Fluorescent nano- and microparticles for sensing cellular microenvironment: past, present and future applications.

The tumor microenvironment (TME) demonstrates distinct hallmarks, including acidosis, hypoxia, reactive oxygen species (ROS) generation, and altered ion fluxes, which are crucial targets for early cancer biomarker detection, tumor diagnosis, and therapeutic strategies. Various imaging and sensing techniques have been developed and employed in both research and clinical settings to visualize and monitor cellular and TME dynamics. Among these, ratiometric fluorescence-based sensors have emerged as powerful analytical tools, providing precise and sensitive insights into TME and enabling real-time detection and tracking of dynamic changes. In this comprehensive review, we discuss the latest advancements in ratiometric fluorescent probes designed for the optical mapping of pH, oxygen, ROS, ions, and biomarkers within the TME. We elucidate their structural designs and sensing mechanisms as well as their applications in in vitro and in vivo detection. Furthermore, we explore integrated sensing platforms that reveal the spatiotemporal behavior of complex tumor cultures, highlighting the potential of high-resolution imaging techniques combined with computational methods. This review aims to provide a solid foundation for understanding the current state of the art and the future potential of fluorescent nano- and microparticles in the field of cellular microenvironment sensing.

Probing Single-Cell Fermentation Fluxes and Exchange Networks via pH-Sensing Hybrid Nanofibers.

The homeostatic control of their environment is an essential task of living cells. It has been hypothesized that, when microenvironmental pH inhomogeneities are induced by high cellular metabolic activity, diffusing protons act as signaling molecules, driving the establishment of exchange networks sustained by the cell-to-cell shuttling of overflow products such as lactate. Despite their fundamental role, the extent and dynamics of such networks is largely unknown due to the lack of methods in single-cell flux analysis. In this study, we provide direct experimental characterization of such exchange networks. We devise a method to quantify single-cell fermentation fluxes over time by integrating high-resolution pH microenvironment sensing via ratiometric nanofibers with constraint-based inverse modeling. We apply our method to cell cultures with mixed populations of cancer cells and fibroblasts. We find that the proton trafficking underlying bulk acidification is strongly heterogeneous, with maximal single-cell fluxes exceeding typical values by up to 3 orders of magnitude. In addition, a crossover in time from a networked phase sustained by densely connected "hubs" (corresponding to cells with high activity) to a sparse phase dominated by isolated dipolar motifs (i.e., by pairwise cell-to-cell exchanges) is uncovered, which parallels the time course of bulk acidification. Our method addresses issues ranging from the homeostatic function of proton exchange to the metabolic coupling of cells with different energetic demands, allowing for real-time noninvasive single-cell metabolic flux analysis.

Pediatric Cervical Spine Injuries.

Objective Pediatric cervical spine injuries are rare and account for 1 to 2% of all pediatric spine injuries. There is a paucity of data on pediatric cervical spine injuries in developing countries like India. The purpose of this study is to review and analyze our 5 years of experience with pediatric cervical spine injuries. Methods All the available medical records over the 5 years were reviewed retrospectively. The data was analyzed to know the epidemiology, mechanism of injury, injury patterns, management, and outcome. The patients were divided into two groups: 0 to 9 years and 10 to 18 years. Results Seventy-five eligible records were included in our study. The incidence of cervical spine injuries was significantly lower in younger children than the older ones ( p < 0.042). The most common mechanism of injury was fall from height: 33 (44%) patients followed by road traffic accidents: 27 (36%) patients. The involvement of the upper cervical spine was significantly higher in younger children ( p < 0.001). Fractures with subluxation were the most common pattern of injury, observed in 35 (47%) patients. However, fractures with subluxation were uncommon in younger children compared with older children ( p < 0.04). Spinal cord injury without radiographic abnormality (SCIWORA) was observed in 42% of younger children compared with 8% of older children ( p < 0.02). Thirty (40%) patients were managed surgically; anterior cervical corpectomy with fusion was the most commonly performed procedure in 19 (63%) patients. The overall mortality was 20%. Conclusion The results of our study revealed predominant involvement of the upper cervical spine in children younger than 10 years of age. SCIWORA was documented in both the age groups with a significantly higher incidence in younger children. The instrumentation and fusion techniques in children are safe; however, developing pediatric spine needs special considerations.

Dynamic change in cortisol levels associated with severity, progression, and survival of patients with traumatic brain injury.

BACKGROUND: Cortisol levels are elevated in severe traumatic brain injury (TBI) and gradually decrease during patient recovery. Thus, dynamic changes in cortisol levels may serve as a prognostic biomarker of TBI. AIM: This study aimed to examine the relationship between serum cortisol levels and outcomes in TBI patients. METHODS: In this prospective case-cohort study, 238 patients with TBI were enrolled. Demographic, clinical, and radiological data were recorded within the first 24 h of hospitalization. Serum cortisol levels were measured using chemiluminescence assay (Immunoassay i1000). The association between cortisol levels and outcome (Glasgow Outcome Scale score) was evaluated at discharge from the hospital and after six months of follow-up. RESULTS: The mean age of the patients was 35.03 ± 17.68 years and the male: female ratio was 4.3:1. At the time of admission(day-1), cortisol levels in the TBI patients were significantly higher than those on day-7 (9.81 ± 4.20 µg/dl versus 23.41 ± 11.83 µg/dl; p<0.001). There was a significant relationship between cortisol levels and Glasgow Coma Scale (GCS) (p = 0.018). Moderate head injury (GCS;9-12) was observed in 108(45.4 %) patients and 130(54.6 %) patients with severe head injury (GCS;3-8) at presentation. CGS was significantly associated with the survival of patients with TBI; alive(n = 143) vs. dead (n = 77); p < 0.001. At 6 months follow-up of patients (n = 184), the findings revealed that the Glasgow Outcome Scale (GOS) and GCS score were significantly associated(p = 0.018). One-way ANOVA showed a significant difference in cortisol levels on day-1, day-7 and six months of sampling (p < 0.0001). Based on the GOS E score, the cortisol levels in the unfavorable and favorable groups significantly differed from those in the GOSE groups (p = 0.05). Similarly, cortisol levels were significantly associated with survival in patients with TBI (p = 0.04). With increasing cortisol levels, the GOSE score was poor and at > 50 µg/dl, no patient has survived. CONCLUSIONS: Day 1 and 7 cortisol, correlated with the outcomes at 6 months, had predictive value post-TBI.

A pH-sensor scaffold for mapping spatiotemporal gradients in three-dimensional in vitro tumour models.

The detection of extracellular pH at single cell resolution is challenging and requires advanced sensibility. Sensing pH at high spatial and temporal resolution might provide crucial information in understanding the role of pH and its fluctuations in a wide range of physio-pathological cellular processes, including cancer. Here, a method to embed silica-based fluorescent pH sensors into alginate-based three-dimensional (3D) microgels tumour models, coupled with a computational method for fine data analysis, is presented. By means of confocal laser scanning microscopy, live-cell time-lapse imaging of 3D alginate microgels was performed and the extracellular pH metabolic variations were monitored in both in vitro 3D mono- and 3D co-cultures of tumour and stromal pancreatic cells. The results show that the extracellular pH is cell line-specific and time-dependent. Moreover, differences in pH were also detected between 3D monocultures versus 3D co-cultures, thus suggesting the existence of a metabolic crosstalk between tumour and stromal cells. In conclusion, the system has the potential to image multiple live cell types in a 3D environment and to decipher in real-time their pH metabolic interplay under controlled experimental conditions, thus being also a suitable platform for drug screening and personalized medicine.

Fully Automated Computational Approach for Precisely Measuring Organelle Acidification with Optical pH Sensors.

pH balance and regulation within organelles are fundamental to cell homeostasis and proliferation. The ability to track pH in cells becomes significantly important to understand these processes in detail. Fluorescent sensors based on micro- and nanoparticles have been applied to measure intracellular pH; however, an accurate methodology to precisely monitor acidification kinetics of organelles in living cells has not been established, limiting the scope of this class of sensors. Here, silica-based fluorescent microparticles were utilized to probe the pH of intracellular organelles in MDA-MB-231 and MCF-7 breast cancer cells. In addition to the robust, ratiometric, trackable, and bioinert pH sensors, we developed a novel dimensionality reduction algorithm to automatically track and screen massive internalization events of pH sensors. We found that the mean acidification time is comparable among the two cell lines (ΔTMCF-7 = 16.3 min; ΔTMDA-MB-231 = 19.5 min); however, MCF-7 cells showed a much broader heterogeneity in comparison to MDA-MB-231 cells. The use of pH sensors and ratiometric imaging of living cells in combination with a novel computational approach allow analysis of thousands of events in a computationally inexpensive and faster way than the standard routes. The reported methodology can potentially be used to monitor pH as well as several other parameters associated with endocytosis.

Electrospun polyvinyl-alcohol/gum arabic nanofibers: Biomimetic platform for in vitro cell growth and cancer nanomedicine delivery.

The design of powerful in vitro cell culture platforms to support precision medicine can contribute to predict therapeutic success of cancer patients. Electrospun nanofibers applied to cell culture can mimic extracellular matrix and improve in vitro cell behavior. Here, we describe biocompatible blended polyvinyl-alcohol (PVA)/gum arabic (GA) extracellular matrix (ECM)-like nanofibers for in vitro cell cultures capable of delivering nanocomposite for desired biomedical application. Therefore, PVA/GA ECM-like electrospun nanofibers were developed and characterized. Heat treatment was used to crosslink the nanofibers and biocompatibility was evaluated, which demonstrated the ability of developed platform to provide a cell culture-friendly environment. Previous work demonstrated that GA-gold nanoparticles (GA-AuNPs) in non-cytotoxic concentrations can reduce key metastatic cellular events such as invasion and colony formation of metastatic melanoma cells. Thus, crosslinked nanofibers were functionalized with GA-AuNPs and its cellular delivery was evaluated. GA-AuNPs were efficiently adsorbed onto the PVA/GA nanofibers surface and the system effectively delivered the nanocomposites to metastatic melanoma cells. In conclusion, the described biocompatible system could be prospected as a valuable in vitro tool for precision medicine.

Highly Sensitive Fluorescent pH Microsensors Based on the Ratiometric Dye Pyranine Immobilized on Silica Microparticles.

Invited for the cover of this issue are Anil Chandra, Loretta L. del Mercato and co-workers at the Institute of Nanotechnology of National Research Council and the University of Salento. The image depicts how negatively charged pH-sensitive pyranine (HPTS) molecules were successfully immobilized on silica microparticles (SMPs) without compromising the molecules' pH sensitivity. These resulting sensors can be used to measure pH in vitro and in vivo due to the cytocompatibility of HPTS molecules and SMPs. Read the full text of the article at 10.1002/chem.202101568.

Highly Sensitive Fluorescent pH Microsensors Based on the Ratiometric Dye Pyranine Immobilized on Silica Microparticles.

Pyranine (HPTS) is a remarkably interesting pH-sensitive dye that has been used for plenty of applications. Its high quantum yield and extremely sensitive ratiometric fluorescence against pH change makes it a very favorable for pH-sensing applications and the development of pH nano-/microsensors. However, its strong negative charge and lack of easily modifiable functional groups makes it difficult to use with charged substrates such as silica. This study reports a methodology for noncovalent HPTS immobilization on silica microparticles that considers the retention of pH sensitivity as well as the long-term stability of the pH microsensors. The study emphasizes the importance of surface charge for governing the sensitivity of the immobilized HPTS dye molecules on silica microparticles. The importance of the immobilization methodology, which preserves the sensitivity and stability of the microsensors, is also assessed.

Stereomicroscopic Evaluation of Microcrack Formation in Dentin by ProTaper Next, Revo S, and WaveOne Gold File System.

BACKGROUND: During the biomechanical preparation of the root canal by rotary file systems, stress is generated within the canal, which leads to dentinal microcrack formation. Such defects are evaluated only under the microscope. AIM: The present study aimed to evaluate dentinal microcrack formation after instrumentation with ProTaper Next (M-wire), Revo S (conventional NiTi), and WaveOne Gold (Gold-wire) file systems under the stereomicroscope. MATERIALS AND METHODS: Forty single-rooted teeth free from any defect were selected and divided into four groups (Control, PTN, RS, and WOG). Experimental groups were instrumented with file system used in the study, and the control group were left unprepared. Samples were further decoronated and sectioned at 3 mm, 6 mm, and 9 mm from the apex perpendicular to the long axis and examined under a stereomicroscope at ×25 for different types of microcracks (no fracture, complete fracture, partial fracture, and other fracture). STATISTICAL ANALYSIS USED: Data analysis was done by the Chi-square test using the SPSS version 17.0 software. A two-sided (α = 2) P < 0.05 (P < 0.05) was considered statistically significant. RESULTS: The null hypothesis was rejected. There was a statistically significant difference between the groups. All file systems created microcracks in the root dentin. WOG group showed an incidence of 66.7% for no fracture, PTN group had 40% for other fracture, and RS group had 60% for complete fracture. CONCLUSION: Single file system WOG with gold wire technology proves to be the best choice for canal preparation among the tested groups in terms of least dentinal crack formation.

Complications Encountered with ETV in Infants with Congenital Hydrocephalus.

BACKGROUND: Hydrocephalus is an abnormal excessive accumulation of cerebrospinal fluid (CSF) in the cavity and spaces of the brain. Endoscopic third ventriculostomy (ETV) has been an established treatment modality for congenital hydrocephalus. However, in very young infants, the results are challenging. In our study, we have evaluated whether ETV really offers an acceptable complication-free postoperative course. OBJECTIVE: To study the complication and mortality rate in infants having congenital hydrocephalus treated with ETV. MATERIALS AND METHODS: This is a single-center prospective study conducted at the Department of Neurosurgery, K. G. M. U, Lucknow, from January 2019 to February 2020. We studied 40 infants presenting with clinical and radiological features suggestive of congenital hydrocephalus. Follow-up was done at the first, third, and sixth months after discharge. RESULTS: Nineteen infants (47.5%) required a second CSF diversion procedure at 6 months of follow-up. The failure rate was significantly higher in infants less than 3 months of age (P value of 0.04). The ETV site bulge was the most frequent complication encountered in the postoperative period, occurring in 20% of the cases. Eventually, all these infants required a ventriculoperitoneal shunt; 15% developed clinical features consistent with the diagnosis of post-ETV meningitis. The ETV site CSF leak occurred in 10% of the patients. Subdural hygroma developed in 7.5% of the patients; 17.5% of the patients contributed to mortality with a mean time of expiry of 22 days post-procedure. All these deaths had multifactorial causes and could not be said as a complication or failure of ETV. CONCLUSION: We do not recommend ETV for infants less than 3 months because of a high failure rate. The ETV site bulge was the most reliable and earliest marker of failure and a second CSF diversion surgery should be immediately considered.

Optical and magnetic resonance imaging approaches for investigating the tumour microenvironment: state-of-the-art review and future trends.

The tumour microenvironment (TME) strongly influences tumorigenesis and metastasis. Two of the most characterized properties of the TME are acidosis and hypoxia, both of which are considered hallmarks of tumours as well as critical factors in response to anticancer treatments. Currently, various imaging approaches exist to measure acidosis and hypoxia in the TME, including magnetic resonance imaging (MRI), positron emission tomography and optical imaging. In this review, we will focus on the latest fluorescent-based methods for optical sensing of cell metabolism and MRI as diagnostic imaging tools applied both in vitro and in vivo. The primary emphasis will be on describing the current and future uses of systems that can measure intra- and extra-cellular pH and oxygen changes at high spatial and temporal resolution. In addition, the suitability of these approaches for mapping tumour heterogeneity, and assessing response or failure to therapeutics will also be covered.

Beyond gold nanoparticles cytotoxicity: Potential to impair metastasis hallmarks.

Gold nanoparticle (AuNP)-based systems have been extensively investigated as diagnostic and therapeutic agents due to their tunable properties and easy surface functionalization. Upon cell uptake, AuNPs present an inherent cell impairment potential based on organelle and macromolecules damage, leading to cell death. Such cytotoxicity is concentration-dependent and completely undesirable, especially if unspecific. However, under non-cytotoxic concentrations, internalized AuNPs could potentially weaken cells and act as antitumor agents. Therefore, this study aimed to investigate the antitumor effect of ultrasmall AuNPs (~3 nm) stabilized by the anionic polysaccharide gum arabic (GA-AuNPs). Other than intrinsic cytotoxicity, the focus was downregulation of cancer hallmarks of aggressive tumors, using a highly metastatic model of melanoma. We first demonstrated that GA-AuNPs showed excellent stability under biological environment. Non-cytotoxic concentrations to seven different cell lines, including tumorigenic and non-tumorigenic cells, were determined by standard 2D in vitro assays. Gold concentrations ≤ 2.4 mg L-1 (16.5 nM AuNPs) were non-cytotoxic and therefore chosen for further analyses. Cells exposed to GA-AuNPs were uptaken by melanoma cells through endocytic processes. Next we described remarkable biological properties using non-cytotoxic concentrations of this nanomaterial. Invasion through an extracellular matrix barrier as well as 3D growth capacity (anchorage-independent colony formation and spheroids growth) were negatively affected by 2.4 mg L-1 GA-AuNPs. Additionally, exposed spheroids showed morphological changes, suggesting that GA-AuNPs could penetrate into the preformed tumor and affect its integrity. All together these results demonstrate that side effects, such as cytotoxicity, can be avoided by choosing the right concentration, nevertheless, preserving desirable effects such as modulation of key tumor cell malignancy features.

A randomized controlled trial of endodontic treatment using ultrasonic irrigation and laser activated irrigation to evaluate healing in chronic apical periodontitis.

BACKGROUND: Aim of this trial was to evaluate the combined clinical and radiographic success rate of endodontic treatment using passive ultrasonic irrigation (PUI) and laser activated irrigation (LAI) as compared to conventional syringe irrigation. MATERIAL AND METHODS: Permanent incisors and single rooted premolars were assessed for eligibility and 69 patients were randomly divided into three treatment groups (n=23) by allocation concealment method and irrigation was performed in accordance with the allocated group. Teeth were evaluated clinically and radiographically with CBCT after 6 months and 12 months of the treatment. RESULTS: A significant difference was observed in the radiographic healing rates among three groups (χ2=12.29, p=0.05). On comparing the final outcome among the three groups (n=19), it was found that 2 (10.5%) cases of group I(Conventional Syringe irrigation), 7 (36.8%) cases of group II (Passive ultrasonic irrigation) and 8 (42.1%) cases of group III(Laser activated irrigation) were healed while under healing category 13 (68.4%) cases of group I, 12 (63.2%) cases of group II and 11 (57.9%) of group III were observed whereas 4 (21.1%) cases were categorised as diseased in group I only. CONCLUSIONS: LAI and PUI can increase the predictability of the endodontic treatment success in cases of chronic apical periodontitis. Key words:Cone-beam computed tomography, CBCT-PAI, Irrigation, LASER, Radiographic healing, Root canal treatment, Ultrasonic.

"Effect of local application of simvastatin in bone regeneration of peri-apical defects-a clinico-radiographic study.

OBJECTIVE: The present study evaluates and compares the effectiveness of Simvastatin (SIM), Hydroxyapatite (HA), and platelet-rich fibrin (PRF) in bone regeneration of periapical defects. MATERIAL& METHOD: Thirty-nine patients were selected and randomized into three groups, Group 1: HA (n = 13), Group 2: PRF (n = 13), Group 3: SIM (n = 13). After completion of RCT and apicoectomy, the grafts were placed locally in the defect and sutured. RESULTS: At the end of twelve months, postoperative symptoms and radiographic analysis assessed the outcome of the treatment. CONCLUSION: Intragroup analysis of CBCT- Periapical Index (PAI) scores at 6th and 12th month revealed a significant change in the SIM group (p = 0.018 and 0.001 respectively), compared to PRF (p = 0.026 and 0.001 respectively) and HA (p = 0.053 and 0.039 respectively). Intergroup analysis of change in the level of CBCT-PAI score was highly significant (p = 0.003).SIM caused a more considerable change in the level of CBCT-PAI score compared to other groups, thereby indicating a faster rate of bone regeneration.

Probing the pH Microenvironment of Mesenchymal Stromal Cell Cultures on Additive-Manufactured Scaffolds.

Despite numerous advances in the field of tissue engineering and regenerative medicine, monitoring the formation of tissue regeneration and its metabolic variations during culture is still a challenge and mostly limited to bulk volumetric assays. Here, a simple method of adding capsules-based optical sensors in cell-seeded 3D scaffolds is presented and the potential of these sensors to monitor the pH changes in space and time during cell growth is demonstrated. It is shown that the pH decreased over time in the 3D scaffolds, with a more prominent decrease at the edges of the scaffolds. Moreover, the pH change is higher in 3D scaffolds compared to monolayered 2D cell cultures. The results suggest that this system, composed by capsules-based optical sensors and 3D scaffolds with predefined geometry and pore architecture network, can be a suitable platform for monitoring pH variations during 3D cell growth and tissue formation. This is particularly relevant for the investigation of 3D cellular microenvironment alterations occurring both during physiological processes, such as tissue regeneration, and pathological processes, such as cancer evolution.

Quantitative evaluation of masking effect of resin infiltration on developmental defects of enamel.

OBJECTIVE: Qualitative developmental defects of enamel (DDEs) are characterized by an intact hypermineralized surface enamel layer and a porous subsurface hypomineralized layer. This structure is similar to natural early caries lesions, which are effectively arrested by resin infiltration, a micro-invasive clinical procedure. An additional reported effect in early enamel caries is recovery of enamel translucency. Due to structural similarities, a similar mechanism is expected in qualitative DDEs. The objective of the present study was to observe quantitative color masking of qualitative DDEs when subjected to resin infiltration. METHOD AND MATERIALS: A total of 70 selected enamel defects were subjected to resin infiltration in a nonrandomized study design. Each defect was subjected to photographic imaging using a digital camera both pre- and post-intervention. Each image was assessed using ImageJ digital software (National Institutes of Health) for CIE L*a*b coordinates. RESULTS: There were significant changes observed in L*a*b coordinates of all DDEs, when subjected to resin infiltration. CONCLUSION: The resin infiltration intervention was successful in masking the "white" opaque discolorations of developmentally affected enamel.

Management of multiloculated hydrocephalus in children with emphasis on role of CT ventriculography.

OBJECTIVE: The authors performed a retrospective review of children diagnosed with multiloculated hydrocephalus (MLH) in our institute. The goal was to analyze the different diagnostic and therapeutic modalities used with special emphasis on CT ventriculography (CTV). METHODS: Male and female patients below the age of 18 years diagnosed with MLH were included. Cases of uniloculated hydrocephalus like entrapped temporal horn or isolated fourth ventricle were excluded. We used iohexol for CTV and gadodiamide for MR ventriculography. Neuroendoscopic procedures performed were endoscopic fenestration, endoscopic third ventriculostomy (ETV), endoscopic septostomy, endoscopic aqueductoplasty, or a combination of the above. The cohort was divided into two groups (endoscopic or shunt) based on initial surgical intervention. RESULTS: A total of 52 patients were included, with 43 boys and 9 girls. The average age of presentation was 7.7 months. The most common predisposing factor for MLH was neonatal meningitis seen in 30 patients. Mean duration of follow-up was 39 months. CTV was used in 26 patients and MR ventriculography in three patients. In one patient, the diagnosis of MLH was ruled out after ventriculography. Patients who underwent ETV only had the best outcome with 71.4% success rate. At the end of follow-up, 14 patients (27%) were shunt independent. CONCLUSIONS: The present study indicates that CTV helps to accurately define the anatomy of the ventricles and determine the site of physiological CSF obstruction. This helps in therapeutic planning and in avoiding misdiagnoses. Further, neuroendoscopy has the potential to lead to shunt independence in some patients.

Effect of laser-activated irrigation on smear layer removal and sealer penetration: An in vitro study.

INTRODUCTION: The present study was carried out with the objective of evaluation of efficacy of two root canal irrigants (LARGAL ULTRA and Biopure mixture of tetracycline, acid, and detergent [MTAD]) in smear layer removal and sealer penetration along the canal walls and to apprise any change in their efficacy when they were activated with two different types of lasers (neodymium-doped yttrium aluminum garnet [Nd:YAG] and erbium-doped yttrium aluminum garnet [Er:YAG]). The analysis was done with the help of confocal laser scanning microscope. MATERIALS AND METHODS: The curved root canals (>20°) from 140 extracted human molars (negative control, n = 20) were prepared to size #25.06 with NaOCl irrigation. Teeth were divided into two experimental groups with sample size 60 each (Group I, II). Groups further divided into subgroups IA, IB, and IC and IIA, IIB, and IIC with sample size twenty each. Group IA, Largal Ultra irrigation without LAI; Group IB, Largal Ultra irrigation with Nd:YAG; Group IC, Largal ultra with Er:YAG; Group IIA, MTAD irrigation without LAI; Group IIB, MTAD irrigation with Nd:YAG; Group IIC, MTAD irrigation with Er:YAG. Teeth were obturated with gutta-percha and fluorescent dye labeled sealer. Transverse sections at 3 mm from root apex were examined with confocal laser scanning microscopy, and the percentage of sealer penetration into dentinal tubules was measured. STATISTICAL ANALYSIS: Analysis of variance calculated (e.g., one-way between-subjects ANOVA) and Tukey's test was performed for each mean comparison. RESULTS: Difference between control and Group IIc (65.48 ± 0.57) was highest, followed by control and Group Ic (54.92 ± 0.57) and minimum between Group Ib and Group IIa (0.23 ± 0.57). Order of percent diffusion in the present study was: Group IIc > Group Ic > Group IIb > Group Ib ≈ Group IIa > Group Ia > Control (P <.001). CONCLUSIONS: It was inferred that, among the two irrigating solutions tested, Biopure MTAD is more proficient than Largal Ultra. Laser activation of final irrigating solution has been shown to allow better sealer penetration, with Er:YAG laser being more potent than Nd:YAG laser.