Tridimensional quantitative porosity characterization of three set calcium silicate‐based repair cements for endodontic use

The aim of the this study was to quantitatively evaluate in three‐dimensional (3D), the porosity degree of three improved silicate‐based endodontic repair cements (iRoot BP Plus^®, Biodentine^®, and Ceramicrete) compared to a gold‐standard calcium silicate bioactive cement (Pro Root^® MTA). From each tested cement, four samples were prepared by a single operator following the manufacturer's instructions in terms of proportion, time, and mixing method, using cylindrical plastic split‐ring moulds. The moulds were lubricated and the mixed cements were inserted with the aid of a cement spatula. The samples were scanned using a compact micro‐CT device (Skyscan 1174, Bruker micro‐CT, Kontich, Belgium) and the projection images were reconstructed into cross‐sectional slices (NRecon v.1.6.9, Bruker micro‐CT). From the stack of images, 3D models were rendered and the porosity parameters of each tested material were obtained after threshold definition by comparison with standard porosity values of Biodentine^®. No statistically significant differences in the porosity parameters among the different materials were seen. Regarding total porosity, iRoot BP Plus^® showed a higher percentage of total porosity (9.58%), followed by Biodentine^® (7.09%), Pro Root^® MTA (6.63%), and Ceramicrete (5.91%). Regarding closed porosity, Biodentine^® presented a slight increase in these numbers compared to the other sealers. No significant difference in porosity between iRoot BP Plus^®, Biodentine^®, and Ceramicrete were seen. In addition, no significant difference in porosity between the new calcium silicate‐containing repair cements and the gold‐standard MTA were found. Microsc. Res. Tech., 76:1093–1098, 2013. © 2013 Wiley Periodicals, Inc.     

Chemical and morphological evaluation of enamel and dentin near cavities restored with conventional and zirconia modified glass ionomer subjected to erosion‐abrasion

Microenergy dispersive X‐ray fluorescence (μ‐EDXRF) spectroscopy and scanning electron microscopy (SEM) were used to test the hypothesis that zirconia modified glass ionomer cement (GIC) could improve resistance to erosion‐abrasion to a greater extent than conventional cement. Bovine enamel (n = 40) and dentin (n = 40) samples were prepared with cavities, filled with one of the two restorative materials (GIC: glass‐ionomer cement or ZrGIC: zirconia‐modified GIC). Furthermore, the samples were treated with abrasion‐saliva (AS) or abrasion‐erosion cycles (AE). Erosive cycles (immersion in orange juice, three times/day for a duration of 1 min over a 5 day period) and/or abrasive challenges (electric toothbrush, three times/day for a duration of 1 min over a 5 day period) were performed. Positive mineral variation (MV%) on the enamel after erosion‐abrasion was observed for both materials (p < 0.05), whereas a negative MV% on the dentin was observed for both materials and treatments (p < 0.05). The SEM images showed clear enamel loss after erosion‐abrasion treatment and material degradation was greater in GIC_AE compared to those of the other groups. Toothbrush abrasion showed a synergistic effect with erosion on substance loss of bovine enamel, dentin, GIC, and ZrGIC restorations. Zirconia addition to the GIC powder improved the resistance to abrasive‐erosive processes. The ZrGIC materials may find application as a restorative material due to improved resistance as well as in temporary restorations and fissure sealants.     

Two‐photon laser scanning microscopy as a useful tool for imaging and evaluating macrophage‐, IL‐4 activated macrophage‐ and osteoclast‐based In Vitro degradation of beta‐tricalcium phosphate bone substitute material

Two‐photon microscopy is an innovative technology that has high potential to combine the examination of soft and hard tissues in vitro and in vivo. Calcium phosphates are widely used substitutes for bone tissue engineering, since they are degradable and consequently replaced by newly formed tissue. It is well known that osteoclasts are responsible for the resorption processes during bone remodelling. We hypothesize that also macrophages are actively involved in the resorption process of calcium phosphate scaffolds and addressed this question in in vitro culture systems by two‐photon laser scanning microscopy. Beta‐tricalcium phosphate specimens were incubated with (1) macrophages, (2) interleukin‐4 activated macrophages, and (3) osteoclasts for up to 21 days. Interestingly, macrophages degraded beta‐tricalcium phosphate specimens in an equivalent fashion compared to osteoclasts and significantly more than IL‐4 activated macrophages. An average of ～32% of the macrophages was partially filled with ceramic material while this was 18% for osteoclasts and 9% for IL‐4 activated macrophages. For the first time by applying two‐photon microscopy, our studies show the previously unrecognized potential of macrophages to phagocytose ceramic material, which is expected to have implication on osteoconductive scaffold design. Microsc. Res. Tech. 77:143–152, 2014. © 2013 Wiley Periodicals, Inc.     

Adhesive interface and bond strength of endodontic sealers to root canal dentine after immersion in phosphate‐buffered saline

This study evaluated the bond strength (BS) and the adhesive interface of four endodontic sealers to root canal dentine, before, and after immersion in phosphate buffered saline (PBS) to simulate an in vivo environment. Eighty roots were instrumented using ProTaper rotatory files, under irrigation with 17% EDTA and 1% NaOCl. Posteriorly were divided into four groups (n = 20) according to the sealer used: Endofill, AH Plus, Sealapex, and MTA Fillapex. Each group was divided into two subgroups (n = 10) and stored at 37°C immersed in water for 7 days and in PBS for 60 days. From each subgroup, 1 mm thick sections were obtained. One section of each region (coronal, middle, and apical) was submitted to the push‐out test and failures were observed. Twelve sections of each subgroup (four from each region) were evaluated under SEM. Three‐way ANOVA evaluation for BS showed significant differences between groups and regions (P < 0.0001), but not between subgroups (P > 0.05). AH Plus had significantly higher BS than the others sealers, regardless of the analyzed subgroup (Tukey's test, P < 0.5). The most common failures were adhesive to dentine and cohesive of the sealer. The SEM evaluation (Kruskal–Wallis, Mann–Whitney) showed homogeneous adhesive interface formed and sealer tags in all groups with significant statistical differences with AH Plus, regardless of PBS immersion. AH Plus was superior to the other sealers for both BS and quality of interface formation. Immersion in PBS did not interfere on BS or adhesive interface of the sealers tested. Microsc. Res. Tech. 77:1015–1022, 2014. © 2014 Wiley Periodicals, Inc.     

Erosion effects on chemical composition and morphology of dental materials and root dentin

Purpose: This work aims to study the erosion on restorative materials and on surrounding dentin. Fifty root dentin samples were obtained from bovine incisors. Methods: Twenty samples were not restored and thirty received cavity preparations. Samples were assigned to five groups: G1, G2: sound dentin (D); G3: composite resin (CR); G4: resin‐modified glass‐ionomer cement (RMGIC); G5: glass‐ionomer cement (GIC). The samples of groups 2–5 were submitted to six cycles (demineralization–remineralization). Samples were analyzed by micro energy‐dispersive X‐ray fluorescence spectrometry (μ‐EDXRF) and by scanning electron microscopy (SEM). Results: Mineral loss was greater in G2 samples than in RMGI > CR > GIC > D (control). SEM images showed pronounced dentin demineralization in groups 2 and 4. The acid erosion has a significant effect on mineral loss (Ca and P) of root dentin without restoration. Conclusions: Composite resin had the best chemical resistance to erosion among all the materials. Fluoride contained in GIC seemed to cause some protection, however, with material degradation. Chemical interaction of tooth‐colored dental materials with root dentin could be assessed by μ‐EDXRF. Microsc. Res. Tech. 75:703–710, 2012. © 2011 Wiley Periodicals, Inc.     

The effect of gastric acid on chitosan modified glass ionomer cement: SEM‐EDS

This study aimed to use scanning electron microscopy with energy dispersive spectroscopy (SEM‐EDS) to examine the elements that passed into the gastric acid solution after the application of a gastric acid erosive cycle to chitosan modified glass ionomer cement (GIC). Chitosan modified GIC samples were obtained by adding chitosan (vol/vol) of 5 and 10% to GIC for the experimental groups. These two experimental groups and a control group were subjected to gastric acid erosive treatment for 60 s six times a day for 10 days. The sample surfaces were coated with approximately 1 nm of gold to increase conductivity with the Q 150R ES device (Quorum Technologies, East Sussex, England). Surface topography images were obtained with a SEM. Besides, EDS analysis was also determined the number of elements graphically in the region where the fast electron beam hit. In the samples examined, the amount of element was determined. After gastric acid application, cracks and voids were observed on the surfaces of the samples. In the EDS analysis of the 5 and 10% chitosan modified GIC and control groups, Si, Al, Na, and F was found. It is necessary to investigate the antibacterial properties and physical properties of chitosan modified glass ionomer‐free elements and fluorine ions using advanced techniques.     

Effects of Er:YAG laser on bond strength of self‐etching adhesives to caries‐affected dentin

The erbium:yttrium–aluminum–garnet (Er:YAG) laser may be effective the bond strength of adhesive systems on dentine surfaces, the chemical composition and aggressiveness of adhesive systems in clinical practice. The purpose of this study was to evaluate the effects of the Er:YAG laser system with the bonding ability of two different self‐etching adhesives to caries‐affected dentine in primary molars. Ninety mid‐coronal flat dentine surfaces obtained from sound and caries‐affected human primary dentine were treated with an Er:YAG laser or a bur. The prepared surfaces were restored with an adhesive system (Xeno V; Clearfil S³) and a compomer (Dyract Extra). The restored teeth were sectioned with a low‐speed saw and 162 samples were obtained. The bond strength of the adhesive systems was tested using the micro‐tensile test method. The data were statistically analyzed. A restored tooth in each group was processed for scanning electron microscopy evaluation. The values of the highest bond strength were obtained from the Clearfil S³‐Er:YAG laser‐sound dentine group in all groups. (24.57 ± 7.27 MPa) (P > 0.05). The values of the lowest bond strength were obtained from the Xeno V‐Er:YAG laser‐sound dentine group in all groups (11.01 ± 3.89 MPa). It was determined that the Clearfil S³ increased the bond strength on the surface applied with Er:YAG laser according to the Xeno V. Microsc. Res. Tech. 77:282–288, 2014. © 2014 Wiley Periodicals, Inc.     

Label‐free optical detection of age‐related and diabetic oxidative damage in human aqueous humors

In this study, we investigate the biochemical characteristics of oxidative stress in age‐related macular degeneration (AMD) and diabetic retinopathy (DR) by analyzing aqueous humors. Nondiabetic cataract aqueous humor was used as the control. The level of oxidative damage was evaluated based on changes in Raman spectral intensity. Seven prominent peaks were detected at 1002, 1043, 1062, 1352, 1419, 1454, and 1656 cm^?1. We proposed four multimodal biomarkers to distinguish these peaks based on the ratios of Raman intensities in two wavelengths, including CHO (C–O stretching or C–O–H bending modes), AG (adenine and guanine), PRO‐AG (protein and AG), and PHEα (phenylalanine symmetric ring breath and amide I α‐helix) markers. The presence of oxidative damage was detected by CHO and AG markers associated with C–O stretching, C–O–H bending modes in carbohydrates (1043 cm^?1), and the nucleic acids adenine and guanine (1352 cm^?1), respectively. DR‐related oxidative damage was identified by PRO‐AG and PHEα markers associated with adenine, guanine, and protein components (1419 and 1454 cm^?1) and amide I α‐helix protein structure (1656 cm^?1), respectively. AMD‐related oxidative damage was identified by four biomarkers. Four multimodal biomarkers with simple linear threshold values achieved high sensitivity of 100% and high specificity of 100% for classifying oxidative stress‐induced AMD and DR diseases. Therefore, Raman‐based label‐free optical detection is effective for detecting the presence of age‐related or diabetic oxidative damage in aqueous humor.     

Analysis of efficacy of the self‐assembling peptide‐based remineralization agent on artificial enamel lesions

The objective of this in vitro study was to analyze and compare the biomimetic remineralizing efficacy of the self‐assembling peptide (P11‐4) with agents containing casein phoshopeptide‐amorhous calcium phosphate fluoride (CPP‐ACFP) and sodium fluoride (NaF) on artificial caries lesions using DIAGNOdent and micro‐computed tomography (μCT). Artificial enamel lesions were prepared on extracted impacted sound mandibular third molars. The samples were randomly allocated to four groups (n = 8): Group 1, P11‐4 (Curodont Repair, Credentis AG, Switzerland); Group 2, CPP‐ACFP (MI Varnish, GCCo., Japan); Group3, NaF (Duraphat Varnish, Colgate, Colgate‐Palmolive, NY, USA); Group 4, artificial saliva (control). The agents were applied to demineralized surfaces according to manufacturers' instructions; all specimens were stored in artificial saliva for 1 month. Demineralization and remineralization on enamel surfaces were analyzed and quantified by DIAGNOdent (KaVo, Germany) and μCT (SkyScan1174, Belgium) for lesion depth/area/volume/mineral density (MD). The remineralization efficacy of the agents was evaluated by DIAGNOdent on 1st, 7th, 30th days and by μCT on 30th day. Data were statistically analyzed by ANOVA, Kruskal–Wallis, T test, and Wilxocon tests. The highest remineralization efficacy findings in all periods were determined in Group 1, followed by Groups 2, 3, and 4. The remineralization findings for fluorescence, MD, lesion depth in Group 1 were found significantly higher (p < 0.01) than Group 3; and no significant differences (p > 0.05) were found between Groups 1–2 and Groups 2–3. The area and volume change values in Groups 1, 2, and 3 have shown no significancy (p > 0.05). A significant correlation (p < 0.01) was found between μCT and DIAGNOdent methods. The data of this study have demonstrated that P11‐4 has showed the best remineralization efficacy, followed by CPP‐ACFP and NaF. It is concluded that self‐assembling peptide‐based remineralization agent can be used successfully for biomimetic remineralization of enamel subsurface lesions.     

Fibre‐optical microendoscopy

Microendoscopy has been an essential tool in exploring micro/nano mechanisms in vivo due to high‐quality imaging performance, compact size and flexible movement. The investigations into optical fibres, micro‐scanners and miniature lens have boosted efficiencies of remote light delivery to sample site and signal collection. Given the light interaction with materials in the fluorescence imaging regime, this paper reviews two classes of compact microendoscopy based on a single fibre: linear optical microendoscopy and nonlinear optical microendoscopy. Due to the fact that fluorescence occurs only in the focal volume, nonlinear optical microendoscopy can provide stronger optical sectioning ability than linear optical microendoscopy, and is a good candidate for deep tissue imaging. Moreover, one‐photon excited fluorescence microendoscopy as the linear optical microendoscopy suffers from severe photobleaching owing to the linear dependence of photobleaching rate on excitation laser power. On the contrary, nonlinear optical microendoscopy, including two‐photon excited fluorescence microendoscopy and second harmonic generation microendoscopy, has the capability to minimize or avoid the photobleaching effect at a high excitation power and generate high image contrast. The combination of various nonlinear signals gained by the nonlinear optical microendoscopy provides a comprehensive insight into biophenomena in internal organs. Fibre‐optical microendoscopy overcomes physical limitations of traditional microscopy and opens up a new path to achieve early cancer diagnosis and microsurgery in a minimally invasive and localized manner.     

Biochemical fingerprints of human papillomavirus infection and cervical dysplasia using cervical fluids: Spectral pattern investigation

The Pap smear is the primary screening tool for invasive cervical cancer resulting from a persistent infection with oncogenic human papillomavirus (HPV); however, there are the problems such as the inability to distinguish between HPV infection and cervical dysplasia and a low sensitivity remain. We present preliminary findings of a label‐free method to detect and classify HPV infection and cervical dysplasia using human cervical fluids. Three experimental groups, defined as normal, HPV‐positive, and cervical dysplasia, were evaluated through their Raman spectral patterns for noise‐independence, high reproducibility, and uniformity. Clinical diagnosis was performed through liquid‐based cervical cytology, HPV test, and cervical histologic examination. Healthy cervical fluids showed a strong Raman intensity at 877 cm^?1 (symmetric C–C stretching), and at 963 cm^?1 (phosphate), compared to a reference Raman peak at 1003 cm^?1 (phenylalanine symmetric ring breath). The HPV‐positive cervical fluids showed a strong intensity of a Raman peak at 1448 cm^?1 corresponding to C–H deformation vibration mode and the highest similarity between the central and ring zones among the three groups. The cervical dysplasia fluids showed the presence of strong peaks compared to the control and HPV‐positive groups. In addition, different Raman spectra were acquired according to HPV type. Therefore, all ranges of cervical fluid‐induced Raman spectra could be used to detect the presence of cervical pre‐cancer. Raman peak‐gated assessment provides a label‐free and nondestructive tool for the clinical diagnosis of HPV infection and cervical precancerous changes.     

Zinc as an essential trace element in the acceleration of matrix vesicles-mediated mineral deposition

BACKGROUND: Zinc (Zn) has a potent stimulatory effect on osteoblastic bone formation and an inhibitory effect on osteoclastic bone resorption. PURPOSE: The effect of Zn on the function of matrix vesicles (MVs) remains controversial. The purpose of this study was to investigate the effect of Zn on alkaline phosphatase (ALP) activity of osteoblasts and in the initial biological MVs‐mediated mineral deposition. STUDY DESIGN: Osteoblasts were treated with varying concentrations of Zn dissolved in culture medium. After three, five, and seven days of culture, ALP activity was assayed. For the detection of a low level of calcium concentration in MVs, X‐ray fluorescence (XRF) analyses were applied. The effect of Zn for the transformation of calcium phosphate was analyzed using a scanning electron microscope fitted with an energy dispersive X‐ray microanalysis (EDX) system. RESULTS: The ALP activity of osteoblasts in culture medium supplemented with 1 × 10^?5M of Zn was significantly increased at both five and seven days. XRF data demonstrated higher levels of calcium concentration over time in the Zn‐supplemented group. EDX data showed that mineral deposits beginning on day 3 were transformed from whitlockite to calcium phosphate near hydroxyapatite, and that Zn accelerated this transformation. CONCLUSIONS: The proper concentration of Zn increased the ALP activity of osteoblasts after five and seven days of incubation. The present XRF and EDX data suggest that the increase of mineral deposition with Zn exposure for one to five days might be mediated by the activation of ALP and calcium‐binding proteins. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.     

Label‐free identification of antibiotic resistant isolates of living Escherichia coli: Pilot study

We introduce a label‐free spectroscopic method to classify subtypes of quinolone‐nonsusceptible Escherichia coli (E. coli ) isolates obtained from human blood cultures. Raman spectroscopy with a 30‐nm gold‐deposited, surface‐enhanced Raman scattering (SERS) substrate was used to evaluate three multilocus sequencing typing (MLST)‐predefined groups including E . coli ATCC25922, E . coli ST131:O75, and E . coli ST1193:O25b. Although there was a coffee‐ring effect, the ring zone was selected at the ideal position to screen E. coli isolates. Strong Raman peaks were present at 1001–1004 cm^?1 (C? C aromatic ring breathing stretching vibrational mode of phenylalanine), 1447–1448 cm^?1 (C? H₂ scissoring deformation vibrational mode), and 1667 cm^?1 (amide I α‐helix). Although the three MLST‐predefined E . coli isolates had similar Raman spectral patterns, a support vector machine (SVM) learning algorithm‐assisted principal component analysis (PCA) analysis had superior performance in detecting the presence of quinolone‐nonsusceptible E. coli isolates as well as classifying similar microbes, such as quinolone‐nonsusceptible E . coli ST131:O75 and E . coli ST1193:O25b isolates. Therefore, this label‐free and nondestructive technique is likely to be useful for clinically diagnosing quinolone‐nonsusceptible E. coli isolates with the MLST method.     

Spatially resolved recording of transient fluorescence‐lifetime effects by line‐scanning TCSPC

We present a technique that records transient changes in the fluorescence lifetime of a sample with spatial resolution along a one‐dimensional scan. The technique is based on scanning the sample with a high‐frequency pulsed laser beam, detecting single photons of the fluorescence light, and building up a photon distribution over the distance along the scan, the arrival times of the photons after the excitation pulses and the time after a stimulation of the sample. The maximum resolution at which lifetime changes can be recorded is given by the line scan period. Transient lifetime effects can thus be resolved at a resolution of about one millisecond. We demonstrate the technique for recording photochemical and nonphotochemical chlorophyll transients in plants and transient changes in free Ca²⁺ in cultured neurons. Microsc. Res. Tech. 77:216–224, 2014. © 2014 Wiley Periodicals, Inc.     

The effectiveness of glass ionomer cement as a fiber post cementation system in endodontically treated teeth

This study compared the performance of a glass ionomer (GC Gold Label 1, GIC) as a fiber post cementation system for glass fiber posts with a self‐adhesive resin cement (Relyx U200, RUC) and a conventional resin cement system (Scotchbond Muli‐Purpose and Relyx ARC, RAC). Thirty endodontically treated canines were randomly divided in three groups (n = 10), according to the fiber post cementation system: (RAC)—Scotchbond Multi‐Purpose and Relyx X ARC; (RUC)—Relyx U200 and (GIC)—GC Gold Label 1 Luting & Lining. Rhodamine was incorporated into the cementation system prior to the fiber post cementation. After glass fiber post cementation, roots were incubated in artificial saliva for 6 months. After that, specimens from the cervical, middle, and apical thirds of the post space were prepared and analyzed using a push‐out bond strength test and confocal laser microscopy. ANOVA one way and Tukey tests showed that GIC and RUC demonstrated similar push‐out bond strength values, independently of the post space third (p > .05); however, values were greater than those shown by RAC (p < .05). For dentin penetrability, GIC and RUC also had similar results (p > 0.05) and lower than RAC (p < 0.05). Inside the root canal, the cementation system using glass ionomer cement (GC Gold Label 1 Luting & Lining) has similar push‐out bond strength to the self‐adhesive resin cement (Relyx U200) and these were higher than the conventional resin (Relyx ARC), despite its higher dentin penetrability.     

Quantitative evaluation of bone resorption activity of osteoclast‐like cells by measuring calcium phosphate resorbing area using incubator‐facilitated and video‐enhanced microscopy

Quantitative evaluation of the ability of bone resorption activity in live osteoclast‐like cells (OCLs) has not yet been reported on. In this study, we observed the sequential morphological change of OCLs and measured the resorbing calcium phosphate (CP) area made by OCLs alone and with the addition of elcatonin utilizing incubator facilitated video‐enhanced microscopy. OCLs, which were obtained from a coculture of ddy‐mouse osteoblastic cells and bone marrow cells, were cultured on CP‐coated quartz cover slips. The CP‐free area increased constantly in the OCLs alone, whereas it did not increase after the addition of elcatonin. This study showed that analysis of the resorbed areas under the OCL body using this method enables the sequential quantitative evaluation of the bone resorption activity and the effect of several therapeutic agents on bone resorption in vitro. Microsc. Res. Tech, 2009. © 2008 Wiley‐Liss, Inc.     

Photo‐ and bio‐physical characterization of novel violet and near‐infrared lipophilic fluorophores for neuronal tracing

Lipophilic fluorescent dyes have been used to trace neuronal connections because of their ability to diffuse laterally within nerve cell membranes. Given the hundreds to thousands of connections that a typical neuron makes with its neighbours, a diffusion‐matched set of spectrally distinct dyes is desirable. To extend a set of these dyes to obtain six independent labels, we have characterized the properties of novel violet and near‐infrared candidates. By combining two‐photon and confocal microscopy all of these candidates can be imaged using a single Titanium Sapphire laser. Here we present measurements of the two‐photon action cross‐sections and diffusion properties of the dyes, using either the relative diffusion distance or fluorescence recovery after photobleaching techniques, and demonstrate six‐colour neuronal tracing within the spinal cord and brain tissue.     

Label‐free monitoring of inflammatory tissue conditions using a carrageenan‐induced acute inflammation rat model

Although the confirmation of inflammatory changes within tissues at the onset of various diseases is critical for the early detection of disease and selection of appropriate treatment, most therapies are based on complex and time‐consuming diagnostic procedures. Raman spectroscopy has the ability to provide non‐invasive, real‐time, chemical bonding analysis through the inelastic scattering of photons. In this study, we evaluate the feasibility of Raman spectroscopy as a new, easy, fast, and accurate diagnostic method to support diagnostic decisions. The molecular changes in carrageenan‐induced acute inflammation rat tissues were assessed by Raman spectroscopy. Volumes of 0 (control), 100, 150, and 200 µL of 1% carrageenan were administered to rat hind paws to control the degree of inflammation. The prominent peaks at [1,062, 1,131] cm^?1 and [2,847, 2,881] cm^?1 were selected as characteristic measurements corresponding to the C–C stretching vibrational modes and the symmetric and asymmetric C–H (CH₂) stretching vibrational modes, respectively. Principal component analysis of the inflammatory Raman spectra enabled graphical representation of the degree of inflammation through principal component loading profiles of inflammatory tissues on a two‐dimensional plot. Therefore, Raman spectroscopy with multivariate statistical analysis represents a promising method for detecting biomolecular responses based on different types of inflammatory tissues.     

Two‐frequency CARS imaging by switching fiber laser excitation

To fully exploit the power of coherent Raman imaging, techniques are needed to image more than one vibrational frequency simultaneously. We describe a method for switching between two vibrational frequencies based on a single fiber‐laser source. Stokes pulses were generated by soliton self‐frequency shifting in a photonic crystal fiber. Pump and Stokes pulses were stretched to enhance vibrational resolution by spectral focusing. Stokes pulses were switched between two wavelengths on the millisecond time scale by a liquid‐crystal retarder. Proof‐of‐principle is demonstrated by coherent anti‐Stokes Raman imaging of polystyrene beads embedded in a poly(methyl methacrylate) (PMMA) matrix. The Stokes shift was switched between 3,050 cm^?1, where polystyrene has a Raman transition, and 2,950 cm^?1, where both polystyrene and PMMA have Raman resonances. The method can be extended to multiple vibrational modes.     

FLIM and FCS detection in laser‐scanning microscopes: Increased efficiency by GaAsP hybrid detectors

Photon counting detectors currently used in fluorescence lifetime microscopy have a number of deficiencies that result in less‐than‐ideal signal‐to‐noise ratio of the lifetimes obtained: Either the quantum efficiency is unsatisfactory or the active area is too small, and afterpulsing or tails in the temporal response contribute to overall timing inaccuracy. We have therefore developed a new FLIM detector based on a GaAsP hybrid photomultiplier. Compared with conventional PMTs and SPADs, GaAsP hybrid detectors have a number of advantages: The detection quantum efficiency reaches or surpasses the efficiency of fast SPADs, and the active area is on the order of 5 mm², compared with 2.5 10^?3 mm² for a SPAD. The TCSPC response is clean, without the bumps and the diffusion tails typical for PMTs and SPADs. Most important, the hybrid detector is intrinsically free of afterpulsing. FLIM results are therefore free of signal‐dependent background, and FCS curves are free of the known afterpulsing peak. We demonstrate the performance of the new detector for multiphoton NDD FLIM and for FCS. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.