The availability of the Airyscan detector in the Zeiss LSM 880 has made possible the development of a new concept in fluctuation correlation spectroscopy using super-resolution. The Airyscan unit ...acquires data simultaneously on 32 detectors arranged in a hexagonal array. This detector opens up the possibility to use fluctuation methods based on time correlation at single points or at a number of points simultaneously, as well as methods based on spatial correlation in the area covered by the detector. Given the frame rate of this detector, millions of frames can be acquired in seconds, providing a robust statistical basis for fluctuation data. We apply the comprehensive analysis to the molecular fluctuations of free GFP diffusing in live cells at different subcellular compartments to show that at the nanoscale different cell environments can be distinguished by the comprehensive fluctuation analysis.
Single Particle Tracking (SPT) is a technique used to locate fluorescent particles with nanometer precision. In the orbital tracking method the position of a particle is obtained analyzing the ...distribution of intensity along a circular orbit scanned around the particle. In combination with an active feedback this method allows tracking of particles in 2D and 3D with millisecond temporal resolution. Here we describe a SPT setup based on a feedback approach implemented with minimal modification of a commercially available confocal laser scanning microscope, the Zeiss LSM 510, in combination with an external piezoelectric stage scanner. The commercial microscope offers the advantage of a user-friendly software interface and pre-calibrated hardware components. The use of an external piezo-scanner allows the addition of feedback into the system but also represents a limitation in terms of its mechanical response. We describe in detail this implementation of the orbital tracking method and discuss advantages and limitations. As an example of application to live cell experiments we perform the 3D tracking of acidic vesicles in live polarized epithelial cells.
The delayed luminescence (DL) (i.e. the photo-induced photon emission long after the illumination is switched off) of unicellular green algae samples has been measured when different concentrations ...of heavy metals are added to the standard culture medium, with the aim of assessing the DL as a promising approach for assaying the toxicity of contaminants such as metals. In particular, samples of freshwater green micro-algae Selenastrum capricornutum have been used. Concentrations of cadmium, chromium, lead and copper, ranging from 10-5 to 10-2 M, have been tested. The analysis of the decay trends, in the time interval from tens of microseconds to seconds, of the DL spectral components demonstrates that the DL parameters are sensitive to the presence of such pollutants. More precisely, the performed analysis allowed us to determine phenomenological relationships between the DL parameters and the metal concentration that could be used in view of the possibility of realizing a biosensor for water pollution detection. Attempts to distinguish between different contaminants are also described. Results of this preliminary study show that the DL measure based technique is suitable as a general bioassay of metal contamination and it could also be used to test the efficiency in bioavailability studies.
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► When formation of LDW domains is foreseen in literature, salt solutions exhibit measurable Delayed Luminescence (DL) signals. ► DL intensity decreases with increasing temperature ...according to an Arrhenius trend characterized by an activation energy ΔE=14±2kJmol−1. ► The probability distribution of the DL decay times shows a maximum in the microsecond range.
Liquid water is described in two-state theory as mixture of localised molecular arrangements having different structure and density, the low-density (LDW) and high-density water (HDW). Theoretical calculations describe the formation of LDW and/or HDW domains, while their lifetime remains unknown. As ions partition selectively into LDW or HDW, modifying the water structuring, time-resolved photo-induced Delayed Luminescence (DL) of different salts solutions was measured. When formation of LDW domains is foreseen, significant DL signal was found, whose decay-time probability distribution is maximum in microsecond range. This result suggests the existence of such long lifetimes structures, which can participate in biological processes.
Design and characterization of single photon avalanche diodes arrays Neri, L.; Tudisco, S.; Lanzanò, L. ...
Nuclear instruments & methods in physics research. Section A, Accelerators, spectrometers, detectors and associated equipment,
05/2010, Letnik:
617, Številka:
1
Journal Article
Recenzirano
During the last years, in collaboration with ST-Microelectronics, we developed a new avalanche photo sensor, single photon avalanche diode (SPAD) see Ref.S. Privitera, et al., Sensors 8 (2008) 4636
...1;S. Tudisco et al., IEEE Sensors Journal 8 (2008) 1324
2. Such sensor is able to detect and count, with excellent performance, single photons. Today, design and monolithic integration of many elements represents the most challenging goal in this field. In the present contribution we report on design and realization of a bi-dimensional array of SPADs. Such array is realized in standard planar technology and is the first prototype designed for imaging applications. The final aim is to identify the position and the arrival time of the impinging photons. Lay-out, readout strategy, characterization test are discussed.
Bi-dimensional arrays of SPAD for time-resolved single photon imaging Tudisco, S.; Lanzanò, L.; Musumeci, F. ...
Nuclear instruments & methods in physics research. Section A, Accelerators, spectrometers, detectors and associated equipment,
10/2009, Letnik:
610, Številka:
1
Journal Article
Recenzirano
Many scientific areas like astronomy, biophysics, biomedicine, nuclear and plasma science, etc. are interested in the development of a new time-resolved single photon imaging device. Such a device ...represents today one of the most challenging goals in the field of photonics. In collaboration with Catania R&D staff of ST-Microelectronics (STM) we created, during the last few years, a new avalanche photosensor—Single Photon Avalanche Diode (SPAD) able to detect and count, with excellent performance, single photons. Further we will discuss the possible realization of a single photon imaging device through the many elements integration (bi-dimensional arrays) of SPADs. In order to achieve the goal, it is also important to develop an appropriate readout strategy able to address the time information of each individual sensor and in order to read a great number of elements easily.
First prototypes were designed and manufactured by STM and the results are reported here. In the paper we will discuss in particular: (i) sensor performance (gain, photodetection efficiency, timing, after-pulsing, etc.); (ii) array performance (layout, cross-talk, etc.); (iii) readout strategy (quenching, electronics), and (iv) first imaging results (general performance).
We describe a novel method to detect very low concentrations of bacteria in water. Our device consists of a portable horizontal geometry small confocal microscope with large pinhole and a holder for ...cylindrical cuvettes containing the sample. Two motors provide fast rotational and slow vertical motion of the cuvette so the device looks like a simplified flow cytometer without flow. To achieve high sensitivity, the design has two detection channels. Bacteria are stained by two different nucleic acid dyes and excited with two different lasers. Data are analyzed with a correlation filter based on particle passage pattern recognition. The passage of a particle through the illumination volume is compared with a Gaussian pattern in both channels. The width of the Gaussian correlates with the time of passage of the particle so one particle is counted when the algorithm finds a match with a Gaussian in both channels. The concentration of particles in the sample is deduced from the total number of coincident hits and the total volume scanned. This portable setup provides higher sensitivity, low-cost advantage, and it can have a wide use ranging from clinical applications to pollution monitors and water and air quality control.
Design and characterization of a new generation of single-photon avalanche diodes (SPAD) array, manufactured by ST-Microelectronics in Catania, Italy, are presented. Device performances, investigated ...in several experimental conditions and here reported, demonstrate their suitability in many applications. SPADs are thin p-n junctions operating above the breakdown condition in Geiger mode at low voltage. In this regime a single charged carrier injected into the depleted layer can trigger a self-sustaining avalanche, originating a detectable signal. Dark counting rate at room temperature is down to 10 s -1 for devices with an active area of 10 mum in diameter, and 10 3 s -1 for those 50 mum wide. SPAD quantum efficiency, measured in the range 350-1050 nm, can be comparable to that of a typical silicon based detector and reaches the values of about 50% at 550 nm. Finally, the low production costs and the integration possibility are other favorable features in sight of highly dense integrated 1-D or 2-D arrays.
Optical spectroscopy is an effective tool to explore the laser-produced plasma. The complex interaction processes occurring in the plasma could be, in principle, analysed using time-resolved studies ...of its spectral emission. Nevertheless, it is difficult to realize this possibility due to the inadequacy of currently available photon detectors, as photomultipliers or change-coupled devices, which does not have enough time resolution and are not rugged enough to be used in applications in which very high intensity pulses of visible light are produced. To explore new solutions, two prototypic arrays of single photon avalanche diodes obtained by ST-microelectronics, working in silicon photomultiplier configuration, have been used to obtain information on the space and time evolution of a plasma plume generated by a 500 mJ Nd:YAG laser at 1064 nm wavelength and 9 ns pulse duration. The obtained results are shown and the further potentialities of these detectors in this field are discussed.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Stimulated emission depletion (STED) microscopy is a powerful bioimaging technique that theoretically provides molecular spatial resolution while preserving the most important assets of fluorescence ...microscopy. When combined with two-photon excitation (2PE) microscopy (2PE-STED), subdiffraction resolution may be achieved for thick biological samples. The most straightforward implementation of 2PE-STED microscopy entails introduction of an STED beam operating in continuous wave (CW) into a conventional Ti:sapphire-based 2PE microscope (2PE CW-STED). In this implementation, resolution enhancement is typically achieved using time-gated detection schemes, often resulting in drastic signal-to-noise/-background ratio (SNR/SBR) reductions. Herein, we employ a pixel-by-pixel phasor approach to discard fluorescence photons lacking super-resolution information to enhance image SNR/SBR in 2PE CW-STED microscopy. We compare this separation of photons by lifetime tuning approach against other postprocessing algorithms and combine it with image deconvolution to further optimize image quality.
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