Confocal Laser Scanning Microscopy System Research Articles

Margination of platelet-sized particles in red blood cell suspensions flowing through a Y-shaped confluence microchannel

In blood flow through microvessels, platelets are known to be distributed in high concentrations near the vessel wall, termed ‘margination’ or ‘near-wall excess’. At the confluence of two vessels, this preferential distribution of platelets is thought to be compromised and reconstituted in the downstream main vessel. The present study aimed to investigate the distance of this margination reconstruction from the confluence by in vitro experiments using platelet-sized fluorescent particles as a platelet surrogate and a Y-shaped confluence microchannel with rectangular cross sections. Fluorescence microscopy was performed using a confocal laser scanning microscope system to measure the distribution of particles in the red blood cell suspension flow. Immediately after confluence, particles were highly concentrated along a narrow band in the middle of the channel width, where particles located near the inner wall of the daughter channels flowed in. This dense band of particles faded downstream and disappeared less than 5 mm from the confluence. This margination distance is comparable to or smaller than the margination development distance in straight channels, but much smaller than that after bifurcation.

Confocal Laser Scanning Microscopy Based on a Silicon Photomultiplier for Multicolor In Vivo Imaging in Near-Infrared Regions I and II.

Confocal laser scanning microscopy (CLSM) is expected to exhibit a better imaging performance in the second near-infrared (NIR-II) windows with weak tissue scattering and autofluorescence. However, the indium gallium arsenide (InGaAs) detectors currently used for imaging in the NIR-II region are prohibitively expensive, hampering its extensive biomedical applications. In this study, a novel NIR-II CLSM system is developed by using the inexpensive silicon photomultiplier (SiPM) that can perform the multicolor biological imaging in vivo. Using IR-780 iodide as the contrast agent, the NIR-II imaging capability of constructed CLSM is inspected, demonstrating a spatial resolution of 1.68 µm (close to the diffraction limit) and a fluorophore detection sensitivity as low as 100 nm. In particular, it is discovered that the multicolor imaging performance in both NIR-I and NIR-II windows is comparable to those from multialkali and InGaAs photomultiplier tubes. In addition, 3D NIR-II CLSM is also conducted for in vivo imaging of the vascular structure in mouse ear and subcutaneous tumors. To the best of authors' knowledge, this is the first time that a low-cost detector based on a SiPM has been used for microscopic imaging of trailing fluorescence signals in the NIR-II region of an NIR fluorescent probe.

Active Aberration Correction with Adaptive Coefficient SPGD Algorithm for Laser Scanning Confocal Microscope.

A laser scanning confocal microscope (LSCM) is an effective scientific instrument for studying sub-micron structures, and it has been widely used in the field of biological detection. However, the illumination depth of LSCMs is limited due to the optical aberrations introduced by living biological tissue, which acts as an optical medium with a non-uniform refractive index, resulting in a significant dispersion of the focus of LSCM illumination light and, hence, a loss in the resolution of the image. In this study, to minimize the effect of optical aberrations, an image-based adaptive optics technology using an optimized stochastic parallel gradient descent (SPGD) algorithm with an adaptive coefficient is applied to the optical path of an LSCM system. The effectiveness of the proposed aberration correction approach is experimentally evaluated in the LSCM system. The results illustrate that the proposed adaptive optics system with an adaptive coefficient SPGD algorithm can effectively reduce the interference caused by aberrations during depth imaging.

Margination of Platelet-Sized Particles in the Red Blood Cell Suspension Flow through Square Microchannels.

In the blood flow through microvessels, platelets show high concentrations near the vessel wall. This phenomenon is called margination of platelets and is closely associated with hemostasis and thrombosis. In the present study, we conducted in vitro experiments using platelet-sized fluorescent particles as platelet substitutes to investigate the cross-sectional distribution of these particles in the red blood cell suspension flowing through microchannels with a square cross section. Fluorescence observations were performed to measure the transverse distribution of particles at various heights from the bottom face with the use of a confocal laser scanning microscope system. In downstream cross sections of the channel, particles showed focusing near the four corners rather than uniform margination along the entire circumference of the cross section. The focusing of particles near the corners was more enhanced for higher hematocrits. On the other hand, particles in circular channel flows showed nearly axisymmetric uniform accumulation adjacent to the channel wall. The present result suggests that the segregation of suspended particles in the flow of multicomponent suspensions could have such heterogeneous 2D features of particle distribution in the cross section of channels, especially for rectangular channels often used in microfluidics.

Confocal-based fluorescence fluctuation spectroscopy with a SPAD array detector

The combination of confocal laser-scanning microscopy (CLSM) and fluorescence fluctuation spectroscopy (FFS) is a powerful tool in studying fast, sub-resolution biomolecular processes in living cells. A detector array can further enhance CLSM-based FFS techniques, as it allows the simultaneous acquisition of several samples–essentially images—of the CLSM detection volume. However, the detector arrays that have previously been proposed for this purpose require tedious data corrections and preclude the combination of FFS with single-photon techniques, such as fluorescence lifetime imaging. Here, we solve these limitations by integrating a novel single-photon-avalanche-diode (SPAD) array detector in a CLSM system. We validate this new implementation on a series of FFS analyses: spot-variation fluorescence correlation spectroscopy, pair-correlation function analysis, and image-derived mean squared displacement analysis. We predict that the unique combination of spatial and temporal information provided by our detector will make the proposed architecture the method of choice for CLSM-based FFS.

Tracing Carbon Nanotubes (CNTs) in Rat Peripheral Nerve Regenerated with Conductive Conduits Composed of Poly(lactide-co-glycolide) and Fluorescent CNTs.

Nerve regeneration can be promoted using nerve guide conduits (NGCs). Carbon nanotubes (CNTs) are often used to prepare conductive NGCs, however, the major concern for their applications is the final location of the implanted CNTs in vivo. Herein, photoluminescent multiwalled CNTs (MWCNTs) were prepared and electrospun with poly(lactide-co-glycolide) (PLGA), followed by shaping into multichannel NGCs for repairing of injured rat sciatic nerve, thereby the distribution of CNTs in vivo could be detected via bioimaging. Photoluminescent MWCNTs (MWCNT-FITC) were prepared by functionalization with poly(glycidyl methacrylate) (PGMA) and fluorescein-isothiocyanate-isomer I (FITC) subsequently. The conductivity of the PLGA/MWCNT-FITC fibers was approx. 10-4 S/cm at 3 wt % MWCNTs. Compared with PLGA fibers, Schwann cells on PLGA/MWCNT-FITC fibers matured at a faster rate, accordingly, nerve regeneration was promoted by the PLGA/MWCNT-FITC NGC. With a confocal laser scanning microscope and small-animal imaging system, the location of MWCNTs was detected. Alongside the degradation of PLGA, MWCNTs intended to aggregate and were entrapped in the regenerated nerve tissue without migrating into surrounding tissues and other organs (liver, kidneys, and spleen). This study provides a useful characterization method for MWCNTs and the guidance for in vivo applications of MWCNTs in tissue engineering.

Synthesis and biological evaluation of novel pyrazoline derivatives containing indole skeleton as anti-cancer agents targeting topoisomerase II

In order to develop potent anticaner agents, a novel series of 3-(1H-indol-3-yl)-2,3,3a,4-tetrahydrothiochromeno[4,3-c]pyrazole derivatives were synthesized. Structures of all compounds were confirmed. MTT assay has been employed to study antiproliferative activity of these compounds with four human cancer cell lines (MGC-803, Hela, MCF-7 and Bel-7404) and a normal cell line L929. Most of these compounds showed potential anticancer activity and low cytotoxicity on normal cell in vitro. 7d and 7f showed the best anticancer activity, whose IC50 value is 15.43 μM and 20.54 μM towards MGC-803, respectively. Most of them exhibited topoisomerase II selective inhibitory. Cleavage reaction assay and DNA unwinding assay showed that 7f was a nonintercalative Topo II catalytic inhibitor, which was consistent with the docking results. Laser scanning confocal microscopy system tracks the location of representative compounds 7d and 7f which can be abundantly entering the nucleus. In particular, the most potent compounds 7d and 7f were shown to be able to induce G2/M cell cycle arrest and apoptosis in MGC-803 cells.

Lysosome targeting carbon dots-based fluorescent probe for monitoring pH changes in vitro and in vivo

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.

Microcrack propagation in transverse surface from heartwood to sapwood during drying

Microcracks, which are difficult to be observed with naked eyes, occur on the transverse surface during the first stage of wood drying. The precise investigation of their appearance is critical for understanding the mechanism of crack occurrence and propagation, which influence the properties of wood. In this study, the location and time difference of microcrack occurrence on the transverse surfaces of outer heartwood to sapwood via the intermediate wood of Cryptomeria japonica were investigated. Using the developed confocal laser scanning microscope system and a digital microscope, the microcrack propagation was captured dynamically with the wide range of high-quality images; microcracks appeared initially in the heartwood region, immediately after drying. On the other hand, in sapwood, microcrack generation occurred only after microcracks appeared in intermediate wood, and the ones in heartwood started to close or were closed. Finally, most of the microcracks almost closed and some completely disappeared towards the end of the drying process. From this result, it can be established that appropriate drying conditions should be prepared during the early stage of drying to produce high-quality wooden products because microcracks can appear in heartwood, even though the moisture content of the specimen is high.

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.

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.

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.

Modern Laser Scanning Confocal Microscopy.

Since its commercialization in the late 1980's, confocal laser scanning microscopy (CLSM) has since become one of the most prevalent fluorescence microscopy techniques for three-dimensional structural studies of biological cells and tissues. The flexibility of the approach has enabled its application in a diverse array of studies, from the fast imaging of dynamic processes in living cells, to meticulous morphological analyses of tissues, and co-localization of protein expression patterns. In this chapter, we introduce the principles of confocal microscopy and discuss how the approach has become a mainstay in the biological sciences. We describe the components of a CLSM system and assess how modern implementations of the approach have further expanded the use of the technique. Finally, we briefly outline some practical considerations to take into account when acquiring data using a CLSM system. © 2018 by John Wiley & Sons, Inc.

Root canal adaptation and intra-tubular penetration of three fiber-post cementation systems

BackgroundTo measure the penetration of three bonding systems for the luting of fiber glass posts in endodontically treated teeth, using confocal laser scanning microscopy (CLSM).Material and MethodsA total of 30 maxillary incisors were shaped with the Mtwo system and filled with gutta-percha and Top Seal cement. The sample was divided into three groups (ni=10) according to the bonding system used to cement the posts: Group 1 (Prime&Bond NT and Rebilda DC with a total-etch technique); Group 2 (Futurabond DC and Rebilda DC with a dentin self-etch technique); and Group 3 (BisCem self-adhesive cement). Rhodamine B was added to the bonding systems to allow visualization by CLSM. Three 1-mm thick cross-sections were obtained of each root at a distance of 2, 5 and 8 mm from the coronal limit of the root. The specific software of the CLSM system was used to measure the percentage of the root canal perimeter showing penetration of the bonding system in the dentinal tubules, together with the maximum depth of penetration. Comparison between groups were made by Kruskal Wallis test, and comparison two by two groups with Mann-Whitney U-test.ResultsDepth of penetration of the resin tags, were distributed from greater to lesser depth as follows: BisCem > Prime&Bond NT > Futurabond DC. BisCem showed significantly greater penetration in the middle and apical thirds than the rest of the systems (906.14±67.42 and 699.27±76.26 µm, respectively). The percentage perimeter exhibiting penetration in the coronal third was significantly greater with BisCem versus Futurabond DC (56.08±7.24 and 44.38±5.23%, respectively). No significant differences were recorded in the middle and apical thirds among the three systems.ConclusionsBisCem resulted in greater depth of intratubular penetration at all studied levels. The percentage perimeter of the canal showing penetration was similar for all the bonding systems. Key words:Fiber post luting, sealer adaptation, confocal laser scanning microscope.

Direct imaging of tunable photonic nanojets from a self-assembled liquid crystal microdroplet.

We report the direct experimental observation of electrically tunable photonic nanojets (PNJs) generated from self-assembled liquid crystal (LC) microdroplets formed by dispersing nematic LCs in polymer matrix. Optical measurements were performed with a home-built laser-scanning confocal microscope system. PNJs with subwavelength beam waists were successfully obtained from LC microdroplets of 5μm diameter, similar to those from SiO2 microspheres of the same size. By applying external voltage of a few volts, some of major properties of PNJs, such as beam length and brightness, are tuned. Electro-tunable PNJs from self-assembled LC microdroplets may open the way for the development of novel micro-optical devices.

In vivo cellular-level real-time pharmacokinetic imaging of free-form and liposomal indocyanine green in liver.

Indocyanine green (ICG) is a near-infrared fluorophore approved for human use which has been widely used for various clinical applications. Despite the well-established clinical usage, our understanding about the microscopic in vivo pharmacokinetics of systemically administered ICG has been relatively limited. In this work, we successfully visualized real-time in vivo pharmacokinetic dynamics of the intravenously injected free-form and liposomal ICG in cellular resolution by utilizing a custom-built video-rate near infrared laser-scanning confocal microscopy system. Initial perfusion and clearance from blood stream, diffusion into perisinusoidal space, and subsequent absorption into hepatocyte were directly visualized in vivo. The quantification analysis utilizing the real-time image sequences revealed distinct dynamic in vivo pharmacokinetic behavior of free-form and liposomal ICG.

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.

Characterizing depth-dependent refractive index of articular cartilage subjected to mechanical wear or enzymic degeneration.

Utilizing a laser scanning confocal microscope system, the refractive indices of articular cartilage (AC) with mechanical or biochemical degenerations were characterized to investigate whether potential correlations exist between refractive index (RI) and cartilage degeneration. The cartilage samples collected from the medial femoral condyles of kangaroo knees were mechanically degenerated under different loading patterns or digested in trypsin solution with different concentrations. The sequences of RI were then measured from cartilage surface to deep region and the fluctuations of RI were quantified considering combined effects of fluctuating frequency and amplitude. The compositional and microstructural alterations of cartilage samples were assessed with histological methods. Along with the loss of proteoglycans, the average RI of cartilage increased and the local fluctuation of RI became stronger. Short-term high-speed test induced little influence to both the depth fluctuation and overall level of RI. Long-term low-speed test increased the fluctuation of RI but the average RI was barely changed. The results substantially demonstrate that RI of AC varies with both compositional and structural alterations and is potentially an indicator for the degeneration of AC.

Advanced oxidative protein products induced human keratinocyte apoptosis through the NOX-MAPK pathway.

Impaired wound healing is a major diabetes-related complication. Keratinocytes play an important role in wound healing. Multiple factors have been proposed that can induce dysfunction in keratinocytes. The focus of present research is at a more specific molecular level. We investigated the role of advanced oxidative protein products (AOPPs) in inducing human immortalized keratinocyte (HaCaT) cell apoptosis and the cellular mechanism underlying the proapoptotic effect of AOPPs. HaCaT cells were treated with increasing concentrations of AOPP-human serum albumin or for increasing time durations. The cell viability was measured using the thiazolyl blue tetrazolium bromide method, and flow cytometry was used to assess the rate of cell apoptosis. A loss of mitochondrial membrane potential (MMP) and an increase in intracellular reactive oxygen species (ROS) were observed through a confocal laser scanning microscope system, and the level of ROS generation was determined using a microplate reader. Nicotinamide adenine dinucleotide phosphate oxidase (NOX)4, extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and apoptosis-related downstream protein interactions were investigated using the Western blot analysis. We found that AOPPs triggered HaCaT cell apoptosis and MMP loss. After AOPP treatment, intracellular ROS generation increased in a time- and dose-dependent manner. Proapoptotic proteins, such as Bax, caspase 9/caspase 3, and poly(ADP-ribose) polymerase (PARP)-1 were activated, whereas anti-apoptotic Bcl-2 protein was downregulated. AOPPs also increased NOX4, ERK1/2, and p38 MAPK expression. Taken together, these findings suggest that extracellular AOPP accumulation triggered NOX-dependent ROS production, which activated ERK1/2 and p38 MAPK, and induced HaCaT cell apoptosis by activating caspase 3 and PARP-1.