Two-photon excitation selective plane illumination microscopy (2PE-SPIM) of highly scattering samples: Characterization and application

In this work we report the advantages provided by two photon excitation (2PE) implemented in a selective plane illumination microscopy (SPIM) when imaging thick scattering samples. In particular, a detailed analysis of the effects induced on the real light sheet excitation intensity distribution is performed. The comparison between single-photon and twophoton excitation profiles shows the reduction of the scattering effects and sample-induced aberrations provided by 2PE-SPIM. Furthermore, uniformity of the excitation distribution and the consequent improved image contrast is shown when imaging scattering phantom samples in depth by 2PE-SPIM. These results show the advantages of 2PE-SPIM and suggest how this combination can further enhance the SPIM performance. Phantom samples have been designed with optical properties compatible with biological applications of interest. © 2013 Optical Society of America

Parkin regulates kainate receptors by interacting with the GluK2 subunit

Although loss-of-function mutations in the PARK2 gene, the gene that encodes the protein parkin, cause autosomal recessive juvenile parkinsonism, the responsible molecular mechanisms remain unclear. Evidence suggests that a loss of parkin dysregulates excitatory synapses. Here we show that parkin interacts with the kainate receptor (KAR) GluK2 subunit and regulates KAR function. Loss of parkin function in primary cultured neurons causes GluK2 protein to accumulate in the plasma membrane, potentiates KAR currents and increases KAR-dependent excitotoxicity. Expression in the mouse brain of a parkin mutant causing autosomal recessive juvenile parkinsonism results in GluK2 protein accumulation and excitotoxicity. These findings show that parkin regulates KAR function in vitro and in vivo, and suggest that KAR upregulation may have a pathogenetic role in parkin-related autosomal recessive juvenile parkinsonism

Marine Toxins: Chemistry, Toxicity, Occurrence and Detection, with Special Reference to the Dutch Situation

Various species of algae can produce marine toxins under certain circumstances. These toxins can then accumulate in shellfish such as mussels, oysters and scallops. When these contaminated shellfish species are consumed severe intoxication can occur. The different types of syndromes that can occur after consumption of contaminated shellfish, the corresponding toxins and relevant legislation are discussed in this review. Amnesic Shellfish Poisoning (ASP), Paralytic Shellfish Poisoning (PSP), Diarrheic Shellfish Poisoning (DSP) and Azaspiracid Shellfish Poisoning (AZP) occur worldwide, Neurologic Shellfish Poisoning (NSP) is mainly limited to the USA and New Zealand while the toxins causing DSP and AZP occur most frequently in Europe. The latter two toxin groups are fat-soluble and can therefore also be classified as lipophilic marine toxins. A detailed overview of the official analytical methods used in the EU (mouse or rat bioassay) and the recently developed alternative methods for the lipophilic marine toxins is given. These alternative methods are based on functional assays, biochemical assays and chemical methods. From the literature it is clear that chemical methods offer the best potential to replace the animal tests that are still legislated worldwide. Finally, an overview is given of the situation of marine toxins in The Netherlands. The rat bioassay has been used for monitoring DSP and AZP toxins in The Netherlands since the 1970s. Nowadays, a combination of a chemical method and the rat bioassay is often used. In The Netherlands toxic events are mainly caused by DSP toxins, which have been found in Dutch shellfish for the first time in 1961, and have reoccurred at irregular intervals and in varying concentrations. From this review it is clear that considerable effort is being undertaken by various research groups to phase out the animal tests that are still used for the official routine monitoring programs

NANOBIOPHOTONICS APPROACHES TOWARDS ADVANCED BIOIMAGING

Due to its nature, imaging is dependent on data visualization. More specifically, when dealing with optical microscopy imaging, diffraction limited spatial resolution and the presence of out-of-focus contributions are two of the key elements for the generation of 4D (x,y,z,t) data sets oriented to study molecular processes at the nanoscale. Within the medical nanotechnology scenario, optical fluorescence techniques have a pivotal role thanks to their non invasive capability to address biological questions in three dimensions with a remarkable capability to distinguish fine details, tremendously improved up to the nanoscale level in the last decade. Within this scenario, this Thesis concerns the study and the development of advanced fluorescence optical methods for the improvement of the imaging capability and a better exploitation of its potentialities. Two approaches have been followed in fluorescence microscopy to gather information, namely: a direct diffraction-limited observation of the sample allowed by the optical architecture and the analysis of those fluorescence signals particularly sensitive to the environmental condition. Consequently, two strategies have been pursued to improve such capabilities. They are related to the design of novel optical arrangements utilizing light interferences pathways to improve the diffraction resolution limit and to the study of novel fluorescent molecules and/or fluorescent techniques which allows the investigations of subresolved molecular interactions. In particular, following a fluorescent probe approach, a nanostructured polyelectrolyte system has been designed to study the fluorescence quenching effect induced by specific quencher molecules on the fluorescence emission process. The realized system allows entrapping the fluorescent molecules and monitoring fluorescence signal variations to probe quencher metal ions at microrange concentrations, significantly higher with respect to the current fluorescent quenching based technologies. As well, following an optical approach, the interferences effect induced by structuring the illumination light with different masks have been studied, in order to improve some features of the imaging capability of the fluorescence microscope. In the Two-Photon Excitation (2PE) and in the Confocal Single Photon Laser Scanning Microscope, the insertion of a ring shaped mask in the illumination pathway is proposed to enhance the signal to noise ratio of the optical system at the high spatial frequencies. Since in these optical systems the transmission of the high spatial frequencies is particularly weak, such features allow to improve the overall practical capability of the confocal and 2PE system to distinguish fine details of the image. In the widefield microscope, the insertion of a periodic grid to structure the light has been investigated in order to confer a 3D optical sectioning capability comparable to that of the confocal microscope, with major advantages in terms of the efficient use of the light, simplicity of construction, speed of imaging acquisition, versatility, and low cost. The proposed scheme allows to quickly collect a pure sectioned image without any computational demodulation thanks to a novel optical architecture where both the illumination and the detection light is structured by a spinning grid

Frequency dependent detection in a STED microscope using modulated excitation light

We present a novel concept adaptable to any kind of STED microscope in order to expand the limited number of compatible dyes for performing super resolution imaging. The approach is based on an intensity modulated excitation beam in combination with a frequency dependent detection in the form of a standard lock-in amplifier. This enables to unmix fluorescence signal originated by the excitation beam from the fluorescence caused by the STED beam. The benefit of this concept is demonstrated by imaging biological samples as well as fluorescent spheres, whose spectrum does not allow STED imaging in the conventional way. Our concept is suitable with CW or pulsed STED microscope and can thereby be seen as a general improvement adaptable to any existing setup

Nanostructured polyelectrolyte-based system as a toolbox for metal ions detection

The capability of certain heavy metal ions to induce fluorescence decrease by a quenching mechanism suggested us to design and build a sensor potentially tunable for different ions at different concentrations. We propose a quenching-based sensor exploiting a nanostructured architecture in which fluorescent molecules (the sensing probe) are entrapped to recognize a specific analyte (heavy metal ions) through an optical transduction. The polyelectrolyte nanostructured system, named nanocapsule, improves the fluorophore-ion quenching sensitivity allowing a micromolar detection. Furthermore we couple our sensor with an electrical device in order to refine the sensing procedure: the electric field created allows a metal ions spatial gradient, necessary to detect a specific element on a single sample solution, avoiding a comparative analysis with an intensity reference value. Results obtained will show the advantages and the potentialities of our system as a smart toolbox for metal ions detectio

Improving image formation from the illumination side: linear and non-linear excitation cases

Diffraction imposes for each optical system a resolution limit which could be described by using the vectorial theory of Richards and Wolf. This theory defines the intensity distribution of a point like source imaged by a lens assuming ideal imaging conditions. Unfortunately, these conditions can not be completely achieved in practical situations as a recorded microscope image is always affected by noise which makes the resolution limit worse. In this work we propose and analyze optical set-up schemes towards an image quality improvement in terms of Signal to Noise Ratio (SNR) in linear and non-linear fluorescence microscopy. In order to reach this purpose we insert, on the illumination arm of the microscope, a proper amplitude ring filter inducing laterally interfering beams. The effect induced by the filter results in a shape engineering of the 3D-PSF and in a redistribution of the spatial frequencies of the OTF. In particular, the high frequencies information are collected at improved SNR. In order to implement such schemes we use a computational simulation mainly based on a vectorial approach analyzing the results in both space and frequency domain to characterize the optical system response. Analysis reveals that, although the theoretical resolution of the system is unchanged, when we impose a certain noise level the practical imaging quality could be improved in the ring filtering scheme. The results suggest that further improvement can be reached by the usage of the proposed annular filers in combination with image restoration. A comparison between linear and non-linear excitation cases is presented

Improving image formation from the illumination side: Linear and non-linear excitation cases

Diffraction imposes for each optical system a resolution limit which could be described by using the vectorial theory of Richards and Wolf. This theory defines the intensity distribution of a point like source imaged by a lens assuming ideal imaging conditions. Unfortunately, these conditions can not be completely achieved in practical situations as a recorded microscope image is always affected by noise which makes the resolution limit worse. In this work we propose and analyze optical set-up schemes towards an image quality improvement in terms of Signal to Noise Ratio (SNR) in linear and non-linear fluorescence microscopy. In order to reach this purpose we insert, on the illumination arm of the microscope, a proper amplitude ring filter inducing laterally interfering beams. The effect induced by the filter results in a shape engineering of the 3D-PSF and in a redistribution of the spatial frequencies of the OTF. In particular, the high frequencies information are collected at improved SNR. In order to implement such schemes we use a computational simulation mainly based on a vectorial approach analyzing the results in both space and frequency domain to characterize the optical system response. Analysis reveals that, although the theoretical resolution of the system is unchanged, when we impose a certain noise level the practical imaging quality could be improved in the ring filtering scheme. The results suggest that further improvement can be reached by the usage of the proposed annular filers in combination with image restoration. A comparison between linear and non-linear excitation cases is presented. © 2009 SPIE

Annular pupil filter under shot-noise condition for linear and non linear microscopy

The imaging performances of multiphoton excitation and confocal laser scanning microscopy are herby considered: in typical experimental imaging conditions, a small finite amount of photon reaches the detector giving shot-noise fluctuations which affects the signal acquired. A significant detriment in the high frequencies transmission capability is obtained. In order to partially recover the high frequencies information lost, the insertion of a pupil plane filter in the microscope illumination light pathway on the objective lens is proposed. We demonstrate high-frequency and resolution enhancement in the case of linear and non linear fluorescence microscope approach under shot-noise condition