Second Harmonic Imaging Research Articles

Deep-learning-assisted identification of sub-diffraction nanodomains in LiNbO3 crystals.

LiNbO3 domain structures have been widely applied in nonlinear beam shaping, quantum light generation, and nonvolatile ferroelectric memory. The recent developments in nanoscale domain engineering techniques make it possible to fabricate sub-diffracted nanodomains in LiNbO3 crystal for high-speed modulation and high-capacity storage. However, it still lacks a feasible and efficient way to characterize these nanoscale domains. In this work, we propose and experimentally demonstrate a deep-learning-assisted identification of sub-diffraction LiNbO3 nanodomain lines. In the experiment, we record the second-harmonic (SH) images of nanodomain lines by using a confocal microscope. The domain linewidths range from 200 nm to 600 nm, which are beyond the spatial resolution of the used microscope (∼800 nm). After training a neural network with 1568 SH images, it is capable of recognizing different nanodomain lines at an accuracy of 81.25%. Our approach leverages the exceptional recognition capability of the neural network, which provides an efficient method to identify sub-diffraction nanodomains from diffraction-limited images.

Research on the second-harmonic focused ultrasonic device based on micro-bubble contrast agents

Non-invasive, accurate diagnosis and treatment have increasingly gained attention in medical research. The nonlinear response mechanism of ultrasound contrast agents and their medical application have become major topics in ultrasound imaging studies. This paper reports on a second-harmonic focused ultrasonic device based on micro-bubble contrast agents, which is designed to solve the problems associated with a weak second-harmonic intensity. A periodic array of circular holes is embedded in the center of a specifically shaped resin plate, and contrast agents are encapsulated in the circular holes using thin resin tape. The functional mechanism is theoretically explained and experimentally verified. This device enables second-harmonic ultrasound imaging with a higher ultrasonic lateral resolution and signal-to-noise ratio than the conventional system without the device.

Suppression of the Tissue Component With the Total Least‐Squares Algorithm to Improve Second Harmonic Imaging of Ultrasound Contrast Agents

ABSTRACTThe second harmonic (SH) of ultrasound contrast agents (UCAs) is widely used in contrast‐enhanced ultrasound imaging; however, is affected by the nonlinearity of surrounding tissue. Suppression of the tissue component based on the total least‐squares (STLS) algorithm is proposed to enhance the SH imaging of UCAs. The image blocks of pulse‐inversion‐based SH images before and after UCA injections are set as the reference and input of the total least‐squares model, respectively. The optimal coefficients of the model are obtained by minimizing the Frobenius norm of perturbations in the input and output signals. After processing all image blocks, the complete SH image of UCAs is obtained by subtracting the optimal output of the model (i.e., the estimated tissue SH image) from the SH image after UCA injection. Simulation and in vivo experiments confirm that the STLS approach offers clearer capillaries. For in vivo experiments, the STLS‐based contrast‐to‐tissue ratios and contrasts increase by 26.90% and 56.27%, as well as 26.99% and 56.43%, respectively, compared with those based on bubble‐echo deconvolution and pulse inversion bubble‐wavelet imaging methods. The STLS approach enhances the SH imaging of UCAs by effectively suppressing more tissue SH components, having the potential to provide more accurate diagnostic information for clinical applications.

Wide-Field Polarimetric Second-Harmonic Imaging for Rapid and Nondestructive Investigation of Laser-Induced Crystallization Phenomena.

The selective and controlled formation of nanocrystals in glass is emerging as a versatile method to achieve functional photonics, optoelectronics, and quantum devices, such as single-photon emitters. Here, we investigate the use of wide-field polarimetric second-harmonic (SH) microscopy as a method to rapidly and nondestructively examine nanoscale crystal arrangements in laser-processed glass. As a case study, we investigate tellurite glass, where the formation of a trigonal tellurium (t-Te) nanocrystalline phase after femtosecond laser exposure was recently demonstrated. Combined with theoretical models, we show that wide-field polarimetric SH microscopy offers comprehensive information on the nanocrystals' orientation, distribution, and chirality. With its high imaging throughput and spatial resolution, this method has the potential not only to significantly accelerate investigations on laser-induced glass crystallization processes but also to provide a valuable tool for in situ process monitoring.

Extraction of collagen morphological features from second-harmonic generation microscopy images via GLCM and CT analyses: A cross-laboratory study.

Second-harmonic generation (SHG) microscopy provides a high-resolution label-free approach for noninvasively detecting collagen organization and its pathological alterations. Up to date, several imaging analysis algorithms for extracting collagen morphological features from SHG images-such as fiber size and length, order and anisotropy-have been developed. However, the dependence of extracted features on experimental setting represents a significant obstacle for translating the methodology in the clinical practice. We tackled this problem by acquiring SHG images of the same kind of collagenous sample in various laboratories using different experimental setups and imaging conditions. The acquired images were analyzed by commonly used algorithms, such as gray-level co-occurrence matrix or curvelet transform; the extracted morphological features were compared, finding that they strongly depend on some experimental parameters, whereas they are almost independent from others. We conclude with useful suggestions for comparing results obtained in different labs using different experimental setups and conditions.

Factors Influencing the Visualization of Fallopian Tubes in Hysterosalpingo-Contrast Sonography (HyCoSy)-The Value of Multimodal HyCoSy in Visualizing the Fallopian Tubes.

Four-dimensional hysterosalpingo-contrast sonography (4D-HyCoSy) can non-invasively evaluate the patency of the fallopian tubes and is increasingly used in clinical practice. However, some factors may lead to false-positive diagnoses. This study aims to analyze the factors affecting clear imaging of the fallopian tubes in 4D-HyCoSy and explore methods to improve the quality of fallopian tube imaging. A total of 118 patients were enrolled in this retrospective study. After injecting the SonoVue into the uterine cavity, three modes of HyCoSy were completed in sequence: 4D-HyCoSy, 2D-HyCoSy, and second harmonic imaging (SHI). Participants were divided into two groups: the easy visualization group (fallopian tubes could be visualized using only 4D-HyCoSy) and the difficult visualization group (a multimodal combination was required for visualization). The position of the uterus, the relationship between the ovaries and the uterus, endometrial thickness, time of catheterization in the uterine cavity, presence or absence of lesions in the uterine cavity, whether intestinal gas covers the fallopian tubes and the imaging effect of different modes on the fallopian tubes was analyzed, to determine the key factors affecting the clear imaging of the fallopian tubes. The positional relationship between the ovary and the uterus (OR = 4.711, 95% CI: 1.322-19.77, P = 0.023), the positioning of the uterus (OR = 3.843, 95% CI: 1.129-15.26, P = 0.04), endometrial thickness (OR = 3.985, 95% CI: 1.168-15.99, P = 0.036), and the duration of intrauterine catheter placement (OR = 3.547, 95% CI: 1.042-13.52, P = 0.05) were independent factors that affecting difficulty in visualizing the fallopian tubes. Uterine position, the positional relationship between the ovary and the uterus, endometrial thickness, and the time of catheter insertion are factors that affect visualizing the fallopian tubes during 4D-HyCoSy. The combination of multimodal imaging, especially the combination of 4D-HyCoSy with SHI mode, can help improve the quality of fallopian tube visualization.

Dynamic Second Harmonic Imaging of Proton Translocation Through Water Needles in Lipid Membranes.

Proton translocation through lipid membranes is a fundamental process in the field of biology. Several theoretical models have been developed and presented over the years to explain the phenomenon, yet the exact mechanism is still not well understood. Here, we show that proton translocation is directly related to membrane potential fluctuations. Using high-throughput wide-field second harmonic (SH) microscopy, we report apparently universal transmembrane potential fluctuations in lipid membrane systems. Molecular simulations and free energy calculations suggest that H+ permeation proceeds predominantly across a thin, membrane-spanning water needle and that the transient transmembrane potential drives H+ ions across the water needle. This mechanism differs from the transport of other cations that require completely open pores for transport and follows naturally from the well-known Grotthuss mechanism for proton transport in bulk water. Furthermore, SH imaging and conductivity measurements reveal that the rate of proton transport depends on the structure of the hydrophobic core of bilayer membranes.

Tissue ultrasound imaging based on wavelet correlation analysis and pulse-inversion technique.

Pulse-inversion-based tissue harmonic imaging has been utilized for many years because it can effectively eliminate the harmonic leakage and produce low side-lobe. However, the pulse inversion method is sensitive to imaging object movements, which may result in motion artifacts. Spatial resolution and contrast were limited. To improve ultrasound image quality by a new pulse-inversion-based tissue harmonic imaging technique. Continuous wavelet transform is applied to investigate the correlation between mother wavelet and the received echoes from two opposite pulses. To get a better correlation, a novel mother wavelet named 'tissue wavelet' is designed based on the Khokhlov-Zabolotskaya- Kuznetsov (KZK) wave equation. Radio frequency data were obtained from open Ultrasonix SonixTouch imaging system. Experiments were carried on ultrasonic tissue phantom, human carotid artery and human liver. The average improvement of lateral spatial resolution is 49.52% compared to pulse-inversion-based tissue second-harmonic Imaging (PIHI). Contrast ratio (CR) and contrast-to-noise ratio (CNR) increased by 5.55 dB and 1.40 dB over PIHI. Tissue wavelet performs better than Mexh and Morl wavelet in lateral spatial resolution, CR, and CNR. The proposed technique effectively improves the imaging quality in lateral spatial resolution, CR, and CNR.

Impact of ultrasonic inertial cavitation on the pancreatic tumor stiffness in mice

Pancreatic ductal adenocarcinoma (PDAC) is among the fastest-growing cancers and is predicted to become the second leading cause of cancer-related deaths worldwide by 2030. Ultrasound-induced inertial cavitation (IC) is an emerging therapeutic strategy with promise for treating patients with unresectable tumors. However, the physical impact of IC on tumors remains unclear. In this study, we used orthotopic murine models of pancreatic cancer and employed passive elastography to measure elasticity before and after applying IC to the whole tumor volume. The IC was generated using two focused confocal transducers (center frequency, 1.1 MHz). The output level was adjusted with a real-time feedback loop based on broadband signal acquired with a passive cavitation detector. Cavitation clouds were visualized during treatment using an imaging probe. The results from eight treated mice showed a 24% decrease (p < 0.05) in tumor stiffness after the IC treatment, indicating that the chosen IC sequence can soften the tumor environment. After sacrifice, we utilized second-harmonic imaging microscopy to visualize the collagen network within the tumor, a known contributor to tissue stiffness, and correlated various features of this network (length, width, curvature) with the measured elasticity.

Ion-Induced Transient Potential Fluctuations Facilitate Pore Formation and Cation Transport through Lipid Membranes.

Unassisted ion transport through lipid membranes plays a crucial role in many cell functions without which life would not be possible, yet the precise mechanism behind the process remains unknown due to its molecular complexity. Here, we demonstrate a direct link between membrane potential fluctuations and divalent ion transport. High-throughput wide-field non-resonant second harmonic (SH) microscopy of membrane water shows that membrane potential fluctuations are universally found in lipid bilayer systems. Molecular dynamics simulations reveal that such variations in membrane potential reduce the free energy cost of transient pore formation and increase the ion flux across an open pore. These transient pores can act as conduits for ion transport, which we SH image for a series of divalent cations (Cu2+, Ca2+, Ba2+, Mg2+) passing through giant unilamellar vesicle (GUV) membranes. Combining the experimental and computational results, we show that permeation through pores formed via an ion-induced electrostatic field is a viable mechanism for unassisted ion transport.

Multiscale anisotropy analysis of second-harmonic generation collagen imaging of human pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a minority (< 10%) of patients surviving five years past diagnosis. This could be improved with the development of new imaging modalities for early differentiation of benign and cancerous fibrosis. This study intends to explore the application of a two-photon microscopy technique known as second harmonic generation to PDAC using the 2D Wavelet Transform Modulus Maxima (WTMM) Anisotropy method to quantify collagen organization in fibrotic pancreatic tissue. Forty slides from PDAC patients were obtained and eight images were captured per each tissue category on each slide. Brownian surface motion and white noise images were generated for calibration and testing of a new variable binning approach to the 2D WTMM Anisotropy method. The variable binning method had greater resistance to wavelet scaling effects and white noise images were found to have the lowest anisotropy factor. Cancer and fibrosis had greater anisotropy factors (Fa) at small wavelet scales than normal and normal adjacent tissue. At a larger scale of 21 μm this relationship changed with normal tissue having a higher Fa than all other tissue groups. White noise is the best representative image for isotropy and the 2D WTMM anisotropy method is sensitive to changes induced in collagen by PDAC.

160-LB: Age-Related Adipose Tissue Fibrosis and Neuropathy in the Genetically Diverse HET3 Mouse

Changes in adipose tissue's extracellular matrix, as with fibrosis, inhibits the tissue's ability to shrink and expand as needed, resulting in sheer stress on adipocytes and chronic inflammation, as well as decreased insulin sensitivity. With aging, a state of metabolic dysregulation and increased incidence of insulin resistance, we have observed a reduction in adipose innervation. This ‘adipose neuropathy' likely poses a physiological challenge for tissue metabolic function and glucose homeostasis, as has been observed in adipose denervation studies. Using second-harmonic generation label-free imaging of adipose collagen and advanced image co-localization techniques, we found that obese adipose tissue exhibits higher co-localization of nerves with collagen fibers, indicating that changes in tissue fibrosis may tie mechanistically with tissue neuropathy. We compared C57BL/6J mice with the more genetically diverse HET3 mouse model that is used by the NIA's intervention testing program, to investigate age-related neuropathy and adipose fibrosis. We found that HET3 mice displayed mitigated neuropathy phenotypes in skin, muscle and adipose, compared to inbred C56BL/6J mice. Tissue fibrosis was significantly increased with age in subcutaneous and visceral white adipose depots of both strains of mice. Using birefringence imaging, we found that the relative distribution of type I / thick filament collagen increased with age while type III / thin filament collagen decreased. Gene expression by qPCR revealed significant increases in collagen genes Col1a1, Col3a1, and Col5a1 in adipose with age. Despite its success as a longevity treatment, the mTOR inhibitor rapamycin had little to no effect on reducing or preventing the onset of neuropathy in HET3 aged mice, and importantly it worsened the fibrotic phenotype with increased type 1 collagen in adipose. Together, we found that rapamycin's longevity effects are uncoupled from healthspan producing effects, with detrimental impacts on adipose fibrosis that may impact age-related insulin resistance. Disclosure J. Willows: None. G. Mishra: None. M. Blaszkiewicz: None. K. L. Townsend: Other Relationship; Neuright, Inc. Funding American Heart Association (AHA) Collaborative Sciences Award (18CSA34090028)

Feasibility investigation of logarithmic Nakagami parametric imaging in recovering underestimated perfusion metrics of DCEUS in the uneven acoustic field.

Owing to acoustic-pressure dependence, amplitudes of backscattered-echoes of encapsulated microbubbles (MBs) are unavoidably regulated by an uneven acoustic field, resulting in the misestimation of hemodynamics in conventional amplitude-coding dynamic contrast-enhanced ultrasound (DCEUS) with focused pulse transmission. This study aimed to investigate the feasibility and performance of Nakagami statistical-feature parametric imaging to recover the above misestimation. Logarithmic Nakagami parameter (m)-coding DCEUS scheme was investigated via simulation and in vitro MB phantoms as well as in vivo kidney-perfusion experiments of four rabbits in the uneven acoustic fields with two different focal depths. In vivo tissue artifacts for m estimation were suppressed by pulse-inversion second-harmonic imaging and its robustness was enhanced by multiscale moment-estimation strategy. Time-Nakagami-m curves and the corresponding perfusion metrics of intensity and volume were calculated from the logarithmic m-coding DCEUS images within the prefocal and focal regions. These curves and metrics were further compared with the perfusion curves and metrics estimated from the conventional amplitude-coding images within the same regions. Compared with amplitudes of nonlinear scattering MB echoes, their logarithmic m values were relatively independent of the changes in acoustics pressures. Compared with the fixed-scale moment-estimation, the perfusion intensity estimated from logarithmic m-coding DCEUS scheme using multiscale statistical moment-estimation had smaller differences between the prefocal and focal regions. The differences of perfusion intensity induced by an uneven acoustic field decreased to 3.47% ± 1.58 %. The differences decreased by the logarithmic m-coding DCEUS scheme were further regulated by threshold values of m estimation. The logarithmic m-coding DCEUS scheme could recover the underestimated MB backscattered-echoes and the misestimated perfusion intensity induced by the uneven acoustic field. The scheme had the potential to weaken the limitation of microvasculature identification and hemodynamic characterization marked by MBs within tissues or tumors in the uneven acoustic field.

Orthogonally-polarized excitation for improved two-photon and second-harmonic-generation microscopy, applied to neurotransmitter imaging with GPCR-based sensors.

Fluorescent proteins are excited by light that is polarized parallel to the dipole axis of the chromophore. In two-photon microscopy, polarized light is used for excitation. Here we reveal surprisingly strong polarization sensitivity in a class of genetically encoded, GPCR-based neurotransmitter sensors. In tubular structures such as dendrites, this effect led to a complete loss of membrane signal in dendrites running parallel to the polarization direction of the excitation beam. To reduce the sensitivity to dendritic orientation, we designed an optical device that generates interleaved pulse trains of orthogonal polarization. The passive device, which we inserted in the beam path of an existing two-photon microscope, removed the strong direction bias from fluorescence and second-harmonic (SHG) images. We conclude that for optical measurements of transmitter concentration with GPCR-based sensors, orthogonally polarized excitation is essential.

Multimodal analysis of the differential effects of cyclic strain on collagen isoform composition, fibril architecture and biomechanics of tissue engineered tendon

Tendon is predominantly composed of aligned type I collagen, but additional isoforms are known to influence fibril architecture and maturation, which contribute to the tendon’s overall biomechanical performance. The role of the less well-studied collagen isoforms on fibrillogenesis in tissue engineered tendons is currently unknown, and correlating their relative abundance with biomechanical changes in response to cyclic strain is a promising method for characterising optimised bioengineered tendon grafts. In this study, human mesenchymal stem cells (MSCs) were cultured in a fibrin scaffold with 3%, 5% or 10% cyclic strain at 0.5 Hz for 3 weeks, and a comprehensive multimodal analysis comprising qPCR, western blotting, histology, mechanical testing, fluorescent probe CLSM, TEM and label-free second-harmonic imaging was performed. Molecular data indicated complex transcriptional and translational regulation of collagen isoforms I, II, III, V XI, XII and XIV in response to cyclic strain. Isoforms (XII and XIV) associated with embryonic tenogenesis were deposited in the formation of neo-tendons from hMSCs, suggesting that these engineered tendons form through some recapitulation of a developmental pathway. Tendons cultured with 3% strain had the smallest median fibril diameter but highest resistance to stress, whilst at 10% strain tendons had the highest median fibril diameter and the highest rate of stress relaxation. Second harmonic generation exposed distinct structural arrangements of collagen fibres in each strain group. Fluorescent probe images correlated increasing cyclic strain with increased fibril alignment from 40% (static strain) to 61.5% alignment (10% cyclic strain). These results indicate that cyclic strain rates stimulate differential cell responses via complex regulation of collagen isoforms which influence the structural organisation of developing fibril architectures.

All-Atom Quantum Mechanical Calculation of the Second-Harmonic Generation of Fluorescent Proteins.

Fluorescent proteins (FPs) are biotags of choice for second-harmonic imaging microscopy (SHIM). Because of their large size, computing their second-harmonic generation (SHG) response represents a great challenge for quantum chemistry. In this contribution, we propose a new all-atom quantum mechanics methodology to compute SHG of large systems. This is now possible because of two recent implementations: the tight-binding GFN2-xTB method to optimize geometries and a related version of the simplified time-dependent density functional theory (sTD-DFT-xTB) to evaluate quadratic response functions. In addition, a new dual-threshold configuration selection scheme is introduced to reduce the computational costs while retaining overall similar accuracy. This methodology was tested to evaluate the SHG of the proteins iLOV and bacteriorhodopsin (bR). In the case of bR, quantitative agreement with respect to experiment was reached for the out-of-resonance low-energy part of the βHRS frequency dispersion. This work paves the way toward an accurate prediction of the SHG of large structures-a requirement for the design of new and improved SHIM biotags.

Ultrafast Charge-Transfer Dynamics in Twisted MoS2/WSe2 Heterostructures.

Two-dimensional transition metal dichalcogenides offer a fascinating platform for creating van der Waals heterojunctions with exciting physical properties. Because of their typical type-II band alignment, photoexcited electrons and holes can separate via interfacial charge transfer. Furthermore, the relative crystallographic alignment of the individual layers in these heterostructures represents an important degree of freedom. Based on both effects, various fascinating ideas for applications in optoelectronics and valleytronics have been suggested. Despite its utmost importance for the design and efficiency of potential devices, the nature and the dynamics of ultrafast charge transfer are not yet well understood. This is mainly because the charge transfer can be surprisingly fast, usually faster than the temporal resolution of previous experimental approaches. Here, we apply time- and polarization-resolved second-harmonic imaging microscopy to investigate the charge-transfer dynamics for three MoS2/WSe2 heterostructures with different stacking angles at a previously unattainable time resolution of ≈10 fs. For 1.70 eV excitation energy, electron transfer from WSe2 to MoS2 is found to depend considerably on the stacking angle with the fastest transfer time observed to be as short as 12 fs. At 1.85 eV excitation energy, ultrafast hole transfer from MoS2 to hybridized states at the Γ-point and to the K-points of WSe2 has to be considered. Surprisingly, the corresponding decay dynamics show only a minor stacking-angle dependence indicating that radiative recombination of momentum-space indirect Γ-K excitons becomes the dominant decay route for all samples.

Quasi-phase-matching-division multiplexing holography in a three-dimensional nonlinear photonic crystal

Nonlinear holography has recently emerged as a novel tool to reconstruct the encoded information at a new wavelength, which has important applications in optical display and optical encryption. However, this scheme still struggles with low conversion efficiency and ineffective multiplexing. In this work, we demonstrate a quasi-phase-matching (QPM) -division multiplexing holography in a three-dimensional (3D) nonlinear photonic crystal (NPC). 3D NPC works as a nonlinear hologram, in which multiple images are distributed into different Ewald spheres in reciprocal space. The reciprocal vectors locating in a given Ewald sphere are capable of fulfilling the complete QPM conditions for the high-efficiency reconstruction of the target image at the second-harmonic (SH) wave. One can easily switch the reconstructed SH images by changing the QPM condition. The multiplexing capacity is scalable with the period number of 3D NPC. Our work provides a promising strategy to achieve highly efficient nonlinear multiplexing holography for high-security and high-density storage of optical information.

Multiscale anisotropy analysis of second-harmonic generation collagen imaging of mouse skin.

.Significance: Morphological collagen signatures are important for tissue function, particularly in the tumor microenvironment. A single algorithmic framework with quantitative, multiscale morphological collagen feature extraction may further the use of collagen signatures in understanding fundamental tumor progression.Aim: A modification of the 2D wavelet transform modulus maxima (WTMM) anisotropy method was applied to both digitally simulated collagen fibers and second-harmonic-generation imaged collagen fibers of mouse skin to calculate a multiscale anisotropy factor to detect collagen fiber organization.Approach: The modified 2D WTMM anisotropy method was initially validated on synthetic calibration images to establish the robustness and sensitivity of the multiscale fiber organization tool. Upon validation, the algorithm was applied to collagen fiber organization in normal wild-type skin, melanoma stimulated skin, and integrin skin.Results: Normal wild-type skin collagen fibers have an increased anisotropy factor at all sizes scales. Interestingly, the multiscale anisotropy differences highlight important dissimilarities between collagen fiber organization in normal wild-type skin, melanoma stimulated, and integrin skin. At small scales ( to ), the integrin skin was vastly different than normal skin (), whereas the melanoma stimulated skin was vastly different than normal at large scales ( to , ).Conclusions: This objective computational collagen fiber organization algorithm is sensitive to collagen fiber organization across multiple scales for effective exploration of collagen morphological alterations associated with melanoma and the lack of integrin binding.

Tacrolimus action pathways in an ointment base for hypertrophic scar prevention in a rabbit ear model

BackgroundTacrolimus is used to prevent unaesthetic scars due to its action on fibroblast activity and collagen production modulation. ObjectivesTo evaluate the action pathways, from the histopathological point of view and in cytokine control, of tacrolimus ointment in the prevention of hypertrophic scars. MethodsTwenty-two rabbits were submitted to the excision of two 1-cm fragments in each ear, including the perichondrium. The right ear received 0.1% and 0.03% tacrolimus in ointment base twice a day in the upper wound and in the lower wound respectively. The left ear, used as the control, was treated with petrolatum. After 30 days, collagen fibers were evaluated using special staining, and immunohistochemistry analyses for smooth muscle actin, TGF-β and VEGF were performed. ResultsThe wounds treated with 0.1% tacrolimus showed weak labeling and a lower percentage of labeling for smooth muscle actin, a higher proportion of mucin absence, weak staining, fine and organized fibers for Gomori's Trichrome, strong staining and organized fibers for Verhoeff when compared to controls. The wounds treated with 0.03% tacrolimus showed weak labeling for smooth muscle actin, a higher proportion of mucin absence, strong staining for Verhoeff when compared to the controls. There was absence of TGF-β and low VEGF expression. Study limitationsThe analysis was performed by a single pathologist. Second-harmonic imaging microscopy was performed in 2 sample areas of the scar. ConclusionsBoth drug concentrations were effective in suppressing TGF-β and smooth muscle actin, reducing mucin, improving the quality of collagen fibers, and the density of elastic fibers, but only the higher concentration influenced elastic fiber organization.