Electron multiplying CCD – based detection in Fluorescence Correlation Spectroscopy and measurements in living zebrafish embryos

Fluorescence correlation spectroscopy (FCS) is an ultra-sensitive optical technique to investigate the dynamic properties of ensembles of single fluorescent molecules in solution. It is in particular suited for measurements in biological samples. High sensitivity is obtained by employing confocal microscopy setups with diffraction limited small detection volumes, and by using single-photon sensitive detectors, for example avalanche photo diodes (APD). However, fluorescence signal is hence typically collected from a single focus position in the sample only, and several measurements at different positions have to be performed successively. To overcome the time-consuming successive FCS measurements, we introduce electron multiplying CCD (EMCCD) camera-based spatially resolved detection for FCS. With this new detection method, multiplexed FCS measurements become feasible. Towards this goal, we perform FCS measurements with two focal volumes. As an application, we demonstrate spatial cross-correlation measurements between the two detection volumes, which allow to measure calibration-free diffusion coefficients and direction-sensitive processes like molecular flow in microfluidic channels. FCS is furthermore applied to living zebrafish embryos, to investigate the concentration gradient of the morphogen fibroblast growth factor 8 (Fgf8). It is shown by one-focus APD-based and two-focus EMCCD-based FCS, that Fgf8 propagates largely by random diffusion through the extracellular space in developing tissue. The stable concentration gradient is shown to arise from the equilibrium between a local morphogen production and the sink function of the receiving cells by receptor-mediated removal from the extracellular space. The study shows the applicability of FCS to whole model organisms. Especially in such dynamically changing systems in vivo, the perspective of fast parallel FCS measurements is of great importance. In this work, we exemplify parallel, spatially resolved FCS by utilizing an EMCCD camera. The approach, however, can be easily adapted to any other class of two-dimensional array detector. Novel generations of array detectors might become available in the near future, so that multiplexed spatial FCS could then emerge as a standard extension to classical one-focus FCS.Fluoreszenz-Korrelations-Spektroskopie (FCS) ist eine hochempfindliche optische Methode, um die dynamischen Eigenschaften eines Ensembles von einzelnen, fluoreszierenden Molekülen in Lösung zu erforschen. Sie ist insbesondere geeignet für Messungen in biologischen Proben. Die hohe Empfindlichkeit wird erreicht durch Verwendung konfokaler Mikroskop-Aufbauten mit beugungsbegrenztem Detektionsvolumen, und durch Messung der Fluoreszenz mit Einzelphotonen-empfindlichen Detektoren, zum Beispiel Avalanche-Photodioden (APD). Dadurch wird das Fluoreszenzsignal allerdings nur von einer einzelnen Fokusposition in der Probe eingesammelt, und mehrfache Messungen an verschiedenen Positionen in der Probe müssen nacheinander durchgeführt werden. Um die zeitaufwendigen, aufeinanderfolgenden FCS-Einzelmessungen zu überwinden, entwickeln wir in dieser Arbeit Elektronenvervielfachungs-CCD (EMCCD) Kamera-basierte räumlich aufgelöste Detektion für FCS. Mit dieser neuartigen Detektionsmethode werden Multiplex-FCS Messungen möglich. Darauf abzielend führen wir FCS Messungen mit zwei Detektionsvolumina durch. Als Anwendung nutzen wir die räumliche Kreuzkorrelation zwischen dem Signal beider Fokalvolumina. Sie ermöglicht die kalibrationsfreie Bestimmung von Diffusionskoeffizienten und die Messung von gerichteter Bewegung, wie zum Beispiel laminarem Fluss in mikrostrukturierten Kanälen. FCS wird darüber hinaus angewendet auf Messungen in lebenden Zebrafischembryonen, um den Konzentrationsgradienten des Morphogens Fibroblasten-Wachstumsfaktor 8 (Fgf8) zu untersuchen. Mit Hilfe von APD-basierter ein-Fokus FCS und EMCCD-basierter zwei-Fokus FCS zeigen wir, dass Fgf8 hauptsächlich frei diffffundiert im extrazellulären Raum des sich entwickelnden Embryos. Der stabile Konzentrationsgradient entsteht durch ein Gleichgewicht von lokaler Morphogenproduktion und globalem Morphogenabbau durch Rezeptor vermittelte Entfernung aus dem extrazellulären Raum. Die Studie zeigt die Anwendbarkeit von FCS in ganzen Modell-Organismen. Gerade in diesen sich dynamisch ändernden Systemen in vivo ist die Perspektive schneller, paralleler FCS-Messungen von großer Bedeutung. In dieser Arbeit wird räumlich aufgelöste FCS am Beispiel einer EMCCD Kamera durchgeführt. Die Herangehensweise ist jedoch einfach übertragbar auf jede andere Art von zwei-dimensionalem Flächendetektor. Neuartige Flächendetektoren könnten in naher Zukunft verfügbar sein. Dann könnte räumlich aufgelöste Multiplex-FCS eine standardisierte Erweiterung zur klassischen ein-Fokus FCS werden

High Frequency Sampling of TTL Pulses on a Raspberry Pi for Diffuse Correlation Spectroscopy Applications

Diffuse Correlation Spectroscopy (DCS) is a well-established optical technique that has been used for non-invasive measurement of blood flow in tissues. Instrumentation for DCS includes a correlation device that computes the temporal intensity autocorrelation of a coherent laser source after it has undergone diffuse scattering through a turbid medium. Typically, the signal acquisition and its autocorrelation are performed by a correlation board. These boards have dedicated hardware to acquire and compute intensity autocorrelations of rapidly varying input signal and usually are quite expensive. Here we show that a Raspberry Pi minicomputer can acquire and store a rapidly varying time-signal with high fidelity. We show that this signal collected by a Raspberry Pi device can be processed numerically to yield intensity autocorrelations well suited for DCS applications. DCS measurements made using the Raspberry Pi device were compared to those acquired using a commercial hardware autocorrelation board to investigate the stability, performance, and accuracy of the data acquired in controlled experiments. This paper represents a first step toward lowering the instrumentation cost of a DCS system and may offer the potential to make DCS become more widely used in biomedical applications.Radiation Monitoring Devices, Inc

Monte Carlo Simulations of Single-Molecule Fluorescence Detection Experiments

Several Monte Carlo simulations of single-molecule fluorescence systems are developed to help evaluate and improve ongoing experiments. In the first simulation, trapping of a single molecule in a nanochannel is studied. Molecules move along the nanochannel by diffusion and electrokinetic flow. Single-molecule fluorescence signals excited by two spatially offset laser beams are detected and the direction of the flow is adjusted to try to equalize the signals and center the molecule between the beams. An algorithm is evaluated for trapping individual molecules in succession by rapidly reloading the trap after a molecule photobleaches or escapes. This is shown to be effective for trapping fast-diffusing single-chromophore molecules in succession within a micron-sized confocal region while accommodating the limited electrokinetic speed and the finite latency of feedback imposed by experimental hardware. In the second simulation, trapping of a molecule in a two-dimensional fluidic device consisting of sub-micron-separated glass plates is studied. Two different illumination schemes for sensing the molecule\u27s position are compared: (i) a single continuous laser spot circularly scanned at 40 KHz or 240 KHz in the plane of the device; and (ii) four pulsed laser spots arranged in a square and temporally alternated at 304 MHz In either case, the times of detected photons are used by algorithms to control the electrokinetic flow in two dimensions to compensate diffusion and achieve single-molecule trapping. However each scheme is found to have limitations, as circular scanning produces a modulation in the fluorescence signal and in the autocorrelation function, whereas the four-pulse scheme becomes ineffective if the fluorescence lifetime of the molecule is greater than the time between laser pulses, The third simulation investigates appropriate conditions for detection of single molecules flowing through an array of fluidic channels for an application to high-throughput screening for pharmaceutical drug discovery. For parallelized single-molecule detection, illumination is provided by a continuous laser focused to a line intersecting all channels and fluorescence is imaged to a single row of pixels of an electron-multiplying CCD with sufficient gain for single-photon detection. The simulation separately models each channel to determine laser, flow, and camera operating conditions suitable for efficient detection

Development of fluorescence lifetime measurement techniques for use in microfluidic channels

Fluorescence lifetime measurements are a powerful tool in biomedical research and advances in detection technology make them ideally suited for the study of biomolecular interactions. Time-resolved techniques, compared to more conventional methods, provide improved precision and contrast in the monitoring of complex biological processes. Fluorescence lifetimes are extracted by using time-correlated single-photon counting, which offers single photon sensitivity, high temporal resolution and excellent signal to noise ratio. Furthermore, combining this technique with microfluidics offers unprecedented advantages. For example, in analytical applications, apart from the high sensitivity required, the study of analytes often demands low sample consumption and short mixing times to allow for the monitoring of quick reactions. These parameters can nicely be achieved with the use of microfluidics. Hydrodynamic focusing within 3-inlet 1-outlet continuous flow microfluidic devices can be used as a molecular confinement mechanism to improve the detection efficiency as well as a means to enhance mixing within microchannels for the study of fast reaction kinetics. In this work, a powerful combination of confocal microscopy and microfluidics was used to perform fluorescence lifetime measurements on freely diffusing and freely flowing molecules. For this purpose, a home-built scanning confocal system was developed to ensure sufficient reduction in background levels, enabling the detection of fluorescence signal that arises from single molecules. Fluorescence lifetime imaging along with a maximum likelihood estimator adapted from single molecule studies was performed to visualise hydrodynamic focusing and characterise mixing within microfluidic devices. Time-resolved methods were also employed to detect single molecules freely flowing within microchannels. A novel fluorescence lifetime approach was developed to perform Förster resonance energy transfer measurements on freely diffusing molecules and subsequently applied for the study of streptavidin-biotin binding and protein conformational changes upon unfolding

The COMPASS Experiment at CERN

The COMPASS experiment makes use of the CERN SPS high-intensitymuon and hadron beams for the investigation of the nucleon spin structure and the spectroscopy of hadrons. One or more outgoing particles are detected in coincidence with the incoming muon or hadron. A large polarized target inside a superconducting solenoid is used for the measurements with the muon beam. Outgoing particles are detected by a two-stage, large angle and large momentum range spectrometer. The setup is built using several types of tracking detectors, according to the expected incident rate, required space resolution and the solid angle to be covered. Particle identification is achieved using a RICH counter and both hadron and electromagnetic calorimeters. The setup has been successfully operated from 2002 onwards using a muon beam. Data with a hadron beam were also collected in 2004. This article describes the main features and performances of the spectrometer in 2004; a short summary of the 2006 upgrade is also given.Comment: 84 papes, 74 figure

The BrightEyes-TTM: an open-source time-tagging module for fluorescence lifetime imaging microscopy applications

The aim of this Ph.D. work is to reason and show how an open-source multi-channel and standalone time-tagging device was developed, validated and used in combination with a new generation of single-photon array detectors to pursue super-resolved time-resolved fluorescence lifetime imaging measurements. Within the compound of time-resolved fluorescence laser scanning microscopy (LSM) techniques, fluorescence lifetime imaging microscopy (FLIM) plays a relevant role in the life-sciences field, thanks to its ability of detecting functional changes within the cellular micro-environment. The recent advancements in photon detection technologies, such as the introduction of asynchronous read-out single-photon avalanche diode (SPAD) array detectors, allow to image a fluorescent sample with spatial resolution below the diffraction limit, at the same time, yield the possibility of accessing the single-photon information content allowing for time-resolved FLIM measurements. Thus, super-resolved FLIM experiments can be accomplished using SPAD array detectors in combination with pulsed laser sources and special data acquisition systems (DAQs), capable of handling a multiplicity of inputs and dealing with the single-photons readouts generated by SPAD array detectors. Nowadays, the commercial market lacks a true standalone, multi-channel, single-board, time-tagging and affordable DAQ device specifically designed for super-resolved FLIM experiments. Moreover, in the scientific community, no-efforts have been placed yet in building a device that can compensate such absence. That is why, within this Ph.D. project, an open-source and low-cost device, the so-called BrightEyes-TTM (time tagging module), was developed and validated both for fluorescence lifetime and time-resolved measurements in general. The BrightEyes-TTM belongs to a niche of DAQ devices called time-to-digital converters (TDCs). The field-gate programmable array (FPGA) technology was chosen for implementing the BrightEyes-TTM thanks to its reprogrammability and low cost features. The literature reports several different FPGA-based TDC architectures. Particularly, the differential delay-line TDC architecture turned out to be the most suitable for this Ph.D. project as it offers an optimal trade-off between temporal precision, temporal range, temporal resolution, dead-time, linearity, and FPGA resources, which are all crucial characteristics for a TDC device. The goal of the project of pursuing a cost-effective and further-upgradable open-source time-tagging device was achieved as the BrigthEyes-TTM was developed and assembled using low-cost commercially available electronic development kits, thus allowing for the architecture to be easily reproduced. BrightEyes-TTM was deployed on a FPGA development board which was equipped with a USB 3.0 chip for communicating with a host-processing unit and a multi-input/output custom-built interface card for interconnecting the TTM with the outside world. Licence-free softwares were used for acquiring, reconstructing and analyzing the BrightEyes-TTM time-resolved data. In order to characterize the BrightEyes-TTM performances and, at the same time, validate the developed multi-channel TDC architecture, the TTM was firstly tested on a bench and then integrated into a fluorescent LSM system. Yielding a 30 ps single-shot precision and linearity performances that allows to be employed for actual FLIM measurements, the BrightEyes-TTM, which also proved to acquire data from many channels in parallel, was ultimately used with a SPAD array detector to perform fluorescence imaging and spectroscopy on biological systems. As output of the Ph.D. work, the BrightEyes-TTM was released on GitHub as a fully open-source project with two aims. The principal aim is to give to any microscopy and life science laboratory the possibility to implement and further develop single-photon-based time-resolved microscopy techniques. The second aim is to trigger the interest of the microscopy community, and establish the BrigthEyes-TTM as a new standard for single-photon FLSM and FLIM experiments

Detectors for Super-Resolution & Single-Molecule Fluorescence Microscopies

The resolution of light microscopy was thought to be limited to 250–300 nanometers based on the work of Ernest Abbe. This Abbe diffraction limit was believed to be insurmountable until the invention of Super-resolution microscopic techniques in the late 20th century. These techniques remove this limit and have provided unprecedented detail of cellular structures and dynamics down to several nanometers. An emerging goal in this field is to quantitatively measure individual molecules. Measurement of single-molecule dynamics, such as diffusion coefficients and complex stoichiometries, can be accomplished using fluorescence fluctuation techniques to reveal nanosecond-to-microsecond temporal reactions. These powerful complimentary experimental approaches are made possible by sensitive low-light photodetectors. In this chapter, an overview of the principles of super-resolution and single-molecule microscopies are provided. The different types of photodetectors employed in these techniques are explained. In addition, the advantages and disadvantages for these detectors are discussed, as well as the development of next generation detectors. Finally, example super-resolution and single-molecule cellular studies that take advantage of these detector technologies are presented