Confocal Microscope System Research Articles

Optimising performance of a confocal fluorescence microscope with a differential pinhole

The signal-to-noise ratio (SNR)-resolution trade-off is of great importance to bio-imaging applications where the aim is to image the sample using as little light as possible without significantly sacrificing image quality. In this paper the inherent SNR-resolution tradeoff in Confocal Fluorescence Microscopy (CFM) systems is presented by means of an effective tradeoff curve. A CFM system that employs a differential pinhole detection scheme has recently been shown to offer increased resolution, but at the expense of SNR. An optimum profile for the differential pinhole is identified in this paper that offers improved performance over a conventional (circular pinhole) system. The performance enhancement is illustrated through computer simulation.

Ethanol extract of Sanguisorba officinalis L. inhibits biofilm formation of methicillin-resistant Staphylococcus aureus in an ica-dependent manner

Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen that shows resistance to many antibiotics and is usually associated with serious infections. Having the ability for biofilm formation increases resistance to antibiotics. Sanguisorba officinalis L. is a perennial plant that is distributed in the northern districts of China and has been used as a traditional Chinese medicine. In this study, the effect of S. officinalis on MRSA strain SA3 isolated from a dairy cow with mastitis was evaluated by testing the growth and biofilm formation ability of MRSA cultured with or without ethanol extracts of S. officinalis. The results showed that the ethanol extract of S. officinalis strongly inhibited the biofilm formation of MRSA. With a confocal laser scanning microscope system, we observed that the biofilm structure of the test group with the addition of S. officinalis appeared looser and had less biomass compared with the control group without S. officinalis. Furthermore, we found that the transcript levels of the icaADBC operon remarkably decreased upon addition of the ethanol extract of S. officinalis, indicating that S. officinalis inhibits biofilm formation of MRSA in an ica-dependent manner.

In vivo longitudinal cellular imaging of small intestine by side-view endomicroscopy.

Visualization of cellular dynamics in the gastrointestinal tract of living mouse model to investigate the pathophysiology has been a long-pursuing goal. Especially, for chronic disease such as Crohn's disease, a longitudinal observation of the luminal surface of the small intestine in the single mouse is highly desirable to investigate the complex pathogenesis in sequential time points. In this work, by utilizing a micro-GRIN lens based side-view endomicroscope integrated into a video-rate confocal microscopy system, we successfully performed minimally-invasive in vivo cellular-level visualization of various fluorescent cells and microvasculature in the small intestinal villi. Also, with a transgenic mouse universally expressing photoconvertible protein, Kaede, we demonstrated repetitive cellular-level confocal endoscopic visualization of same area in the small intestinal lumen of a single mouse, which revealed the continuous homeostatic renewal of the small intestinal epithelium.

Parallel detection experiment of fluorescence confocal microscopy using DMD.

Parallel detection of fluorescence confocal microscopy (PDFCM) system based on Digital Micromirror Device (DMD) is reported in this paper in order to realize simultaneous multi-channel imaging and improve detection speed. DMD is added into PDFCM system, working to take replace of the single traditional pinhole in the confocal system, which divides the laser source into multiple excitation beams. The PDFCM imaging system based on DMD is experimentally set up. The multi-channel image of fluorescence signal of potato cells sample is detected by parallel lateral scanning in order to verify the feasibility of introducing the DMD into fluorescence confocal microscope. In addition, for the purpose of characterizing the microscope, the depth response curve is also acquired. The experimental result shows that in contrast to conventional microscopy, the DMD-based PDFCM system has higher axial resolution and faster detection speed, which may bring some potential benefits in the biology and medicine analysis. SCANNING 38:234-239, 2016. © 2015 Wiley Periodicals, Inc.

Measurement of Local Partial Pressure of Oxygen in the Brain Tissue under Normoxia and Epilepsy with Phosphorescence Lifetime Microscopy.

In this work a method for measuring brain oxygen partial pressure with confocal phosphorescence lifetime microscopy system is reported. When used in conjunction with a dendritic phosphorescent probe, Oxyphor G4, this system enabled minimally invasive measurements of oxygen partial pressure (pO2) in cerebral tissue with high spatial and temporal resolution during 4-AP induced epileptic seizures. Investigating epileptic events, we characterized the spatio-temporal distribution of the "initial dip" in pO2 near the probe injection site and along nearby arterioles. Our results reveal a correlation between the percent change in the pO2 signal during the "initial dip" and the duration of seizure-like activity, which can help localize the epileptic focus and predict the length of seizure.

Anti-Apoptotic Gene Delivery with cyclo-(d-Trp-Tyr) Peptide Nanotube via Eye Drop Following Corneal Epithelial Debridement.

Corneal keratocyte apoptosis triggered by cornel debridement is one mechanism of corneal disorders. In this study, the feasibility of cyclo-(d-Trp-Tyr) peptide nanotubes (PNTs) as carriers of caspase 3 silence shRNA delivery was assessed. A model of epithelial injury by epithelial debridement was applied to investigate the feasibility of PNTs as gene delivery carriers on corneal injury. First, the PNTs were found within 2 μm in length and 300 nm in width by an atomic force microscope and confocal laser microscope system. Plasmid DNAs were observed to be associated with PNTs by atomic force microscope and confocal laser scanning microscope. The plasmids were associated with tyrosine of PNTs with a binding constant of 2.7 × 108 M−1. The stability of plasmid DNA with PNTs against the DNase was found at 60 min. Using thioflavin T pre-stained PNTs on the corneal eye drop delivery, the distribution of PNTs was in the epithelial and stroma regions. After corneal debridement, the rhodamine-labeled plasmid DNA and thioflavin T pre-stained PNTs were also delivered and could be observed in the stroma of cornea. PNTs complexed with anti-apoptotic plasmid caspase 3 silencing shRNA eye drop delivery decreased 41% of caspase 3 activity after the first dose by caspase 3 activity and Western blot analysis.

Alpha-phellandrene-induced apoptosis in mice leukemia WEHI-3 cells in vitro.

Although reports have shown that α-phellandrene (α-PA) is one of the monoterpenes and is often used in the food and perfume industry, our previous studies have indicated that α-PA promoted immune responses in normal mice in vivo. However, there is no available information to show that α-PA induced cell apoptosis in cancer cells, thus, we investigated the effects of α-PA on the cell morphology, viability, cell cycle distribution, and apoptosis in mice leukemia WEHI-3 cells in vitro. Results indicated that α-PA induced cell morphological changes and decreased viability, induced G0/G1 arrest and sub-G1 phase (apoptosis) in WEHI-3 cells. α-PA increased the productions of reactive oxygen species (ROS) and Ca2+ and decreased the levels of mitochondrial membrane potential (ΔΨm ) in dose- and time-dependent manners in WEHI-3 cells that were analyzed by flow cytometer. Results from confocal laser microscopic system examinations show that α-PA promoted the release of cytochrome c, AIF, and Endo G from mitochondria in WEHI-3 cells. These results are the first findings to provide new information for understanding the mechanisms by which α-PA induces cell cycle arrest and apoptosis in WEHI-3 cells in vitro. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1640-1651, 2016.

Dictionary-based image reconstruction for superresolution in integrated circuit imaging.

Resolution improvement through signal processing techniques for integrated circuit imaging is becoming more crucial as the rapid decrease in integrated circuit dimensions continues. Although there is a significant effort to push the limits of optical resolution for backside fault analysis through the use of solid immersion lenses, higher order laser beams, and beam apodization, signal processing techniques are required for additional improvement. In this work, we propose a sparse image reconstruction framework which couples overcomplete dictionary-based representation with a physics-based forward model to improve resolution and localization accuracy in high numerical aperture confocal microscopy systems for backside optical integrated circuit analysis. The effectiveness of the framework is demonstrated on experimental data.

In vivo quantitation of injected circulating tumor cells from great saphenous vein based on video-rate confocal microscopy.

The number of circulating tumor cell (CTC) in the peripheral blood of cancer patients can be a valuable biomarker for cancer diagnosis and treatment monitoring. In this study, we implemented a custom-design video-rate confocal microscopy system in capable of direct visualization of fast flowing CTC at great saphenous vein (GSV) of a live animal model in vivo. Continuous acquisition of video-rate images at GSV revealed the highly dynamic time-dependent changes in the number of intravenously injected circulating tumor cells. By extracting a calibration factor through the hemocytometric analysis of intravenously injected long-circulating red blood cells, we established a novel quantitation method for CTC in whole body blood in vivo.

Ab initio calculations of phonon dispersion and lattice dynamics in TlGaTe2

AbstractThis work reports the results of DFT‐based calculations of phonon spectra of TlGaTe2. The dispersion of phonon bands was calculated along the directions of Brillouin zone (BZ) that include symmetry points. The calculated phonon frequencies at the centre of BZ were compared with those obtained by Raman spectroscopy with the aid of a confocal laser microscopy system. A fairly good agreement between the calculated and experimental data was found. Complimentary, molar heat capacity at constant volume and Debye temperature were calculated in the range 5÷500 K on the base of the obtained phonon density of states. The obtained temperature dependencies were compared with available experimental data.The results of comparison were satisfactory. (© 2015 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Evaluation of crystal structure in TlInS2 by optical second- harmonic generation

Second-harmonic generation (SHG) in layered TlInS2 crystals was studied over the temperature range of 77-300 K using a confocal laser microscope system. As expected, the SHG signal was observed in the low temperature ferroelectric phase of the layered compound. In addition, the polarization properties of the SHG signals of TlInS2 were investigated in the 80-180 K range. The results are in good agreement with those of the symmetric space group C32 in the ferroelectric phase.

Temperature dependence of low‐frequency optical phonons in TlInS2

AbstractThe unpolarized Stocks component of the Raman spectra of the layered ternary thallium dichalcogenide, TlInS2 was studied with the aid of a Raman confocal microscope system in the low‐frequency region of 35‐150 cm‐1 over the temperature range that embraced the region of the successive phase transitions in this crystal. The observed spectra were deconvoluted into Lorentzian peaks to single‐out the contribution of each Raman mode. The temperature dependence of the Raman frequency and broadening associated with each mode was then obtained. The irregular temperature behaviour of most modes was disclosed in the proximity of phase transitions. (© 2015 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Fabrication and micro-photoluminescence property of CdSe/CdS core/shell nanowires

Hetero-epitaxial CdSe/CdS core/shell nanowires (NWs) were prepared by a source-controllable chemical vapor deposition method. A two-stage growth mechanism was proposed to the growth process of the core/shell NWs. Micro-photoluminescence (μ-PL) property of individual NW was studied by a confocal microscopy system. The pure CdSe NW emits a red light with peak at 712.3 nm, which is inconsistent with the CdSe band-edge emission. The CdSe/CdS core/shell NW emits two apparent peaks, one is an intensive red emission peak centered at 715.2 nm and the other is a weak green emission peak located at 516.2 nm. The room temperature μ-PL spectrum shows that the PL intensity of CdSe NW was evidently promoted by coating the CdS shell, and this is because CdS improves the surface state optimizing the energy band structure of CdSe NW. The as-synthesized CdSe/CdS core/shell NW has more efficient PL quantum yields than pure CdSe NW and may find potential applications in nanoscale photonic devices.

A series of flexible design adaptations to the Nikon E-C1 and E-C2 confocal microscope systems for UV, multiphoton and FLIM imaging.

Multiphoton microscopy is widely employed in the life sciences using extrinsic fluorescence of low- and high-molecular weight labels with excitation and emission spectra in the visible and near infrared regions. For imaging of intrinsic and extrinsic fluorophores with excitation spectra in the ultraviolet region, multiphoton excitation with one- or two-colour lasers avoids the need for ultraviolet-transmitting excitation optics and has advantages in terms of optical penetration in the sample and reduced phototoxicity. Excitation and detection of ultraviolet emission around 300 nm and below in a typical inverted confocal microscope is more difficult and requires the use of expensive quartz optics including the objective. In this technical note we describe the adaptation of a commercial confocal microscope (Nikon, Japan E-C1 or E-C2) for versatile use with Ti-sapphire and OPO laser sources and the addition of a second detection channel that enables detection of ultraviolet fluorescence and increases detection sensitivity in a typical fluorescence lifetime imaging microscopy experiment. Results from some experiments with this setup illustrate the resulting capabilities.

Rapid Detection of Methicillin Resistant Staphylococcus aureus Based on Surface Enhanced Raman Scattering

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the severe nosocomial infectious agents. The traditional diagnostic methods including biochemical test, antibiotic susceptibility test and PCR amplification are time consuming and require much work. The Surface enhanced Raman spectroscopy (SERS) biosensor is a rapid and powerful tool for analyzing the chemical composition within a single living cell. To identify the biochemical and genetic characterization of clinical MRSA, all isolates from patients were performed with VITEK2 gram positive (gP) bacterial identification and Antibiotic Susceptibility Testing (AST). Virulence genes of MRSA also were identified by DNA based PCR using specific primers. All isolates, which were placed on a gold coated nanochip, were analyzed by a confocal Raman microscopy system. All isolates were identified as S. aureus by biochemical tests. MRSA, which exhibited antibiotic resistance, demonstrated to be positive gene expression of both femA and mecA. Furthermore, Raman shift of S. aureus and MRSA (n=20) was perfectly distinguished by a confocal Raman microscopy system. This novel technique explained that a SERS based confocal Raman microscopy system can selectively isolate MRSA from non-MRSA. The study recommends the SERS technique as a rapid and sensitive method to detect antibiotic resistant S. aureus in a single cell level.

Discrimination of seal inks used for seals by confocal Raman microscopy

Purpose The purpose of this paper is to introduce an effective method to discriminate seal inks with Raman microscopy. Design/methodology/approach Raman spectra could effectively avoid interference from the paper and give extra peak information in the inks discrimination and identification. Thus, a Renishaw invia confocal Raman microscope system was employed for ink analysis in this study. A total of 12 representative seal ink samples, widely used in seven Chinese provinces, were investigated using the latest model of Renishaw Raman microscope. Findings Four types of inks were identified and discriminated successfully. Popular pigments such as Pigment Scarlet Powder, Pigment Yellow 55, phthalocyanine blue, Bronze red C and PbCrO3 were all identified in these seal ink samples. The indicative peaks to identify and discriminate the inks were also summarised and tentatively interpreted. Research limitations/implications More ink samples were needed to establish a useful library. Many other pigments used in inks were still unknown. Practical implications This method was proved to be fast, accurate and non-destructive, and it could be more easily applied in real cases than Fourier transform infrared spectroscopy. Originality/value This method can help scientists discriminate some inks, which can hardly be discriminated by other techniques. The results are useful for the ink analysis and discrimination in forensic (document examination and file source identification), polymer and pigment fields.

Digital differential confocal microscopy based on spatial shift transformation.

Differential confocal microscopy is a particularly powerful surface profilometry technique in industrial metrology due to its high axial sensitivity and insensitivity to noise. However, the practical implementation of the technique requires the accurate positioning of point detectors in three-dimensions. We describe a simple alternative based on spatial transformation of a through-focus series of images obtained from a homemade beam scanning confocal microscope. This digital differential confocal microscopy approach is described and compared with the traditional Differential confocal microscopy approach. The ease of use of the digital differential confocal microscopy system is illustrated by performing measurements on a 3D standard specimen.

Multiscale BerEp4 molecular imaging of microtumor phantoms: toward theranostics for basal cell carcinoma.

Basal cell carcinoma (BCC), the most common cancer in humans, appears macroscopically and microscopically similar to many other skin lesions, which makes differential diagnosis difficult. We are developing an approach for quantitative molecular imaging of BerEP4, a transmembrane biomarker for BCC, with the goal of increasing the precision and accuracy of diagnosis. This pilot study was conducted to assess the affinity and selectivity of BerEp4 antibody and assess its possible use in designing theranostic probes for BCC. We provide evidence that our photon-counting fluorescence macrodetection system can recover specific signal increases from a film/pellet phantom. Additionally, we show that a two-photon excited fluorescence /backscatter confocal microscopy system can image BerEP4 antibody/antigen complex on the surface of BerEP4-expressing cancer cells in three dimensions. Based on the initial results, BerEP4 seems to be a promising biomarker for molecular imaging of BCC. To prepare BerEP4 for eventual theranostic use, we examined the feasibility of a combined macro-/micro-optical approach to imaging BCC with various histologies. These optical methods, endowed with the ability to monitor treatment in real time, may open an opportunity for noninvasive diagnosis, treatments, and follow-up.

ZebraBeat: a flexible platform for the analysis of the cardiac rate in zebrafish embryos

Heartbeat measurement is important in assesssing cardiac function because variations in heart rhythm can be the cause as well as an effect of hidden pathological heart conditions. Zebrafish (Danio rerio) has emerged as one of the most useful model organisms for cardiac research. Indeed, the zebrafish heart is easily accessible for optical analyses without conducting invasive procedures and shows anatomical similarity to the human heart. In this study, we present a non-invasive, simple, cost-effective process to quantify the heartbeat in embryonic zebrafish. To achieve reproducibility, high throughput and flexibility (i.e., adaptability to any existing confocal microscope system and with a user-friendly interface that can be easily used by researchers), we implemented this method within a software program. We show here that this platform, called ZebraBeat, can successfully detect heart rate variations in embryonic zebrafish at various developmental stages, and it can record cardiac rate fluctuations induced by factors such as temperature and genetic- and chemical-induced alterations. Applications of this methodology may include the screening of chemical libraries affecting heart rhythm and the identification of heart rhythm variations in mutants from large-scale forward genetic screens.

In vivo analysis of THz wave irradiation induced acute inflammatory response in skin by laser-scanning confocal microscopy.

The recent development of THz sources in a wide range of THz frequencies and power levels has led to greatly increased interest in potential biomedical applications such as cancer and burn wound diagnosis. However, despite its importance in realizing THz wave based applications, our knowledge of how THz wave irradiation can affect a live tissue at the cellular level is very limited. In this study, an acute inflammatory response caused by pulsed THz wave irradiation on the skin of a live mouse was analyzed at the cellular level using intravital laser-scanning confocal microscopy. Pulsed THz wave (2.7 THz, 4 μs pulsewidth, 61.4 μJ per pulse, 3Hz repetition), generated using compact FEL, was used to irradiate an anesthetized mouse's ear skin with an average power of 260 mW/cm(2) for 30 minutes using a high-precision focused THz wave irradiation setup. In contrast to in vitro analysis using cultured cells at similar power levels of CW THz wave irradiation, no temperature change at the surface of the ear skin was observed when skin was examined with an IR camera. To monitor any potential inflammatory response, resident neutrophils in the same area of ear skin were repeatedly visualized before and after THz wave irradiation using a custom-built laser-scanning confocal microscopy system optimized for in vivo visualization. While non-irradiated control skin area showed no changes in the number of resident neutrophils, a massive recruitment of newly infiltrated neutrophils was observed in the THz wave irradiated skin area after 6 hours, which suggests an induction of acute inflammatory response by the pulsed THz wave irradiation on the skin via a non-thermal process.