Rapid label-free identification of Klebsiella pneumoniae antibiotic resistant strains by the drop-coating deposition surface-enhanced Raman scattering method.

Although many methodologies have been developed to identify unknown bacteria, bacterial identification in clinical microbiology remains a complex and time-consuming procedure. To address this problem, we developed a label-free method for rapidly identifying clinically relevant multilocus sequencing typing-verified quinolone-resistant Klebsiella pneumoniae strains. We also applied the method to identify three strains from colony samples, ATCC70063 (control), ST11 and ST15; these are the prevalent quinolone-resistant K. pneumoniae strains in East Asia. The colonies were identified using a drop-coating deposition surface-enhanced Raman scattering (DCD-SERS) procedure coupled with a multivariate statistical method. Our workflow exhibited an enhancement factor of 11.3×106 to Raman intensities, high reproducibility (relative standard deviation of 7.4%), and a sensitive limit of detection (100 pM rhodamine 6G), with a correlation coefficient of 0.98. All quinolone-resistant K. pneumoniae strains showed similar spectral Raman shifts (high correlations) regardless of bacterial type, as well as different Raman vibrational modes compared to Escherichia coli strains. Our proposed DCD-SERS procedure coupled with the multivariate statistics-based identification method achieved excellent performance in discriminating similar microbes from one another and also in subtyping of K. pneumoniae strains. Therefore, our label-free DCD-SERS procedure coupled with the computational decision supporting method is a potentially useful method for the rapid identification of clinically relevant K. pneumoniae strains.

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.

Evaluation of aneurysm-associated wall shear stress related to morphological variations of circle of Willis using a microfluidic device.

Although microfluidic systems have been important tools in analytical chemistry, life sciences, and medical research, their application was rather limited for drug-screening and biosensors. Here, we described a microfluidic device consisting of a multilayer micro-channel system that represented the hemodynamic cerebral vascular system. We analyzed wall shear stresses related to aneurysm formation in the circle of Willis (CoW) and their morphological variations using this system. This device was controlled by pneumatic valves, which occluded various major arteries by closing the associated channels. The hemodynamic analysis indicated that higher degrees of shear stress occurred in an anterior communicating artery (ACoA), particularly in the hypoplastic region of the posterior communicating artery (PCoA) and the P1 segment. Furthermore, occlusion of a common carotid artery (CCA) or a middle cerebral artery (MCA) increased the shear stress, whereas occlusion of a vertebral artery (VA) decreased the shear stress. These results indicate that the morphological variation of the CoW may affect aneurysm formation resulting from increased wall shear stress. Therefore, the technique described in this paper provides a novel method to investigate the hemodynamics of complex cerebral vascular systems not accessible from previous clinical studies.

Internal-specific morphological analysis of sciatic nerve fibers in a radiofrequency-induced animal neuropathic pain model.

This study investigated the reversible effects of pulsed radiofrequency (PRF) treatment at 42 °C on the ultrastructural and biological changes in nerve and collagen fibers in the progression of neuropathic pain after rat sciatic nerve injury. Assessments of morphological changes in the extracellular matrices by atomic force microscopy and hematoxylin-eosin, Masson's trichrome and picrosirius-red staining as well as the expressions of two fibril-forming collagens, types-I and -III, and two inflammatory cytokines, TNF-α and IL-6, were evaluated on day 30 after RF exposure. There were four groups for different RF thermal treatments: no treatment, no current, PRF, and continuous RF (CRF). An RF procedure similar to that used in human clinical trials was used in this study. The CRF treatment at 82 °C led to neural and collagen damage by the permanent blockage of sensory nociceptors. The PRF treatment led to excellent performance and high expandability compared to CRF, with effects including slight damage and swelling of myelinated axons, a slightly decreased amount of collagen fibers, swelling of collagen fibril diameters, decreased immunoreactivity of collagen types-I and -III, presence of newly synthesized collagen, and recovery of inflammatory protein immunoreactivity. These evidence-based findings suggest that PRF-based pain relief is responsible for the temporary blockage of nerve signals as well as the preferential destruction of pain-related principal sensory fibers like the Aδ and C fibers. This suggestion can be supported by the interaction between the PRF-induced electromagnetic field and cell membranes; therefore, PRF treatment provides pain relief while allowing retention of some tactile sensation.

Nanostructural response of mitomycin C application on human scleral tissues.

This study quantitatively examined short-term effects of 0.02% Mitomycin C (MMC) treatment on the nanostructural changes in human scleral collagen fibrils. Histologic analysis and non-contact mode atomic force microscopy (AFM) were employed to assess the ultrastructural changes in the morphological characteristics of human sclera before and after 0.02% MMC application for 1 and 3 min. The scleral collagen fibrils treated with 0.02% MMC for 1 min showed no significant change in the morphology of collagen fibrils, and a significant change (p < 0.05) in the thickness of scleral tissues and collagen density, compared to the controls. 0.02% MMC application for 3 min led to a significant increase (p < 0.001) in the mean fibril diameter (185.43 +/- 22.64 nm vs. 140.72 +/- 18.06 nm), thickness (0.81 +/- 0.03 mm vs. 0.54 +/- 0.05 mm) and collagen density (1.16 times), compared to the controls This study examined the nanostructural changes in the scleral collagen fibrils before and after MMC application by AFM technique combined with conventional histological analysis (Hematoxylin-eosin and Masson's trichrome). This result indirectly suggests that long-term MMC application might increase the incidence of complications like a scleromalcia.

Inflammatory effect of monopolar radiofrequency treatment on collagen fibrils in rabbit skins.

This study quantitatively examined the effects of monopolar radiofrequency (RF) treatment on the progressive nanostructural changes in the inflammatory effect of in vivo rabbit dermal collagen fibrils during postoperative 7 days. Conventional histologic analysis and atomic force microscopy (AFM) were employed to assess the nanostructural dermal response in 6 RF groups that underwent 2 passes of RF treatments (10 and 20 W), and 1 untreated control rabbit. After monopolar RF treatment, the rabbit skin clearly showed changes in the collagen network structure. The RF-treated group showed regular parallel arrangement of collagen fibrils whereas normal group showed tangled nanostructures. This phenomenon was remarkable at postoperative 7 days. Therefore, monopolar RF treatment leads to underlying collagen contracture and promotes new collagen formation. A multi-pass treatment of low-energy RF led to the highest contraction of collagen fibrils at the nanostructural level, compared to a single pass of high-energy RF.

Structural and biomechanical effects of photooxidative collagen cross-linking with photosensitizer riboflavin and 370 nm UVA light on human corneoscleral tissues.

This study quantitatively investigated the immediate effects of a photooxidative collagen cross-linking treatment with photosensitizer riboflavin (RF) and 370 nm UVA light in in vitro human corneoscleral collagen fibrils using histology, thickness, scanning electron microscopy, and atomic force microscopy analyses. Twenty 8 x 2 mm corneoscleral strips were dissected sagittally from donor tissue using a scalpel. Four parameters were investigated, including the density, thickness, adhesion force, and stiffness of corneoscleral tissues before and after the collagen cross-linking treatment. The RFUVA-catalyzed collagen cross-linking treatment led to an increase in the density of both corneal (8%) and scleral (23%) stromal collagens. However, there was no difference in corneoscleral thickness. Furthermore, RFUVA-catalyzed collagen cross-linking treatment led to an increased biomechanical response of corneosclera: 25 and 8% increases in corneoscleral stiffness, and 24 and 22% increases in corneoscleral adhesion force. The collagen cross-linking treatment through RF-sensitized photoreaction may cause structural and biomechanical changes in the collagen fibril network of the cornea and the sclera. This is due to narrowing of the interfibrillar spacing and the stromal edema.

Label-free and quantitative evaluation of cytotoxicity based on surface nanostructure and biophysical property of cells utilizing AFM.

In this study, the four commonly used cytotoxicity assays and the mechanical properties as evaluated by atomic force microscopy (AFM) were compared in a cellular system. A cytotoxicity assay is the first and most essential test to evaluate biocompatibility of various toxic substances. Many of the cytotoxicity methods require complicated and labor-intensive process, as well as introduce experimental error. In addition, these methods cannot provide instantaneous and quantitative cell viability information. AFM has become an exciting analytical tool in medical, biological, and biophysical research due to its unique abilities. AFM-based force-distance curve measurements precisely measure the changes in the biophysical properties of the cell. Therefore, we observed the morphological changes and mechanical property changes in L929 cells following sodium lauryl sulfate (SLS) treatment utilizing AFM. AFM imaging showed that the toxic effects of SLS changed not only the spindle-like shape of L929 cells into a round shape, but also made a rough cell surface. As the concentration of SLS was increased, the surface roughness of L929 cell was increased, and stiffness decreased. We confirmed that inhibition of proliferation clearly increased with increases in SLS concentration based on results from MTT, WST, neutral red uptake, and LIVE/DEAD viability/cytotoxicity assays. The estimated IC50 value by AFM analysis was similar to those of other conventional assays and was included within the 95% confidence interval range. We suggest that an AFM quantitative analysis of the morphological and biophysical changes in cells can be utilized as a new method for evaluating cytotoxicity.

Short-term response of mitomycin C on the human rectus muscle following strabismus surgery: histological, ultrastructural, and biomechanical evaluation.

This study investigated the inflammatory effect of intraoperative mitomycin C (MMC) on adhesion reformation in human rectus muscles. Ten consecutive patients who underwent medial rectus resection had their postoperative rectus muscles divided into two groups: control group (n = 10) and MMC group (n = 10). In the MMC group, the muscle was soaked for 2 min with MMC, prepared as a 0.2 mg/mL (0.02%) solution. The 0.02% MMC reactions were examined using histological analysis with hematoxylin-eosin (inflammatory response) and Masson's trichrome (collagen fibrils), immunoreactivities of cyclooxygenase-II (inflammatory response), and collagen type I and III, scanning electron microscopy analysis to quantify the diameter and D-periodicity of collagen fibrils, and atomic force microscopy analysis to quantify the diameter, D-periodicity, and adhesion force of collagen fibrils. The rectus muscles treated with 0.02% MMC showed a significantly increased inflammatory response (p < 0.05), increased collagen density (p < 0.0001), increased fibril diameter (p < 0.001 or p < 0.05), and decreased fibril adhesion force (p < 0.005) compared to the rectus muscles in the control group. MMC simultaneously caused an inflammatory response as well as nanostructural and biomechanical property changes in the collagen fibril network.

Postoperative effect of radiofrequency treatments on the rabbit dermal collagen fibrillary matrix.

This study quantitatively examined the short and mid-long term effects of radiofrequency (RF) treatment on the normal dermal collagen fibrils of live rabbits. Effects were evaluated by histology and scanning probe microscopy analysis of dermal tissues treated using three RF energy levels (10, 20, and 30 W) and either a single- or multiple-pass procedure. Progressive changes in the morphology of rabbit dermal collagen fibrils were investigated over a 30-day post-treatment period. All RF-treated groups, except for the low-energy group (10 W), displayed more prominent inflammatory responses compared to the control. This inflammatory response was more prominent a day after treatment. Dermal tissues 30-days after RF treatment exhibited prominent myofibroblast activity associated with collagen contractile activity during wound healing in addition to chronic inflammation. A decrease in the morphology of dermal collagen fibrils after RF treatment continued until seven days postoperatively. The collagen fibril diameter increased to near baseline at 30 days postoperatively. Low-energy and multi-pass treatments resulted in greater collagen fibril contraction and recovery at the nanostructural level at 30 days postoperatively than did a single high-energy treatment.

Short-term effect of cryotherapy on human scleral tissue by atomic force microscopy.

This study investigated the inflammatory effect of cryotherapy application on collagen matrix network in human infant sclera. Donor scleral tissues taken from three infant patients divided into five groups: control group, sham-treated group, and three cryotreated groups. In the cryotherapy groups, the sclera was treated for 5 s, 10 s, and 20 s with -80°C freezing by a cryosurgical system. The cryotreated reactions were examined using double histological analysis with hematoxylin-eosin and Masson's trichrome, and atomic force microscopy analysis to quantify the diameter and D-banding of collagen fibrils. The infant scleral tissues treated with cryotherapy showed a significantly increased collagen density associated with inflammatory response (p < 0.05), increased fibril diameter (p < 0.005) compared to the scleral tissues in the control group. The results directly suggest that the cryotherapy affects the morphology of scleral collagen.

Structural response of human corneal and scleral tissues to collagen cross-linking treatment with riboflavin and ultraviolet A light.

High success rates in clinical trials on keratoconic corneas suggest the possibility of efficient treatment against myopic progression. This study quantitatively investigated the in vitro ultrastructural effects of a photooxidative collagen cross-linking treatment with photosensitizer riboflavin and UVA light in human corneo-scleral collagen fibrils. A total of 30.8 × 2 mm corneo-scleral strips from donor tissue were sagittally dissected using a scalpel. The five analytic parameters namely fibril density, fibril area, corneo-scleral thickness, fibril diameter, and fibril arrangement were investigated before and after riboflavin-UVA-catalyzed collagen cross-linking treatment. Collagen cross-linking effects were measured at the corneo-scleral stroma and were based on clinical corneal cross-linking procedures. The structural response levels were assessed by histology, digital mechanical caliper measurement, scanning electron microscopy, and atomic force microscopy. Riboflavin-UVA-catalyzed collagen cross-linking treatment led to an increase in the area, density, and diameters of both corneal (110, 112, and 103 %) and scleral (133, 133, and 127 %) stromal collagens. It also led to increases in corneal (107 %) and scleral (105 %) thickness. Collagen cross-linking treatment through riboflavin-sensitized photoreaction may cause structural property changes in the collagen fibril network of the cornea and sclera due to stromal edema and interfibrillar spacing narrowing. These changes were particularly prominent in the sclera. This technique can be used to treat progressive keratoconus in the cornea as well as progressive myopia in the sclera. Long-term collagen cross-linking treatment of keratoconic and myopic progression dramatically improves weakened corneo-scleral tissues.

Scanning electron microscopy study of the effect of the brushing time on the human tooth dentin after exposure to acidic softdrinks.

Scanning electron microscopy (SEM) was used to examine the abrasive and erosive potential of the brushing time on the dentin surface eroded by acidic soft drinks to suggest an optimized toothbrushing start time after the consumption of cola (pH 2.52) in children. Thirty-six non-carious primary central incisors were assigned to 12 experimental groups (n = 3) based on the erosive and abrasive treatment protocols. Cola exposure was used as the erosive treatment. Three brushing durations (5, 15, and 30 sec) and four brushing start times (immediately, 30 min, 60 min, and 120 min) after an erosive pre-treatment were used for the abrasive treatment. Toothbrushing after exposure to acidic soft drinks led to an increase in the open-tubule fraction and microstructural changes. Toothbrushing immediately after the erosive pre-treatment showed the largest abrasive and erosive potential on the dentin whereas that 60 and 120 min after the pre-treatment showed the least abrasive and erosive potential on the dentin. Toothbrushing for both 60 and 120 min after the pre-treatment showed similar erosive and abrasive potentials on the dentin. The brushing duration showed no effect on the erosive and abrasive potential on the dentin. Therefore, to achieve the desired tooth surface cleaning and less surface lesion on the dentin surface, toothbrushing should be performed at least 1 hour after cola consumption. Three-minute brushing after cola consumption is sufficient to prevent dental lesions, and prolonged brushing can irritate the gingival tissues.

Ultrastructural effect of self-ligating bracket materials on stainless steel and superelastic NiTi wire surfaces.

Frictional interactions between wires and brackets reduce the efficacy in orthodontic treatments. Self-ligating brackets (SLBs) are now more often used due to lower frictional forces when compared with conventional-ligating brackets. In this study, scanning electron microscopy and atomic force microscopy were used to examine the microstructural effects of stainless steel and ceramic SLBs on the surface roughness of stainless steel and superelastic NiTi wires both after in vivo clinical orthodontic treatment as well as in in vitro three-point bending experiments. A combination of two wires-0.019 in. × 0.025 in. stainless steel wires and 0.016 in. superelastic NiTi wires-and two SLBs-both passive-type stainless steel SLBs and active-type ceramic SLBs-was applied for 4 months (bicuspid-extraction) in an in vivo setting and for 1 month in an in vitro setting (200 g loads). After the SLB treatments, all wires exhibited severe scratches secondary to frictional interactions with the brackets. When used with the stainless steel SLBs (Damon 3MX®), the surfaces of 0.019 in. × 0.025 in. stainless steel (P < 0.0001) and 0.016 in. superelastic NiTi wires (P < 0.05) were significantly smoother than when used with the ceramic SLBs (Clippy-C®). Such results suggest that orthodontic treatments with stainless steel SLBs are more effective than with ceramic SLBs.

AFM study for morphological characteristics and biomechanical properties of human cataract anterior lens capsules.

The aim of this study was to quantitatively investigate the morphologies (surface roughness) and biomechanical properties (Young's modulus) of human anterior lens capsules (ALCs) for noncataract and cataract groups using atomic force microscopy. Eight human ALCs obtained during phacoemulsification from patients with senile cataracts (72 ± 13 years) were investigated in both the hydrated and dehydrated conditions. The cataract group showed clearly the proliferated lens epithelial cells (LECs) with a monomorphic cell structure, a diameter of 12.54 ± 4.31 µm, and a height of 0.23 ± 0.04 µm, whereas the control group showed no LECs. A substantial amount of false-positive calcification was observed caused by the deposition of remnants of dried salt solution. Cataract group showed significantly higher surface roughness (382.06 nm, p ≤ 0.001) than control group in the anterior side of ALCs, whereas cataract group showed significantly lower surface roughness (353.79 nm, p ≤ 0.001) than control group in their posterior side. Cataract group showed significantly higher Young's modulus (69.52 kPa, p ≤ 0.001) compared to the control group, regardless of the ALC side. Therefore, it is significant that this study provides a new method to examine the nanostructural characteristic and biomechanical property of human ALCs through a nanometer-scale resolution microscopy technique.

Short-term nanostructural effects of high radiofrequency treatment on the skin tissues of rabbits.

The aim of this study is to quantitatively investigate the short-term effects of RF tissue-tightening treatment in in vivo rabbit dermal collagen fibrils. These effects were measured at different energy levels and at varying pass procedures on the nanostructural response level using histology and AFM analysis. Each rabbit was divided into one of seven experimental groups, which included the following: control group, and six RF group according to RF energy (20 W and 40 W) and three RF pass procedures. The progressive changes in the diameter and D-periodicity of rabbit dermal collagen fibrils were investigated in detail over a 7-day post-treatment period. The dermal tissues treated with the RF tissue-tightening device showed more prominent inflammatory responses with inflammatory cell ingrowth compared to the control. This effect showed more prominent with the passage of day after treatment. Although an increase in the diameter and D-periodicity of dermal collagen fibrils was identified immediately after the RF treatment, a decrease in the morphology of dermal collagen fibrils continued until post-operative day 7. Furthermore, RF treatment led to the loss of distinct borders. Increases in RF energy with the same pass procedure, as well as an increase in the number of RF passes, increased the occurrence of irreversible collagen fibril injury. A multiple-pass treatment at low energy rather than a single-pass treatment at high energy showed a large amount of collagen fibrils contraction at the nanostructural level.

AFM study for morphological and mechanical properties of human scleral surface.

This study examined the structures and the elastic and viscous properties of human scleral collagen fibrils by atomic force microscopy (AFM). Sample preparation was performed to minimize the sources of artifacts for further imaging. To observe the morphological and property characteristics of human scleral surfaces, AFM was used as a microscopic tool. The AFM topography, phase shift and deflection images of the dehydrated scleral collagen fibrils were obtained. The visco-elasticity of collagen fibrils was determined from the force-distance curves of the AFM. Inspection of the fibril surface in high resolution showed that the D-period spacing along the collagen fibrils was clearly evident. The fibril diameter over a scan size of 5 x 5 microm2 was 145.22 +/- 17.78 nm (n = 178) ranging from 98 to 220 nm, and the D-periodicity was 69.14 +/- 14.15 nm (n = 189), which is similar to the normal 67 nm D-periodicity. Force-distance analysis indicated that human scleral collagen had comparatively high adhesion force and elasticity, to protect the eye from external trauma and to withstand the expansive force made by the intraocular pressure.

Nanostructural investigation of frontalis sling biomaterial surfaces.

This study examined the nanostructural surface of three frontalis sling biomaterials: autogenous fascia lata, preserved fascia lata and silicone rod. The morphological characteristics of the sling biomaterial surfaces were examined qualitatively and quantitatively by scanning electron microscopy and atomic force microscopy, respectively. The autogenous fascia lata showed well-arranged nanostructures of parallel fascia collagen fibrils with clear 67 nm axial periodicity, whereas the preserved fascia lata showed tangled nanostructures of damaged collagen fibril bundles. The silicone rod showed a substantial amount of debris with some scratches and the smoothest roughness compared with the other sling biomaterials, followed by preserved fascia lata. Autogenous fascia lata showed the highest surface roughness. The association between the roughness and cell adhesion suggests that the nanostructure of autogenous fascia lata biomaterials is the best for frontalis sling and that of the silicone rod biomaterials is the worst.

Ultrastructural investigation of intact orbital implant surfaces using atomic force microscopy.

This study examined the surface nanostructures of three orbital implants: nonporous poly(methyl methacrylate) (PMMA), porous aluminum oxide and porous polyethylene. The morphological characteristics of the orbital implants surfaces were observed by atomic force microscopy (AFM). The AFM topography, phase shift and deflection images of the intact implant samples were obtained. The surface of the nonporous PMMA implant showed severe scratches and debris. The surface of the aluminum oxide implant showed a porous structure with varying densities and sizes. The PMMA implant showed nodule nanostructures, 215.56 ± 52.34 nm in size, and the aluminum oxide implant showed crystal structures, 730.22 ± 341.02 nm in size. The nonporous PMMA implant showed the lowest roughness compared with other implant biomaterials, followed by the porous aluminum oxide implant. The porous polyethylene implant showed the highest roughness and severe surface irregularities. Overall, the surface roughness of orbital implants might be associated with the rate of complications and cell adhesion.

Changes in ultrastructure and properties of bracket slots after orthodontic treatment with bicuspid extraction.

This study examined the effects of an orthodontic treatment using a bicuspid extraction on the surface roughness and mechanical properties of stainless steel (SS) brackets adjacent to the extraction space. Four experimental groups were employed; groups 1 and 2 used the Archist(®) SS brackets before and after the extraction treatment, respectively, and groups 3 and 4 used the Victory(®) SS brackets before and after the extraction treatment, respectively. The slot surfaces of the bracket were scanned in air at a resolution of 512 × 512 pixels with a scan speed of 0.8 line/s. The visco-elasticity of the bracket slot was determined from the force-distance curves of atomic force microscopy. The orthodontic treatment with bicuspid extraction led to a significant increase (p<0.0001) in surface roughness in both groups. In particular, the Archist(®) SS brackets showed more changes than the Victory(®) SS brackets (p<0.0005). However, there was no significant difference in properties of the Victory(®) and Archist(®) brackets between before and after treatment. This suggests that the orthodontic treatment with bicuspid extraction is more responsible for the changes in surface roughness than the properties of the brackets.