset of the IRF. After correction for background contributions, the histograms data are binned into increasingly wider time-bins, beginning with 32 ps and doubling every eight bins after the peak of the IRF.

In order to identify all the fluorescent components in the recorded image, several key parameters are computed on a pixel-by-pixel basis. These are the spectrum of the recorded fluorescence, as well as a distinctive description of the fluorescent decays on the basis of their moments m(j) which are defined as m(j)=?Iktkj where Ik is the fluorescence intensity in the kth channel of a fluorescence decay curve (measured with time-correlated single-photon counting), tk is the decay time-value of the kth channel, and the summation runs from the first to the last (Nth) channel. The superscript j denotes the order of the moment. Such a moment calculation allows reducing the whole data set of a fluorescence decay curves to only 3 to 4 relevant first moments, which contain the significant information about the fluorescence decay and can then be processed in a further analysis. This moment-based approach works very similar to a phasor approach, but without the necessity to work with complex-valued algebra. We developed a Matlab-based first version of a software package which has the moment analysis at its core.

Development of fast and efficient algorithms for visualizing the relevant spatial, spectral, and lifetime information contained in the scanned images (UGOE-PQ)
This task is closely related to the previous one, because the efficient visualization of the final data strongly depends on the ability to drastically reduce the amount of raw data acquired by the microscope system.

Broad application of the developed sFLIM system for imaging protein-protein interactions by use of FRET (UGOE)
Due to the aforementioned delay in the finalization of the 8x1 SPAD module with integrated microlens array we have not been able to perform measurements of actual fluorescent samples employing the developed 8x1 SPAD array detector. However, we experimentally verified that the performance of each pixel of the SPAD array detector is identical to that of single SPAD detectors and we performed several measurements based on conventional lifetime imaging using individual SPAD modules with appropriate spectral bandpass filters. In this way we were able to test also the performance and applicability of the developed software. Using this approach we studied protein-protein interaction in focal-adhesions of cells.

Redesign of all initially developed hardware modules and their integration into a first fully functional prototype of the MT200 (UGOE-PQ)
The optical and mechanical hardware described in the first paragraph of this WP6 report was iteratively developed, tested and improved in close collaboration between UGOE and PQ. In the final version it is fully working incorporated in the MicroTime 200 system. The accurate tests carried out with the alternative set-up based on the emCCD detector with integrated microlens array provided experimental confirmation that the apparatus ensures performance fully satisfactory also for operating with the 8x1 SPAD array detector.

WP7 Dissemination and exploitation

Definition of a dissemination plan (All partners)
A dissemination plan, aimed to identify the target groups, communication objectives, activities, tools, timing and partners responsibilities for the dissemination, was discussed by all partners, supported by P9-CFc during the first 6 months of the project. Based on the discussions undertaken, CFc supported the Coordinator in the implementation of the dissemination plan in the project activities and in writing the Deliverable D7.2 "Interim Plan for use and dissemination of the knowledge", submitted at month 12.

Design of a website (P1 MPD)
In order to install a platform for intra and extra dissemination of project results MPD set up the project website, which is accessible through the following URL:
The website is an information channel for the promotion of the project and its results. It assists partners in their dissemination activities by providing updated information on project status, as well as reference documents and materials. It aims at attracting the interest of scientists employing fluorescence measurements in their research.

Publications on peer-reviewed journals
1. I. Rech, A. Gulinatti, M. Crotti, C. Cammi, P. Maccagnani, and M. Ghioni, "Towards picosecond array detector for single-photon time-resolved multispot parallel analysis", Journal of Modern Optics, vol. 58, iss. 3 and 4, pp. 233-243, Jan. 2011. IF = 0.988
2. A. Gulinatti, I. Rech, M. Assanelli, M. Ghioni, and S. Cova, "A physically based model for evaluating the photon detection efficiency and the temporal response of SPAD detectors", Journal of Modern Optics, vol. 58, iss. 3 and 4, pp. 210-224, Jan. 2011. IF = 0.988
3. A. Ingargiola, M. Assanelli, I. Rech, A. Gulinatti, M. Ghioni, "Avalanche Current Measurements in SPADs by Means of Hot-Carrier Luminescence", Photonics Technology Letters, vol. 23, iss. 18, pp. 1319-1321, Sep. 2011. IF = 1.989
4. M. Assanelli, A. Ingargiola, I. Rech, A. Gulinatti, and M. Ghioni, "Photon-Timing Jitter Dependence on Injection Position in Single-Photon Avalanche Diodes", IEEE Journal of Quantum Electronics, vol. 47, iss. 2, pp. 151-159, Feb. 2011. IF = 2.480
5. C. Cammi, A. Gulinatti, I. Rech, F. Panzeri, M. Ghioni, "SPAD Array Module for Multi-Dimensional Photon Timing Applications", Journal of Modern Optics, [in press]. IF = 0.988

Participation in events

Presentations to conferences (without conference proceedings)
1. Oral presentation at Single Photon Workshop 2009, November 3-6, 2009 Boulder (Colorado, USA)
2. Oral presentation at SPIE Conference, 5-9 April 2010, Orlando - Florida
3. Discussion with experts Biomedical Optics (BIOMED) part of Biomedical Optics and 3-D Imaging: OSA Optics and Photonics Congress, April 11-14, 2010, Miami, Fl, USA
4. Lectures at 5th Workshop on "Making Single Molecule Fluorescence (Lifetime) Measurements Simple" April 26-28, 2010, Upton, NY, USA
5. Discussion with experts Single Molecule Approaches To Biology, Gordon Research Conferences, June 27 - July 2, 2010, Lucca, Italy
6. I. Rech, C. Cammi, A. Gulinatti, M. Ghioni, S. Cova, "Parallel fluorescence photon timing module with monolithic SPAD array detector", oral presentation at 17th International Workshop on Single Molecule Spectroscopy and Ultra Sensitive Analysis in the Life Sciences, Berlin, September 7-9, 2011.
7. A. Gulinatti, F. Panzeri, I. Rech, P. Maccagnani, M. Ghioni, S. Cova, "A new Silicon SPAD with improved red efficiency and 100 ps timing resolution", poster presentation at 17th International Workshop on Single Molecule Spectroscopy and Ultra Sensitive Analysis in the Life Sciences, Berlin, September 7-9, 2011.
8. A. Gulinatti and I. Rech, "New Silicon SPAD technology for enhanced red-sensitivity, high-resolution timing and system integration", oral presentation at Single Photon Workshop 2011, Braunschweig, June 27-30, 2011, [Invited].
9. I. Rech, C. Cammi, A. Gulinatti, M. Ghioni, S. Cova, M. Taghizadeh, G. Buller, "Micro-optics system and SPAD array detectors for parallel photon timing applications", oral presentation at European Optical Society Annual Meeting 2010 (EOSAM 2010), Paris, October 26-29, 2010, [Invited].
10. "High Speed ASICs for Picosecond Applications", Presentation of the TDC chip at the Hannover Messe 2011, Hannover, Hall 2 booth A30, April 4-8, 2011.G. Kell, D. Schulz, "Successfully Tested ECL Cells and Macros", submission at the IHP's 10th Workshop High-Performance SiGe:C BiCMOS for Wireless and Broadband Communication, September 21, 2011.
11. I. Gregor (University of Gottingen) "Benefiting from advanced FCS methods in Biology", F. Koberling (PicoQuant GmbH) "Fluorescence Lifetime: a new dimension for confocal imaging and FCS" and R. Erdmann (PicoQuant GmbH) "Single Molecule Microscopy: From Nanodiamonds to Nanomanipulation", given talks to the 6th Workshop on Advanced fluorescence spectroscopy and microscopy: from cells to single molecules, Los Angeles, CA, USA January 20, 2011.
12. F. Koberling "Lifetime-Resolved FRET Microscopy" talk at the „International Bunsen Discussion Meeting: Forster Resonance Energy Transfer in Life Sciences", Gottingen March 27-30, 2011.
13. B. Kramer (Advanced time-resolved fluorescence techniques enable for high-resolution deep-tissue FLIM imaging) and F. Koberling (Single Molecule Microscopy: From Nanodiamonds to Nanomanipulation") talk at the "Focus on Microscopy (FOM)", Konstanz, Germany, April 17-20, 2011.

Participation to fairs and exhibitions
1. SPIE Bios Exhibition, Booth 8910, January 22 - 23, 2011, San Francisco (CA, USA).
2. SPIE Photonics West Exhibition, Booth 4829, January 25 - 27 , 2011, San Francisco (CA, USA).
3. Hannover Messe, hall2 booth A30, April 3-8, 2011, Hannover (Germany).
4. SPIE Defense, Security and Sensing Exhibition, Booth 3414, April 25 - 29, 2011, Orlando (FL, USA).
5. Laser World of Photonics, Hall B2, Booth 466, May 23 - 26, 2011, Munich (Germany).
6. Single Photon Workshop Exhibition, June 27 - 30, 2011, Braunschweig (Germany).

Dissemination seminars at universities and research centres

In addition to what had been initially agreed, during the second period the partners deemed necessary to carry out further actions for promoting contacts with potential users with the aim of establishing a starting base for the exploitation phase. Therefore, a number of dedicated seminars were organized at universities and research centres for enhancing the dissemination, as outlined in the following.

1. Presentation given to Professor Yoshihisa Yamamoto's group, Center for Nanoscale Science and Engineering, Stanford University, San Jose (CA, USA), January 24, 2011.
2. Presentation given to researchers at Sandia National Laboratory, Livermore (CA, USA), January 24, 2011.
3. Presentation given to researchers at Aerospace Corporation, Photonics Technology Department, El Segundo (CA, USA), January 28, 2011. Presentation given to researchers at HRL Laboratories, Malibu (CA, USA), January 28, 2011.
4. Presentation given to researchers at NIST (National Institute of Standards and Technology), Boulder (CO, USA), January 31, 2011.
5. Presentation given to researchers at NREL (National Renewable Energy Laboratory), Golden (CO, USA) , January 31, 2011.
6. Presentation given to Dr. Srinivasan, Kartik's group at NIST (National Institute of Standards and Technology), Gaithersburg, (MD, USA), February 2, 2011.
7. Presentation given to Dr. Alan Migdal's group at NIST (National Institute of Standards and Technology), Gaithersburg, (MD, USA), February 2, 2011.
8. Presentation given to Dr. Glenn Solomon's group at NIST (National Institute of Standards and Technology), Gaithersburg, (MD, USA), February 2, 2011.
9. Presentation given to Dr. Hwang Jeeseong's group at NIST (National Institute of Standards and Technology), Gaithersburg, (MD, USA), February 2, 2011.
10. Presentations to Prof. Dr. Fred S. Wouters at Center for Physiology and Pathophysiology, University Hospital Göttingen, Göttingen (Germany)
11. Presentations to Dr. Ute Resch-Genger at Federal institute for materials research and testing, Berlin (Germany)
12. Study work at the FH Brandenburg, "Metastabile Zustände in digitalen Speichern" (Schulz, D), March 26, 2010.
13. ECL Library and Design Meeting, "Structures and applications of ultrafast ECL cells with SiGe bipolar transistors" (G. Kell), FH Brandenburg, June 17 2010.
14. Public reading at the FH Brandenburg, "Kleiner - schneller - sparsamer: Neuere Tendenzen und Entwicklungen bei der Entwicklung digitaler Halbleiterchips" (G. Kell), October 20, 2010.
15. Examination work for the Master degree at the FH Brandenburg, "Modular strukturiertes Architekturkonzept für ein EDA-Werkzeug mit 3D-Fähigkeiten" (Martin Ahlborg), August 26, 2011.
16. Presentation at the IHP: "Simulation result exports and chip microscopy, (Martin Ahlborg), February 16, 2011.
17. Presentation at the IHP, "Design viewing tools and its developments" (G. Kell, D. Schulz and M. Ahlborg), May 18-25, 2011.
18. Presentation at the IHP, "Design kit evaluation tools and simulation aspects", (Daniel Schulz), June 8-9th, 2011.
19. Scientific discussion at the IHP, "Design conversion tools" (Martin Ahlborg), July 1, 2011.
20. Review meeting at the IHP, "Evaluation of metastable Flipflop effects", (Daniel Schulz), July 14, 2011.

Exploitation (MPD, PQ, HPL, POLIMI, CNR, FHB, UGOE, HWU)
The dissemination activities performed during the project and above outlined have been considered by the project partners to be instrumental to the exploitation, since they constitute a vehicle that facilitates and supports it.
In fact, the target audience identified for the PARAFLUO exploitation was the same as for the dissemination, namely:
- Actors within the research community;
- Licensees or industrial actors.

Visits in Europe
Visit to Paul Dear at MRC Laboratory of Molecular Biology, Cambridge (UK)
Visit to Holger Wegendt at Carl Zeiss AG, Oberkochen (Germany)
Visit at Heidelberg Engineering GmbH, Heidelberg (Germany)
Visit to Dr. Hilmar Gugel at Leica Microsystems CMS GmbH, Mannheim (Germany)
Visit at Max-Planck-Institute for Biophysical Chemistry, Department of NanoBiophotonics, Göttingen (Germany)
Visit to Thomas Kalkbrenner and Mirko Liedtke at Carl Zeiss MicroImaging GmbH, Jena (Germany)
Visit to Dietrich Schweitzer at Experimental Ophthalmology University, University Clinics, Jena (Germany)

Visits in US
Visit at Illumina Inc., Hayward (CA, USA)
Visit at Atwater Research Group, California Institute of Technology, Pasadena, (CA, USA)
Visit to Dr. Xavier Michalet, UCLA (University of California Los Angeles), Los Angeles (CA, USA).
Visit to Dr. Michael Krainak's group at NASA Goddard Space Flight Center, Greenbelt (MD, USA).
Visit at University of Sherbrooke, Faculté de Génie Électrique et Informatique, Sherbrooke (Canada).

Updated view on the prospect of exploitation
The results of the PARAFLUO project are high-end scientific modules and software which are mainly marketed in the academic research community. There is good prospect for them to be incorporated in the next generation of time resolved instrumentation and complete microscope products produced by the SME partners of PARAFLUO. Multi-channel TCSPC in combination with the SPAD array detector will make possible to perform measurements in biological applications much faster than with currently available instrumentation, thus overcoming one of the main limitations to a broader application of FLIM and FCS in the community. The SME partners are confident that they will be able to put on the market fully qualified and tested products ready for use within 18 months after the end of the project.

Transfer of knowledge and exploitation activities (MPD, PQ, HPL, POLIMI, CNR, FHB, UGOE, HWU)
This task referred to the management of all the activities needed to have a complete transfer of the project results from RTD performers to SME participants. The transfer of results from RTD performers to SME participants represents an intrinsic feature of the PARAFLUO project and in general of projects targeted to SMEs. During the first reporting period, therefore, all the project activities have been performed with this scope in mind. This is testified also by the number of meetings held between the partners.

Protection of Intellectual Property Rights (MPD, PQ, HPL, POLIMI, CNR, FHB, UGOE, HWU)
No patent was filed during the project period.
However, during the first reporting period, as agreed in Annex I and in the Consortium Agreement, particular attention has been devoted to avoid publication by RTD Performers of results that may be against the interest of SME partners. For this reason, before publishing the results of the project, RTD Performers obtained the agreement of the SMEs.

Potential Impact:

PARAFLUO combined efforts, know-how and competencies of different partners in an interdisciplinary project. This provided all partners with the opportunity to benefit from each other and strengthen their technological knowledge. Existing knowledge can thus be expanded and leveraged to address opportunities in new areas and markets.

Impact in terms of SMEs competitiveness
In the course of the project, new products have been developed as a result of collaboration between the SMEs and the RTD performers.

The SMEs gained a direct economic benefit in two ways:
(a) thanks to the new products they will enter the respective markets with additional turnover and
(b) these markets will offer higher margins;
c) through the launch of the new products they will enter new market segments. In more detail:
- MPD (the SME with well consolidated experience in the field of photon-counting detectors and associated electronics) will strengthen its market position in the field of photon-counting by offering an innovative linear array detector and associated integrated circuits. According to the marketing experience gained on the field in the past years of MPD activity, there is a significant demand of single photon detectors arrays. The expected impact brought by the availability of such devices will expand the current markets and open new ones.

- PQ (the SME specialised in optoelectronic instrumentation for photon counting and confocal microscopy systems) will profit from being able to use the new electronics and the optical setup of the microscope system as components of their advanced instrumentation systems. The new 8-channel TCSPC module will complete the existing family of TCSPC products and will secure PQ the leading position in this market. The availability of the 8-channel SPAD array will make possible for microscopy customers various new applications and methods and will make PQ more competitive from the standpoint of the leading microscopy companies like Zeiss, Olympus or Leica.
- HPL (the SME with consolidated experience in the field of precision optical coatings for semiconductor devices and optical components) will acquire know-how about microlens production and coating. The knowledge acquired during the project will make HPL more competitive in optical technology; the enhanced competitiveness in the European market and in international scenarios will generate additional turnover and profit growth. The know-how developed by HPL in the project will be exploited also for developing components and processes intended for new markets, for instance for time-of-flight LIDAR, where single photon-detection arrays can enhance the efficiency.

Contribution of the project in addressing Community societal objectives
All biological processes rely on complex interactions of biomolecules such as proteins, DNA, RNA, lipids, sugars etc. These interactions usually take place in large molecular assemblies. Typical examples are the "molecular machines" involved in signal transduction, transcription and translation of genomic information, protein degradation, or intracellular transport processes. A better understanding of these processes is paramount for a better understanding of the (patho)physiology of tissues and organisms and gives a base for gaining a better insight in key medical issues, such as origin and growth mechanisms of tumours.

Main dissemination activities and exploitation
For the activities performed in the frame of dissemination and exploitation, please refer to the description of WP7 above as well as to the Section 4.2 below.

List of Websites: