Two-dimensional Fluorescence Imaging Research Articles

Reconstruction based on adaptive group least angle regression for fluorescence molecular tomography.

Fluorescence molecular tomography can combine two-dimensional fluorescence imaging with anatomical information to reconstruct three-dimensional images of tumors. Reconstruction based on traditional regularization with tumor sparsity priors does not take into account that tumor cells form clusters, so it performs poorly when multiple light sources are used. Here we describe reconstruction based on an "adaptive group least angle regression elastic net" (AGLEN) method, in which local spatial structure correlation and group sparsity are integrated with elastic net regularization, followed by least angle regression. The AGLEN method works iteratively using the residual vector and a median smoothing strategy in order to adaptively obtain a robust local optimum. The method was verified using numerical simulations as well as imaging of mice bearing liver or melanoma tumors. AGLEN reconstruction performed better than state-of-the-art methods with different sizes of light sources at different distances from the sample and in the presence of Gaussian noise at 5-25%. In addition, AGLEN-based reconstruction accurately imaged tumor expression of cell death ligand-1, which can guide immunotherapy.

Proton Bragg peak imaging by colour centre radiophotoluminescence in lithium fluoride thin film radiation detectors on silicon

Optically transparent lithium fluoride (LiF) thin films, thermally evaporated on Si(100) substrates, are under investigation as novel radiation detectors based on radiophotoluminescence for imaging of the full Bragg curves of proton beams produced by a linear accelerator for proton therapy under development at ENEA C.R. Frascati. Proton irradiation induces the formation of stable colour centres in LiF, amongst which the broadband light-emitting F2 and F3+ aggregate defects, whose concentrations are locally proportional to the energy deposited in the material. Their spatial distributions in the irradiated LiF thin films and crystals are carefully measured by acquiring the latent two-dimensional visible fluorescence images with an optical microscope under blue lamp excitation. Several LiF films grown on silicon substrate were irradiated in air at increasing proton energies up to 35 MeV with their surface parallel to the particle beam and a cleaved edge perpendicularly facing it; for each sample, the fluorescence image acquired from the top surface side of the film allows to obtain the depth profile of the energy released by protons. Differences in colour centre distributions detected in LiF films with respect to LiF crystals are presented and discussed. Accurate Monte Carlo simulations allow to fully explain their experimental behaviours, paving the way towards using LiF film radiation detectors on silicon for the advanced diagnostics of proton beams at typical particle energies used for proton therapy.

(Digital Presentation) Color Centers Photoluminescence in Lithium Fluoride Thin-Film-on-Silicon Detectors for Proton Bragg Curves Imaging

The photoluminescence (PL) properties of radiation-induced color centers (CCs) in lithium fluoride (LiF) crystals and thin films find applications in optically-pumped solid-state lasers and photonic light-emitting microdevices operating at room temperature (RT) in the visible and near-infrared spectral range [1]. Among their peculiarities, the broad tunability and high emission quantum efficiency, combined with the wide optical transparency of the hosting LiF matrix.On the other hand, LiF dosimeters based on thermoluminescence (TL) of point defects in LiF crystals and pellets have been the most widely used family of phosphors in TL dosimetry, mainly for their high radiation sensitivity at low doses and the LiF tissue-equivalence, which is essential for meaningful medical applications.The excellent thermal and optical stabilities of the laser-active F2 and F3 + electronic defects, consisting of two electrons bound to two and three adjacent anion vacancies, respectively, whose efficient visible photoluminescence is located in the green-red spectral range under simultaneous excitation with blue-light pumping, make radiation detectors based on LiF crystals and thin films attractive for X-ray imaging [2] at nanoscale, related to the atomic-scale dimensions of such point defects.These passive radiation imaging sensors are based on the optical reading of visible radiophotoluminescence (RPL) of aggregate CCs locally created and stored in LiF, by using conventional and advanced fluorescence microscopy techniques for latent images acquisition.In the last years they were successfully tested for proton beam advanced diagnostics and dosimetry at increasing energies from 1.5 to 35 MeV, showing a linear RPL response as a function of absorbed doses in a wide dynamic range [4], long-term stability against fading and non-destructive reading capability. With suitable irradiation geometries of LiF crystals, it is possible to record the energy that protons deposit in the material (Bragg curve) as a bi-dimensional spatial distribution of luminescent CCs, even at dose values below 50 Gy, typically utilized in protontheraphy [5].We have been investigating the feasibility to extend this approach to optically transparent LiF thin films thermally evaporated on Si(100) substrates. Despite of their low thickness, we take advantage of the enhanced PL response of F2 and F3 + CCs, related to the presence of the flat and smooth Si substrate [6], which is optically reflective at the excitation and emission wavelengths utilized in the fluorescence microscope. The irradiations were performed with proton beams produced by the linear accelerator TOP-IMPLART (Oncological Therapy with Protons - Intensity Modulated Proton Linear Accelerator for RadioTherapy), under development at ENEA C.R. Frascati, with the cut edge perpendicular to the proton beam direction. The latent two-dimensional fluorescence images of the CC distributions generated in the polycrystalline LiF layers show a systematic increase in depth of the Bragg peak with respect to LiF crystals.The results obtained in LiF films at increasing proton energies are presented and discussed, also in comparison with those in LiF crystals, in order to explain the observed behaviors and highlight advantages and limits of versatile LiF film radiation detectors.[1] R. M. Montereali, in Handbook of Thin Film Materials, H. S. Nalwa, Editor, Vol.3: Ferroelectric and Dielectric Thin Films, Ch.7, p. 399, Academic Press, S. Diego (2002).[2] G. Baldacchini, F. Bonfigli, A. Faenov, F. Flora, R. M. Montereali, A. Pace, T. Pikuz, L. Reale, J. Nanosci. Nanotechno., 3, 483 (2003).[3] A. Ustione, A. Cricenti, F. Bonfigli, F. Flora, A. Lai, T. Marolo, R. M. Montereali, G. Baldacchini, A. Faenov, T. Pikuz and L. Reale, Jpn. J. Appl. Phys. 45, 2116 (2006).[4] M. Piccinini, F. Ambrosini, A. Ampollini, L. Picardi, C. Ronsivalle, F. Bonfigli, S. Libera, E. Nichelatti, M. A. Vincenti and R. M. Montereali, Appl. Phys. Lett., 106, 261108 (2015).[5] R.M. Montereali, E. Nichelatti, V. Nigro, M. Piccinini, M.A. Vincenti, ECS J. Solid State Sci. and Technol. 10, 116001 (2021).[6] M. A. Vincenti, M. Leoncini,S. Libera, A. Ampollini, A. Mancini, E. Nichelatti, V. Nigro, L. Picardi, M. Piccinini, C. Ronsivalle, A. Rufoloni, R. M. Montereali, Opt. Mater., 119, 111376 (2021).

Multimodal Imaging-Guided Spatiotemporal Tracking of Photosensitive Stem Cells for Breast Cancer Treatment.

Stem cell therapy has shown great potential in treating a wide range of diseases including cancer. The real-time tracking of stem cells with high spatiotemporal resolution and stable imaging signals remains the bottleneck to evaluate and monitor therapeutic outcomes once transplanted into patients. Here, we developed a photosensitive mesenchymal stem cell (MSC) loaded with mesoporous silica-coated gold nanostars (MGNSs) integrated with indocyanine green for spatiotemporal tracking and imaging-guided photothermal therapy (PTT) in treating breast cancers. The MGNS served as a stable imaging probe with multifunctional properties for photoacoustic imaging (PAI), fluorescence imaging, and PT imaging. Owing to the excellent PT stability of MGNSs, long-term three-dimensional (3D) PAI was achieved to monitor stem cells in real time at the tumor site, while the tumor structure was imaged using 3D B-mode ultrasound imaging. PAI revealed that the photosensitive stem cells reached the widest distribution area at the tumor site post 24 h of intratumoral injection, which was further confirmed via two-dimensional (2D) PT and fluorescence imaging. With this optimal cell distribution window, in vivo studies showed that the photosensitive stem cells via both intratumoral and intravenous injections successfully inhibited breast cancer cell growth and decreased the tumor recurrence rate post PTT. Our results support that this photo-integrated platform with stable optical properties is promising to achieve real-time tracking and measure the cell distribution quantitatively with high spatiotemporal resolution for stem cell therapy.

X-ray fluorescence imaging of laser-driven hydrodynamic instability systems with a Ross Pair Imager

An experiment has been conducted at the Shenguang-Ⅲ prototype laser facility, demonstrating the application of x-ray fluorescence imaging (XRFI) in diagnosing laser-driven hydrodynamic instability systems in planar geometry. An aluminum (Al)-foam target was used, including a modulated Al disk followed by a low density poly-4-methyl-1-pentene foam cylinder which was doped with titanium (Ti) dioxide nanoparticles to serve as the tracer layer. A blast wave was driven through the rippled interface, triggering the development of hydrodynamic instabilities and the growth of the perturbation. Vanadium plasma radiation dominated by He-α x-rays, was generated with a delay in time, inducing the emission of Ti K-shell fluorescent photons from the tracer layer. A Ross Pair Imager with matched scandium and Ti filters was employed to provide two-dimensional quasi-monochromatic fluorescent images, and the temporal evolution of the perturbation structures in the tracer layer was successfully observed. Furthermore, one-dimensional fluorescent intensity profiles across the perturbation structures were extracted and compared with the simulation results via a combination of the MULTI code, a buoyancy-drag model, a self-similar analysis and an imaging code. The boundaries of the measured profiles were shown to be connected together due to the small initial length of the tracer layer and the large boundary width caused by the curved interfaces and blurring effects, which made the absolute positions of the perturbation structures inaccessible. This research suggests that XRFI works well in qualitatively diagnosing the instability evolution and the experimental design has to be modified to further improve its performance in achieving quantitative information.

Restoration of Out-of-Focus Fluorescence Microscopy Images Using Learning-Based Depth-Variant Deconvolution

Image quality is degraded in the out-of-focus region because of the depth-variant (DV) point spread function (DV-PSF) of a fluorescence microscope. Either non-blind or blind deconvolution for restoration results in limited improvement. In this work, we propose a two-step learning-based DV deconvolution (LB-DVD) to restore the out-of-focus image. In the first step, DV-PSF is predicted by a defocus level prediction convolutional neural network (DelpNet). In the second step, the extracted DV-PSF is used for DV deconvolution. To our knowledge, LB-DVD is proposed and demonstrated for the first time. DelpNet achieves an accuracy of 98.2% for predicting defocus levels of image patches ( $84 \times 84$ pixels). The subsequent DV deconvolution gives rise to good performance in peak signal-to-noise ratios and structural similarity index, which are improved by up to 6.6 dB and 11%, respectively, before and after the deconvolution. As for a wide-field image, there exist different DV-PSFs within the two-dimensional fluorescence image due to the surface undulation. An overlapping weighting patch-wise LB-DVD is used in image montage to eliminate patch boundary artifacts. As a result, our LB-DVD shows the feasibility and promise to be applied to typical fluorescence microscopy in practical applications.

Three-dimensional virtual refocusing of fluorescence microscopy images using deep learning.

We demonstrate that a deep neural network can be trained to virtually refocus a two-dimensional fluorescence image onto user-defined three-dimensional (3D) surfaces within the sample. Using this method, termed Deep-Z, we imaged the neuronal activity of a Caenorhabditis elegans worm in 3D using a time sequence of fluorescence images acquired at a single focal plane, digitally increasing the depth-of-field by 20-fold without any axial scanning, additional hardware or a trade-off of imaging resolution and speed. Furthermore, we demonstrate that this approach can correct for sample drift, tilt and other aberrations, all digitally performed after the acquisition of a single fluorescence image. This framework also cross-connects different imaging modalities to each other, enabling 3D refocusing of a single wide-field fluorescence image to match confocal microscopy images acquired at different sample planes. Deep-Z has the potential to improve volumetric imaging speed while reducing challenges relating to sample drift, aberration and defocusing that are associated with standard 3D fluorescence microscopy.

A simple and efficient toolset for analysing mitochondrial trafficking in neuronal cells

Mitochondria are crucial for cells, supplying up to 90% of the energy requirements for neurons. Their correct localisation is crucial and ensured by a transport system. Mitochondrial trafficking in neurons is particularly critical, because mitochondria must leave the soma and travel along the axon and dendritic network to facilitate neuronal function. Abnormal mitochondrial trafficking has been reported in several neurological disorders, therefore the ability to quantify and analyse mitochondrial trafficking is vital to improving our understanding of their pathogenesis. Commercial software currently lacks an automated approach for performing such quantitation. Here we demonstrate the development of the Mitochondrial Trafficking and Distribution (MiTrakD) analysis toolset, which consists of simple and free-to-use instructions for mitochondrial trafficking analysis using time-lapse microscopy. MiTrakD utilises existing Fiji (ImageJ) tools for semi-automated, fast and efficient analysis of mitochondrial trafficking and distribution, including velocity, abundance, localisation and distance travelled in neurons. We document MiTrakD’s efficiency and accuracy by analysing mitochondrial trafficking using two-dimensional fluorescence images of cortical neurons of wild type mice after 6 days (DIV6), 10 days (DIV10) and 14 days (DIV14) of in vitro incubation. Using MiTrakD we have demonstrated that neurons at all developmental stages exhibited the same percentage of mobile mitochondria, all of which travel in equidistance. Interestingly, the mitochondria in neurons at DIV10 were in greater abundance and were faster than those at DIV6 and DIV14. We can also conclude that MiTrakD is more efficient than manual analysis and is an accurate and reliable tool for performing mitochondrial trafficking analysis in neuronal cells.

Volumetric Analysis of 3-D-Cultured Colonies in Wet Alginate Spots Using 384-Pillar Plate.

The volumetric analysis of three-dimensional (3-D)-cultured colonies in alginate spots has been proposed to increase drug efficacy. In a previously developed pillar/well chip platform, colonies within spots are usually stained and dried for analysis of cell viability using two-dimensional (2-D) fluorescent images. Since the number of viable cells in colonies is directly related to colony volume, we proposed the 3-D analysis of colonies for high-accuracy cell viability calculation. The spots were immersed in buffer, and the 3-D volume of each colony was calculated from the 2-D stacking fluorescent images of the spot with different focal positions. In the experiments with human gastric carcinoma cells and anticancer drugs, we compared cell viability values calculated using the 2-D area and 3-D volume of colonies in the wet and dried alginate spots, respectively. The IC50 value calculated using the 3-D volume of the colonies (9.5 μM) was less than that calculated in the 2-D area analysis (121.5 μM). We observed that the colony showed a more sensitive drug response regarding volume calculated from the 3-D image reconstructed using several confocal images than regarding colony area calculated in the 2-D analysis.

Image recovery from defocused 2D fluorescent images in multimodal digital holographic microscopy.

A technique of three-dimensional (3D) intensity retrieval from defocused, two-dimensional (2D) fluorescent images in the multimodal digital holographic microscopy (DHM) is proposed. In the multimodal DHM, 3D phase and 2D fluorescence distributions are obtained simultaneously by an integrated system of an off-axis DHM and a conventional epifluorescence microscopy, respectively. This gives us more information of the target; however, defocused fluorescent images are observed due to the short depth of field. In this Letter, we propose a method to recover the defocused images based on the phase compensation and backpropagation from the defocused plane to the focused plane using the distance information that is obtained from a 3D phase distribution. By applying Zernike polynomial phase correction, we brought back the fluorescence intensity to the focused imaging planes. The experimental demonstration using fluorescent beads is presented, and the expected applications are suggested.

Lithium fluoride thin film detectors for low-energy proton beam diagnostics by photoluminescence of colour centres

Optically transparent LiF thin films thermally evaporated on glass and Si(100) substrates were used for advanced diagnostics of proton beams of energies from 1.4 to 7 MeV produced by a linear accelerator for protontheraphy under development at ENEA C.R. Frascati. The proton irradiation induces the formation of stable colour centres, among them the aggregate F2 and F3+ optically active defects. After exposure of LiF films grown on glass perpendicularly to the proton beams, their accumulated transversal spatial distributions were carefully measured by reading the latent two-dimensional (2-D) fluorescence images stored in the LiF thin layers by local formation of these broad-band visible light-emitting defects with an optical microscope under blue lamp excitation. Taking advantage from the low thickness of LiF thin films and from the linear behaviour of the integrated F2 and F3+ photoluminescence intensities up to the irradiation fluence of ∼5x1015 p/cm2, placing a cleaved LiF film grown on Si substrate with the cutted edge perpendicular to the proton beam, the 2-D fluorescence image of the film surface could allow to obtain the depth profile of the energy released by protons, which mainly lose their energy at the end of the path.

Speckle correlation resolution enhancement of wide-field fluorescence imaging

Fluorescence microscopy is indispensable in nanoscience and biological sciences. The versatility of labeling target structures with fluorescent dyes permits to visualize structure and function at a subcellular resolution with a wide field of view. Due to the diffraction limit, conventional optical microscopes are limited to resolving structures larger than 200 nm. The resolution can be enhanced by near-field and far-field super-resolution microscopy methods. Near-field methods typically have a limited field of view and far-field methods are limited by the involved conventional optics. Here, we introduce a combined high-resolution and wide-field fluorescence microscopy method that improves the resolution of a conventional optical microscope by exploiting correlations in speckle illumination through a randomly scattering high-index medium: Speckle correlation resolution enhancement (SCORE). As a test, we collect two-dimensional fluorescence images of 100-nm diameter dye-doped nanospheres. We demonstrate a deconvolved resolution of 130 nm with a field of view of 10 x 10 $\text{\mu m}^2$.

Molecular imaging needles: dual-modality optical coherence tomography and fluorescence imaging of labeled antibodies deep in tissue.

Molecular imaging using optical techniques provides insight into disease at the cellular level. In this paper, we report on a novel dual-modality probe capable of performing molecular imaging by combining simultaneous three-dimensional optical coherence tomography (OCT) and two-dimensional fluorescence imaging in a hypodermic needle. The probe, referred to as a molecular imaging (MI) needle, may be inserted tens of millimeters into tissue. The MI needle utilizes double-clad fiber to carry both imaging modalities, and is interfaced to a 1310-nm OCT system and a fluorescence imaging subsystem using an asymmetrical double-clad fiber coupler customized to achieve high fluorescence collection efficiency. We present, to the best of our knowledge, the first dual-modality OCT and fluorescence needle probe with sufficient sensitivity to image fluorescently labeled antibodies. Such probes enable high-resolution molecular imaging deep within tissue.

Trans-illumination fluorescence imaging of deep-seated tumors in small animals

AbstractBackground:Fluorescence diffuse tomography (FDT) is the most accurate technique for the imaging of labeled tumors in the small animal body. However, the procedure for reconstruction of the spatial distribution of the fluorophore requires a high signal-to-noise ratio due to the ill-condition of the inverse problem. Therefore, the FDT technique is ineffective for imaging tumors of small size or with dim fluorophores because of the low intensity of their fluorescence compared with the high level of tissue autofluorescence. In these cases, the size and position of a marked tumor in the animal body can be estimated from two-dimensional fluorescence images obtained using trans- or epi-illumination techniques.Material and methods:A versatile system for small animal fluorescence imaging which combines planar epi- and trans-illumination geometries of the light source and of the fluorescence receiver was created and tested. For epi-illumination imaging, light-emitting diode sources were used to provide homogeneous and stable illumination of the experimental animal, in combination with a cooled CCD camera which covers the entire illuminated area. For trans-illumination imaging, mechanical raster-scanning devices modulated at a low frequency were used for the laser source, together with a cooled photomultiplier tube, which provided outstanding sensitivity.Results:Monitoring the orthotopic tumor growth in animal bodies has demonstrated the efficacy of trans-illumination imaging in comparison with the epi-illumination technique. The results obtained also showed that the effective use of the trans-illumination technique requires Born normalization of the fluorescence signal and the exclusion of lateral illumination by surrounding the animal with additional light absorption material using light-absorption pads on both sides of the body.

Molecular intravascular imaging approaches for atherosclerosis.

Coronary artery disease (CAD) is an inflammatory process that results in buildup of atherosclerosis, typically lipid-rich plaque in the arterial wall. Progressive narrowing of the vessel wall and subsequent plaque rupture can lead to myocardial infarction and death. Recent advances in intravascular fluorescence imaging techniques have provided exciting coronary artery-targeted platforms to further characterize the molecular changes that occur within the vascular wall as a result of atherosclerosis and following coronary stent-induced vascular injury. This review will summarize exciting recent developments in catheter-based imaging of coronary arterial-sized vessels; focusing on two-dimensional near-infrared fluorescence imaging (NIRF) molecular imaging technology as an approach to specifically identify inflammation and fibrin directly within coronary artery-sized vessels. Intravascular NIRF is anticipated to provide new insights into the in vivo biology underlying high-risk plaques, as well as high-risks stents prone to stent restenosis or stent thrombosis.

Visualization of the Heterogeneity of Cerium Oxidation States in Single Pt/Ce2Zr2Ox Catalyst Particles by Nano‐XAFS

The cerium oxidation states in single catalyst particles of Pt/Ce2Zr2O(x) (x=7 to 8) were investigated by spatially resolved nano X-ray absorption fine structure (nano-XAFS) using an X-ray nanobeam. Differences in the distribution of the Ce oxidation states between Pt/Ce2Zr2O(x) single particles of different oxygen compositions x were visualized in the obtained two-dimensional X-ray fluorescent (XRF) mapping images and the Ce L(III)-edge nano X-ray absorption near-edge structure (nano-XANES) spectra.

The Bionanoprobe: hard X-ray fluorescence nanoprobe with cryogenic capabilities.

Hard X-ray fluorescence microscopy is one of the most sensitive techniques for performing trace elemental analysis of biological samples such as whole cells and tissues. Conventional sample preparation methods usually involve dehydration, which removes cellular water and may consequently cause structural collapse, or invasive processes such as embedding. Radiation-induced artifacts may also become an issue, particularly as the spatial resolution increases beyond the sub-micrometer scale. To allow imaging under hydrated conditions, close to the `natural state', as well as to reduce structural radiation damage, the Bionanoprobe (BNP) has been developed, a hard X-ray fluorescence nanoprobe with cryogenic sample environment and cryo transfer capabilities, dedicated to studying trace elements in frozen-hydrated biological systems. The BNP is installed at an undulator beamline at sector 21 of the Advanced Photon Source. It provides a spatial resolution of 30 nm for two-dimensional fluorescence imaging. In this first demonstration the instrument design and motion control principles are described, the instrument performance is quantified, and the first results obtained with the BNP on frozen-hydrated whole cells are reported.

Evidence of excited state localization and static disorder in LH2 investigated by 2D-polarization single-molecule imaging at room temperature

Two-dimensional polarization fluorescence imaging of single light harvesting complexes 2 (LH2) of Rps. acidophila was carried out to investigate the polarization properties of excitation and fluorescence emission simultaneously, at room temperature. In two separate experiments we excited LH2 with a spectrally narrow laser line matched to the absorption bands of the two chromophore rings, B800 and B850, thereby indirectly and directly triggering fluorescence of the B850 exciton state. A correlation analysis of the polarization modulation depths in excitation and emission for a large number of single complexes was performed. Our results show, in comparison to B800, that the B850 ring is a more isotropic absorber due to the excitonic nature of its excited states. At the same time, we observed a strong tendency for LH2 to emit with dipolar character, from which preferential localization of the emissive exciton, stable for minutes, is inferred. We argue that the observed effects can consistently be explained by static energetic disorder and/or deformation of the complex, with possible involvement of exciton self-trapping.

A detailed study of gold-nanoparticle loaded cells using X-ray based techniques for cell-tracking applications with single-cell sensitivity

In the present study complementary high-resolution imaging techniques on different length scales are applied to elucidate a cellular loading protocol of gold nanoparticles and subsequently its impact on long term and high-resolution cell-tracking utilizing X-ray technology. Although demonstrated for malignant cell lines the results can be applied to non-malignant cell lines as well. In particular the accumulation of the gold marker per cell has been assessed quantitatively by virtue of electron microscopy, two-dimensional X-ray fluorescence imaging techniques and X-ray CT with micrometric and sub-micrometric resolution. Moreover, utilizing these techniques the three dimensional distribution of the incorporated nanoparticles, which are sequestered in lysosomes as a permanent marker, could be determined. The latter allowed elucidation of the gold partition during mitosis and the cell size, which subsequently enabled us to define the optimal instrument settings of a compact microCT system to visualize gold loaded cells. The results obtained demonstrate the feasibility of cell-tracking using X-ray CT with compact sources.

Automation Highlights from the Literature

Automation Highlights from the Literature