Scanning Microscopy System Research Articles

Development of an auto-interchangeable multi-pinhole array for confocal laser scanning microscopy systems using precision laser processing

Confocal laser scanning microscopy systems rely on a pinhole placed at the confocal point to act as a spatial filter. The diameter of the pinhole critically affects both the brightness and resolution of the imaging. Furthermore, different imaging conditions, such as sample type, depth of the sample, wavelength of the signal, and its inherent emission or reflection strength, require varying balances between the brightness and optical resolution for optimal observability. However, exchanging the pinhole, and its subsequent alignment, is often a time consuming challenge when implemented in customized systems. These systems are important since they are often used in studies for which commonly available ones lack sufficient capabilities. As such, we have developed an auto-positioning interchangeable pinhole array, manufactured via a patented laser machining process and designed for convenient implementation in confocal laser scanning microscopy systems and we demonstrate its effectiveness and versatility.

Multiscanning mode laser scanning confocal microscopy system

In this paper, a table-top, reflective mode, laser scanning confocal microscopy system that is capable of scanning the target specimen alternately through various scanning devices and methods is proposed. We have developed a laser scanning confocal microscopy system to utilize combinations of various scanning devices and methods and to be able to characterize the optical performance of different scanners and micromirrors that are frequently used in scanning microscopy systems such as multiphoton microscopy, optical coherence tomography, or confocal microscopy. By integrating the scanner to be characterized on the same optical path with a galvanometric scan mirror, which is the conventional benchmarking scanning unit in a typical scanning microscope, we obtain two major advantages: (1) microscopy images are automatically acquired from the same location on the target specimen without having any time- consuming alignment problem and accordingly provide a high-quality optical comparison opportunity, and (2) it totally eliminates the utilization of a second scanning microscopy to benchmark the performance of the scanner-based system and considerably reduces the time spent for imaging, which is a crucial factor for a freshly excised tissue, especially under a fluorescence microscope. The system is composed of a 658 nm laser source, collimation optics, a 2D galvanometer, a 2D polymer micro-scanner, an objective lens with a numerical aperture of 0.40, a 100 μm pinhole, a PMT, a DAQ card and peripheral electronics as well as a Matlab software that simultaneously controls the system through a personal computer. Prototype of the proposed flexible LSCM system is first optically characterized using a USAF resolution target. Subsequently, we provided images of red blood and bacteria cells to demonstrate the systems’ capability for clinical diagnostics. It is reported that maximum FOV and lateral resolution of the proposed LSCM are measured to be 420 μm x 360 μm and 1 μm with galvanometer and, and 117 μm x 117 μm and 3.2 μm with the polymer scanner unit, respectively.

Hyperspectral imaging fluorescence excitation scanning (HIFEX) microscopy for live cell imaging.

In the past two decades, spectral imaging technologies have expanded the capacity of fluorescence microscopy for accurate detection of multiple labels, separation of labels from cellular and tissue autofluorescence, and analysis of autofluorescence signatures. These technologies have been implemented using a range of optical techniques, such as tunable filters, diffraction gratings, prisms, interferometry, and custom Bayer filters. Each of these techniques has associated strengths and weaknesses with regard to spectral resolution, spatial resolution, temporal resolution, and signal-to-noise characteristics. We have previously shown that spectral scanning of the fluorescence excitation spectrum can provide greatly increased signal strength compared to traditional emission-scanning approaches. Here, we present results from utilizing a Hyperspectral Imaging Fluorescence Excitation Scanning (HIFEX) microscope system for live cell imaging. Live cell signaling studies were performed using HEK 293 and rat pulmonary microvascular endothelial cells (PMVECs), transfected with either a cAMP FRET reporter or a Ca2+ reporter. Cells were further labeled to visualize subcellular structures (nuclei, membrane, mitochondria, etc.). Spectral images were acquired using a custom inverted microscope (TE2000, Nikon Instruments) equipped with a 300W Xe arc lamp and tunable excitation filter (VF-5, Sutter Instrument Co., equipped with VersaChrome filters, Semrock), and run through MicroManager. Timelapse spectral images were acquired from 350-550 nm, in 5 nm increments. Spectral image data were linearly unmixed using custom MATLAB scripts. Results indicate that the HIFEX microscope system can acquire live cell image data at acquisition speeds of 8 ms/wavelength band with minimal photobleaching, sufficient for studying moderate speed cAMP and Ca2+ events.

Optimising passivation shell thickness of single upconversion nanoparticles using a time-resolved spectrometer

Lanthanide-doped upconversion nanoparticles (UCNPs) are the most efficient multi-photon probe that can be used for deep tissue bio-imaging, fluorescence microscopy, and single molecule sensing applications. Passivating UCNPs with inert shell has been demonstrated to be an effective method to significantly enhance their brightness. However, this method also increases the overall size of the nanoparticles, which limited their cellular applications. Current reports to optimise the thickness of the shell are based on the spectrum measurement of ensembles of UCNPs, which are less quantitative. The characterisation of single UCNPs would be desirable, but is limited by the sensitivity of conventional spectrometers. We developed an optical filter-based spectrometer coupled to a laser scanning microscopy system and achieved a high degree of sensitivity—seven times more than the traditional amount. Through highly controlled syntheses of a range Yb3+ and Tm3+ doped UCNPs with different shell thickness, quantitative characterization of the emission intensity and lifetime on single UCNPs were comprehensively studied using a home-made optical system. We found that the optimal shell thickness was 6.3 nm. We further demonstrated that the system was sensitive enough to measure the time-resolved spectrum from a single UCNP, which is significantly useful for a comprehensive study of the energy transfer process of UCNPs.

Fabrication and verification of a glass-silicon-glass micro-/nanofluidic model for investigating multi-phase flow in shale-like unconventional dual-porosity tight porous media.

Unconventional shale or tight oil/gas reservoirs that have micro-/nano-sized dual-scale matrix pore throats with micro-fractures may result in different fluid flow mechanisms compared with conventional oil/gas reservoirs. Microfluidic models, as a potential powerful tool, have been used for decades for investigating fluid flow at the pore-scale in the energy field. However, almost all microfluidic models were fabricated by using etching methods and very few had dual-scale micro-/nanofluidic channels. Herein, we developed a lab-based, quick-processing and cost-effective fabrication method using a lift-off process combined with the anodic bonding method, which avoids the use of any etching methods. A dual-porosity matrix/micro-fracture pattern, which can mimic the topology of shale with random irregular grain shapes, was designed with the Voronoi algorithm. The pore channel width range is 3 μm to 10 μm for matrices and 100-200 μm for micro-fractures. Silicon is used as the material evaporated and deposited onto a glass wafer and then bonded with another glass wafer. The channel depth is the same (250 nm) as the deposited silicon thickness. By using an advanced confocal laser scanning microscopy (CLSM) system, we directly visualized the pore level flow within micro/nano dual-scale channels with fluorescent-dyed water and oil phases. We found a serious fingering phenomenon when water displaced oil in the conduits even if water has higher viscosity and the residual oil was distributed as different forms in the matrices, micro-fractures and conduits. We demonstrated that different matrix/micro-fracture/macro-fracture geometries would cause different flow patterns that affect the oil recovery consequently. Taking advantage of such a micro/nano dual-scale 'shale-like' microfluidic model fabricated by a much simpler and lower-cost method, studies on complex fluid flow behavior within shale or other tight heterogeneous porous media would be significantly beneficial.

Dual-mode phase and fluorescence imaging with a confocal laser scanning microscope

We present dual-mode phase and fluorescence imaging in a confocal laser scanning microscopy (CLSM) system. For phase imaging, the depth of field of the CLSM system is extended by fast axial scanning with a tunable acoustic gradient index of refraction lens. Under transillumination, intensity images of the sample are recorded at a few different defocusing distances. The phase image is reconstructed from these intensity images by using the transport-of-intensity equation. The 3D fluorescence image is obtained by confocal scanning. The dual-mode images with pixel-to-pixel correspondence yield complementary quantitative structural and functional information. Combination of the two imaging modalities enables standalone determination of the refractive index of live cells.

Antibacterial mechanism of plantaricin LPL-1, a novel class IIa bacteriocin against Listeria monocytogenes

Plantaricin LPL-1, produced by Lactobacillus plantarum LPL-1, is a novel class IIa bacteriocin with bactericidal activity against foodborne bacteria. The aim of this present study was to understand the antibacterial activity and mechanism of plantaricin LPL-1 against Listeria monocytogenes 54002, one of the Gram-positive bacteria sensitive to this bacteriocin and typical foodborne pathogenic bacteria in the food industry. The minimal inhibitory concentration (MIC) was confirmed as 16 μg/mL, and the time-kill kinetics showed that plantaricin LPL-1 significantly decrease the viable cell numbers of L. monocytogenes 54002 in a time-dependent and dose-dependent manner. The antibacterial mechanism was investigated using confocal laser-scanning microscopy, scanning and transmission electron microscopy, spectrofluorimeter, spectrophotometer and Real-Time PCR system, which demonstrated that plantaricin LPL-1 facilitated pores formation, damaged cell membrane integrity, increased membrane permeability, triggered intracellular material leakage, induced collapse of proton motive force, and inhibited expression levels of genes related to virulence factors, biofilm formation factors, and RNA polymerase sigma factor. Hence, the bactericidal mechanism of plantaricin LPL-1 against L. monocytogenes was clarified at cellular and molecular levels.

Advanced oxidation protein products induce M1 activation of BV2 cells: an experimental study

Objective To investigate whether advance oxidation protein products (AOPPs) would induce M1 activation of BV2 cells. Methods AOPPs were prepared by incubation of mouse serum albumin (MSA) with sodium hypochlorite solution. Methyl thiazol tetrazolium (MTT)assay was used to determine the effects of different concentration of AOPPs on BV2 cells activity by incubating the cells for 24 h for selecting optimum working concentration of AOPPs. BV2 cells were treated based on concentration effect. Intracellular reactive oxygen species (ROS) production was determined with dichlorofluorescein diacetate (DCFH-DA) and Confocal laser scanning microscope system. And the protein expression of nicotinamide adenine dinucleotide (NADPH) oxidase (NOX)4 (NOX4), cluster of differentiation molecule 11b (CD11b) and tumor necrosis factor (TNF-α) were examined by Western blotting. Results Viability of BV2 cells treated with 400 μg/ml and 800 μg/ml AOPPs was significantly lowered [(50.052±2.163)%, (36.505±1.589)%, P=0.000]. AOPPs, in a concentration-dependent manner, increased the expression of NOX4, CD11b and TNF-α (NOX4: PLPS=0.001, P50 μg/ml=0.000, P100 μg/ml=0.004, P200 μg/ml=0.000; CD11b: P=0.000; TNF-α: P=0.000); prolonged exposure to AOPPs and increase of AOPPs concentration both led to an increase of intracellular ROS production (P=0.000). Conclusion AOPPs may induce M1 activation of BV2 cells through producing ROS. Key words: Advance oxidation protein products; BV2 cells; Reactive oxygen species; M1 activation

Reliability of wear measurements of CAD-CAM restorative materials after artificial aging in a mastication simulator.

The purpose of this in vitro study was to assess the 2-body wear behavior of computer-aided design and computer-aided manufacturing (CAD-CAM) restorative materials and additionally to validate an automatic approach to wear quantification as provided by newly developed software. Two composite resins (Experimental Composite, Shofu Block HC), 1 polymethyl methacrylate- (PMMA)-based material (Telio CAD), and 1 dual-network material (VITA Enamic) were tested (n = 12). The materials were stored in distilled water at 37 °C for 14 days, then aged in a mastication simulator (cycles: 400 000, 50 N, 1.2 Hz, 5/55 °C) for 2-body wear testing with human molars as antagonists. Software was developed as a freely available function in R for automatic wear quantification and was applied to 2 (vertical and horizontal) images obtained by an optical laser scanner (SDM). Wear measurements were validated with a reference confocal laser scanning microscope (LSM) system. The Kruskal-Wallis with Tukey-Kramer post hoc test (α = 0.05), coefficient of variation, Bland-Altman method, and intraclass correlation were applied. Shofu Block HC showed the greatest wear, and no differences were found among other materials. The automatic wear measurements agreed well with the reference LSM system. The reliability of the SDM scans was 98.5%. The wear resistance caused by two-body wear differed among the restorative materials tested, with the mean wear resistance of the 2 composite resin materials differing significantly. Wear quantification with R software based on SDM scans showed satisfactory agreement with the reference LSM system.

Parallelized fluorescence lifetime imaging microscopy (FLIM) based on photon reassignment

We propose a novel parallelized fluorescence lifetime imaging microscopy (FLIM) method based on photon reassignment with a detector array and time-correlated single photon counting (TCSPC) array. The detector array composed of seven avalanche photon diodes (APDs) substitutes for original pinhole and detector to constitute an imaging scanning microscopy (ISM) system. The introduction of parallel detection can solve the pile-up effect caused by the dead-time in TCSPC measurement, which can enhance the effective photon detection efficiency and notably increase the fluorescence lifetime imaging speed. More importantly, the resolution and signal–noise ratio (SNR) can be enhanced based on photon reassignment. The simulation results embody the superiority of our FLIM system, and the experiments with different samples validate the improvement of the resolution and speed of fluorescence lifetime imaging. The experiment results show that the resolution of our method is enhanced by a factor of 1.5 and the imaging speed can be at least increased by a factor of 2.5 compared with conventional confocal TCSPC FLIM system. We envision that the proposed FLIM method based on parallel detection will allow for super-resolution fluorescence lifetime real-time imaging of live cell.

High contrast imaging and flexible photomanipulation for quantitative in vivo multiphoton imaging with polygon scanning microscope

In this study, we introduce two key improvements that overcome limitations of existing polygon scanning microscopes while maintaining high spatial and temporal imaging resolution over large field of view (FOV). First, we proposed a simple and straightforward means to control the scanning angle of the polygon mirror to carry out photomanipulation without resorting to high speed optical modulators. Second, we devised a flexible data sampling method directly leading to higher image contrast by over 2-fold and digital images with 100 megapixels (10 240 × 10 240) per frame at 0.25 Hz. This generates sub-diffraction limited pixels (60 nm per pixels over the FOV of 512 μm) which increases the degrees of freedom to extract signals computationally. The unique combined optical and digital control recorded fine fluorescence recovery after localized photobleaching (r ~10 μm) within fluorescent giant unilamellar vesicles and micro-vascular dynamics after laser-induced injury during thrombus formation in vivo. These new improvements expand the quantitative biological-imaging capacity of any polygon scanning microscope system.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia.

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon's space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Improve the accuracy of 3D gel printer by incorporating Scanning Microscopic Light Scattering System

Improve the accuracy of 3D gel printer by incorporating Scanning Microscopic Light Scattering System

Cryorolling impacts on microstructure and mechanical properties of AISI 316 LN austenitic stainless steel

Microstructure evolution and mechanical properties of AISI 316 LN austenitic stainless steel (SS) after cryorolling with different strains were investigated by means of optical, scanning and transmission electron microscopy, X-ray diffractometer, microhardness tester, and tensile testing system. The deformation-induced martensite transition and the deformation microstructure occurred during cryorolling process were always composed of high-density dislocations, deformation twins, and deformation-induced martensites. Following the strain, the dislocation density in deformation microstructure approached saturation state and the volume fraction of deformation twins combined with deformation-induced martensites increased significantly. At the 70% strain, original austenite was transformed into martensite completely. Further increasing the strain to 90% would refine the martensitic lamellae to nanoscale. The deformation degree also led to remarkable increase of the strength and hardness of the cryorolled SS, and drastic reductions of the elongation. Due to the cryorolling, the tensile fracture morphology changed from typical ductile rupture to a mixture of quasi-cleavage and ductile fracture.

Single Cell Oxygen Mapping (SCOM) by Scanning Electrochemical Microscopy Uncovers Heterogeneous Intracellular Oxygen Consumption

We developed a highly sensitive oxygen consumption scanning microscopy system using platinized platinum disc microelectrodes. The system is capable of reliably detecting single-cell respiration, responding to classical regulators of mitochondrial oxygen consumption activity as expected. Comparisons with commercial multi-cell oxygen detection systems show that the system has comparable errors (if not smaller), with the advantage of being able to monitor inter and intra-cell heterogeneity in oxygen consumption characteristics. Our results uncover heterogeneous oxygen consumption characteristics between cells and within the same cell´s microenvironments. Single Cell Oxygen Mapping (SCOM) is thus capable of reliably studying mitochondrial oxygen consumption characteristics and heterogeneity at a single-cell level.

Morpholine derivative-functionalized carbon dots as a fluorescent probe for highly selective lysosome imaging in living cells

The anomalous fluctuations of pH in lysosome will cause the disorder of normal activity of cells and even many diseases. Thus, developing a convenient strategy for tracking pH changes of lysosome is highly desired. Herein, carbon dots (CDs)-based fluorescent probe with lysosome-targeting function was fabricated. CDs can monitor pH changes in live cells and organisms, with the aid of confocal laser scanning microscope and noninvasive optical imaging system. The advantages of CDs including robust photostability, high pH sensitivity and selectivity, good reversibility and low toxicity render them a prominent candidate to investigate pH-associated physiological and pathological processes.

Enhanced hydrogen evolution properties obtained by ultrasonic-cyclic voltammetry modification of C-supported PtCu thin film catalyst

Carbon-supported Pt-Cu (Pt-Cu/C) bimetallic catalyst was synthesized by Ion Beam Sputtering technology and subsequently annealed in vacuum and electrochemically etched by Ultrasonic-Cyclic Voltammetry (US-CV). Electrochemical measurements indicate that the sample was modified electrochemically by US-CV shows higher catalytic activity towards hydrogen evolution reaction than pure Pt/C. Scanning and transmission electron microscopy and electronic differential system analysis reveal that the surface of post-processed catalyst produced PtCu@Pt core-shell structure which increasing the efficiency of Pt. Transmission electron microscope analysis displays that on the surface of PtCu@Pt core-shell particles detects lattice compressive strain, the lattice compression variable is around 1.12%. X-ray photoelectron spectroscopy analysis confirms that the Pt4f7/2 binding energy of the post-processed PtCu/C is 71.10 eV, decreased by 0.2 eV compared to pure Pt/C (71.3 eV). It can be inferred that the enhancement of catalytic property attribute to the Cu atoms modified the geometric structure and electronic structure of the Pt atoms.

Few-photon color imaging using energy-dispersive superconducting transition-edge sensor spectrometry

Highly sensitive spectral imaging is increasingly being demanded in bioanalysis research and industry to obtain the maximum information possible from molecules of different colors. We introduce an application of the superconducting transition-edge sensor (TES) technique to highly sensitive spectral imaging. A TES is an energy-dispersive photodetector that can distinguish the wavelength of each incident photon. Its effective spectral range is from the visible to the infrared (IR), up to 2800 nm, which is beyond the capabilities of other photodetectors. TES was employed in this study in a fiber-coupled optical scanning microscopy system, and a test sample of a three-color ink pattern was observed. A red–green–blue (RGB) image and a near-IR image were successfully obtained in the few-incident-photon regime, whereas only a black and white image could be obtained using a photomultiplier tube. Spectral data were also obtained from a selected focal area out of the entire image. The results of this study show that TES is feasible for use as an energy-dispersive photon-counting detector in spectral imaging applications.

Design and construction of a cost-efficient Arduino-based mirror galvanometer system for scanning optical microscopy

Mirror galvanometer systems (galvos) are commonly employed in research and commercial applications in areas involving laser imaging, laser machining, laser-light shows, and others. Here, we present a robust, moderate-speed, and cost-efficient home-built galvo system. The mechanical part of this design consists of one mirror, which is tilted around two axes with multiple surface transducers. We demonstrate the ability of this galvo by scanning the mirror using a computer, via a custom driver circuit. The performance of the galvo, including scan range, noise, linearity, and scan speed, is characterized. As an application, we show that this galvo system can be used in a confocal scanning microscopy system.

Cell adaptations of Rhodococcus rhodochrous IEGM 66 to betulin biotransformation

The findings of a comprehensive study on R. rhodochrous IEGM 66 and triterpenoid betulin interactions during its biotransformation were reported. In the presence of betulin, rhodococci were shown to form heterogeneous cell aggregates. The enhanced size of the aggregates from 12–15 μm to 25– 35 μm was consistent with the increase in betulin concentration from 0.5 to 3.0 g/L. The confocal laser scanning microscopy indicated a high (80.0%) level of rhodococcal viability during betulin biotransformation regardless of the betulin concentration. Experiments employing the combined confocal laser scanning and atomic force microscopy system confirmed that interactions between actinobacterial cells and betulin occur by direct contact. Transforming activities of the crude cell extracts from R. rhodochrous IEGM 66 were compared, and localization of enzymes catalyzing betulin oxidation to betulone was determined. Additionally the effects of betulin on fatty acid composition of rhodococci and their morphometric and morphofunctional characteristics during biotransformation were studied. Our findings could be used to develop approaches for enhanced betulin bioavailability, thus leading to improved biotransformation efficiency.