Journal of Biomedical Optics最新文献

Significance: Lattice light-sheet structured illumination microscopy (latticeSIM) has proven highly effective in producing three-dimensional images with super resolution rapidly and with minimal photobleaching. However, due to the use of two separate objectives, sample-induced aberrations can result in an offset between the planes of excitation and detection, causing artifacts in the reconstructed images.

Aim: We introduce a posterior approach to detect and correct the axial offset between the excitation and detection focal planes in latticeSIM and provide a method to minimize artifacts in the reconstructed images.

Approach: We utilized the residual phase information within the overlap regions of the laterally shifted structured illumination microscopy information components in frequency space to retrieve the axial offset between the excitation and the detection focal planes in latticeSIM.

Results: We validated our technique through simulations and experiments, encompassing a range of samples from fluorescent beads to subcellular structures of adherent cells. We also show that using transfer functions with the same axial offset as the one present during data acquisition results in reconstructed images with minimal artifacts and salvages otherwise unusable data.

Conclusion: We envision that our method will be a valuable addition to restore image quality in latticeSIM datasets even for those acquired under non-ideal experimental conditions.

Significance: Spatial frequency domain imaging (SFDI) applies patterned near-infrared illumination to quantify the optical properties of subsurface tissue. The periocular region is unique due to its complex ocular adnexal anatomy. Although SFDI has been successfully applied to relatively flat in vivo tissues, regions that have significant height variations and curvature may result in optical property inaccuracies.

Aim: We characterize the geometric impact of the periocular region on SFDI imaging reliability.

Approach: SFDI was employed to measure the reduced scattering coefficient ( ${{\mu }_s}^{\text{'}}$ ) and absorption coefficient ( ${\mu }_a$ ) of the periocular region in a cast facial tissue-simulating phantom by capturing images along regions of interest (ROIs): inferior temporal quadrant (ITQ), inferior nasal quadrant (INQ), superior temporal quadrant (STQ), central eyelid margin (CEM), rostral lateral nasal bridge (RLNB), and forehead (FH). The phantom was placed on a chin rest and imaged nine times from an "en face" or "side profile" position, and the flat back of the phantom was measured 15 times.

Results: The measured ${\mu }_a$ and ${{\mu }_s}^{\text{'}}$ of a cast facial phantom are accurate when comparing the ITQ, INQ, STQ, and FH to its flat posterior surface. Paired $t$ tests of ITQ, INQ, STQ, and FH ${\mu }_a$ and ${{\mu }_s}^{\text{'}}$ concluded that there is not enough evidence to suggest that imaging orientation impacted the measurement accuracy. Regions of extreme topographical variation, i.e., CEM and RLNB, did exhibit differences in measured optical properties.

Conclusions: We are the first to evaluate the geometric implications of wide-field imaging along the periocular region using a solid tissue-simulating facial phantom. Results suggest that the ITQ, INQ, STQ, and FH of a generalized face have minimal impact on the SFDI measurement accuracy. Areas with heightened topographic variation exhibit measurement variability. Device and facial positioning do not appear to bias measurements. These findings confirm the need to carefully select ROIs when measuring optical properties along the periocular region.

Significance: The technique of remote focusing (RF) has attracted considerable attention among microscopists due to its ability to quickly adjust focus across different planes, thus facilitating quicker volumetric imaging. However, the difficulty in changing objectives to align with a matching objective in a remote setting while upholding key requirements remains a challenge.

Aim: We aim to propose a customized yet straightforward technique to align multiple objectives with a remote objective, employing an identical set of optical elements to ensure meeting the criteria of remote focusing.

Approach: We propose a simple optical approach for aligning multiple objectives with a singular remote objective to achieve a perfect imaging system. This method utilizes readily accessible, commercial optical components to meet the fundamental requirements of remote focusing.

Results: Our experimental observations indicate that the proposed RF technique offers at least comparable, if not superior, performance over a significant axial depth compared with the conventional RF technique based on commercial lenses while offering the flexibility to switch the objective for multi-scale imaging.

Conclusions: The proposed technique addresses various microscopy challenges, particularly in the realm of multi-resolution imaging. We have experimentally demonstrated the efficacy of this technique by capturing images of focal volumes generated by two distinct objectives in a water medium.

Significance: Fluorescent organic dyes provide imaging capabilities at cellular and sub-cellular levels. However, a common problem associated with some of the existing dyes such as the US FDA-approved indocyanine green (ICG) is their weak fluorescence emission. Alternative dyes with greater emission characteristics would be useful in various imaging applications. Complementing optical imaging, magnetic resonance (MR) imaging enables deep tissue imaging. Nano-sized delivery systems containing dyes with greater fluorescence emission as well as MR contrast agents present a promising dual-mode platform with high optical sensitivity and deep tissue imaging for image-guided surgical applications.

Aim: We have engineered a nano-sized platform, derived from erythrocyte ghosts (EGs), with dual near-infrared fluorescence and MR characteristics by co-encapsulation of a brominated carbocyanine dye and gadobenate dimeglumine (Gd-BOPTA).

Approach: We have investigated the use of three brominated carbocyanine dyes (referred to as BrCy106, BrCy111, and BrCy112) with various degrees of bromination, structural symmetry, and acidic modifications for encapsulation by nano-sized EGs (nEGs) and compared their resulting optical characteristics with nEGs containing ICG.

Results: We find that asymmetric dyes (BrCy106 and BrCy112) with one dibromobenzene ring offer greater fluorescence emission characteristics. For example, the relative fluorescence quantum yield ( $\varphi$ ) for nEGs fabricated using $100\mu M$ of BrCy112 is $\sim 41$ -fold higher than nEGs fabricated using the same concentrations of ICG. The dual-mode nEGs containing BrCy112 and Gd-BOPTA show a nearly twofold increase in their $\varphi$ as compared with their single optical mode counterpart. Cytotoxicity is not observed upon incubation of SKOV3 cells with nEGs containing BrCy112.

Conclusions: Erythrocyte nano-ghosts with dual optical and MR characteristics may ultimately prove useful in various biomedical imaging applications such as image-guided tumor surgery where MR imaging can be used for tumor staging and mapping, and fluorescence imaging can help visualize small tumor nodules for resection.

Significance: Pathologies within the tympanic membrane (TM) and middle ear (ME) can lead to hearing loss. Imaging tools available in the hearing clinic for diagnosis and management are limited to visual inspection using the classic otoscope. The otoscopic view is limited to the surface of the TM, especially in diseased ears where the TM is opaque. An integrated optical coherence tomography (OCT) otoscope can provide images of the interior of the TM and ME space as well as an otoscope image. This enables the clinicians to correlate the standard otoscopic view with OCT and then use the new information to improve the diagnostic accuracy and management.

Aim: We aim to develop an OCT otoscope that can easily be used in the hearing clinic and demonstrate the system in the hearing clinic, identifying relevant image features of various pathologies not apparent in the standard otoscopic view.

Approach: We developed a portable OCT otoscope device featuring an improved field of view and form-factor that can be operated solely by the clinician using an integrated foot pedal to control image acquisition. The device was used to image patients at a hearing clinic.

Results: The field of view of the imaging system was improved to a 7.4 mm diameter, with lateral and axial resolutions of $38\mu m$ and $33.4\mu m$ , respectively. We developed algorithms to resample the images in Cartesian coordinates after collection in spherical polar coordinates and correct the image aberration. We imaged over 100 patients in the hearing clinic at USC Keck Hospital. Here, we identify some of the pathological features evident in the OCT images and highlight cases in which the OCT image provided clinically relevant information that was not available from traditional otoscopic imaging.

Conclusions: The developed OCT otoscope can readily fit into the hearing clinic workflow and provide new relevant information for diagnosing and managing TM and ME disease.

Significance: The multispectral imaging-based tissue oxygen saturation detecting (TOSD) system offers deeper penetration ( $\sim 2$ to 3 mm) and comprehensive tissue oxygen saturation ( ${\mathrm{StO}}_2$ ) assessment and recognizes the wound healing phase at a low cost and computational requirement. The potential for miniaturization and integration of TOSD into telemedicine platforms could revolutionize wound care in the challenging pandemic era.

Aim: We aim to validate TOSD's application in detecting ${\mathrm{StO}}_2$ by comparing it with wound closure rates and laser speckle contrast imaging (LSCI), demonstrating TOSD's ability to recognize the wound healing process.

Approach: Utilizing a murine model, we compared TOSD with digital photography and LSCI for comprehensive wound observation in five mice with 6-mm back wounds. Sequential biochemical analysis of wound discharge was investigated for the translational relevance of TOSD.

Results: TOSD demonstrated constant signals on unwounded skin with differential changes on open wounds. Compared with LSCI, TOSD provides indicative recognition of the proliferative phase during wound healing, with a higher correlation coefficient to wound closure rate (TOSD: 0.58; LSCI: 0.44). ${\mathrm{StO}}_2$ detected by TOSD was further correlated with proliferative phase angiogenesis markers.

Conclusions: Our findings suggest TOSD's enhanced utility in wound management protocols, evaluating clinical staging and therapeutic outcomes. By offering a noncontact, convenient monitoring tool, TOSD can be applied to telemedicine, aiming to advance wound care and regeneration, potentially improving patient outcomes and reducing healthcare costs associated with chronic wounds.

Significance: Traditional diffuse optical tomography (DOT) reconstructions are hampered by image artifacts arising from factors such as DOT sources being closer to shallow lesions, poor optode-tissue coupling, tissue heterogeneity, and large high-contrast lesions lacking information in deeper regions (known as shadowing effect). Addressing these challenges is crucial for improving the quality of DOT images and obtaining robust lesion diagnosis.

Aim: We address the limitations of current DOT imaging reconstruction by introducing an attention-based U-Net (APU-Net) model to enhance the image quality of DOT reconstruction, ultimately improving lesion diagnostic accuracy.

Approach: We designed an APU-Net model incorporating a contextual transformer attention module to enhance DOT reconstruction. The model was trained on simulation and phantom data, focusing on challenges such as artifact-induced distortions and lesion-shadowing effects. The model was then evaluated by the clinical data.

Results: Transitioning from simulation and phantom data to clinical patients' data, our APU-Net model effectively reduced artifacts with an average artifact contrast decrease of 26.83% and improved image quality. In addition, statistical analyses revealed significant contrast improvements in depth profile with an average contrast increase of 20.28% and 45.31% for the second and third target layers, respectively. These results highlighted the efficacy of our approach in breast cancer diagnosis.

Conclusions: The APU-Net model improves the image quality of DOT reconstruction by reducing DOT image artifacts and improving the target depth profile.

Significance: Accurate identification of epidermal cells on reflectance confocal microscopy (RCM) images is important in the study of epidermal architecture and topology of both healthy and diseased skin. However, analysis of these images is currently done manually and therefore time-consuming and subject to human error and inter-expert interpretation. It is also hindered by low image quality due to noise and heterogeneity.

Aim: We aimed to design an automated pipeline for the analysis of the epidermal structure from RCM images.

Approach: Two attempts have been made at automatically localizing epidermal cells, called keratinocytes, on RCM images: the first is based on a rotationally symmetric error function mask, and the second on cell morphological features. Here, we propose a dual-task network to automatically identify keratinocytes on RCM images. Each task consists of a cycle generative adversarial network. The first task aims to translate real RCM images into binary images, thus learning the noise and texture model of RCM images, whereas the second task maps Gabor-filtered RCM images into binary images, learning the epidermal structure visible on RCM images. The combination of the two tasks allows one task to constrict the solution space of the other, thus improving overall results. We refine our cell identification by applying the pre-trained StarDist algorithm to detect star-convex shapes, thus closing any incomplete membranes and separating neighboring cells.

Results: The results are evaluated both on simulated data and manually annotated real RCM data. Accuracy is measured using recall and precision metrics, which is summarized as the $F1$ -score.

Conclusions: We demonstrate that the proposed fully unsupervised method successfully identifies keratinocytes on RCM images of the epidermis, with an accuracy on par with experts' cell identification, is not constrained by limited available annotated data, and can be extended to images acquired using various imaging techniques without retraining.

Significance: Photoacoustic imaging (PAI) is an emerging technology that holds high promise in a wide range of clinical applications, but standardized methods for system testing are lacking, impeding objective device performance evaluation, calibration, and inter-device comparisons. To address this shortfall, this tutorial offers readers structured guidance in developing tissue-mimicking phantoms for photoacoustic applications with potential extensions to certain acoustic and optical imaging applications.

Aim: The tutorial review aims to summarize recommendations on phantom development for PAI applications to harmonize efforts in standardization and system calibration in the field.

Approach: The International Photoacoustic Standardization Consortium has conducted a consensus exercise to define recommendations for the development of tissue-mimicking phantoms in PAI.

Results: Recommendations on phantom development are summarized in seven defined steps, expanding from (1) general understanding of the imaging modality, definition of (2) relevant terminology and parameters and (3) phantom purposes, recommendation of (4) basic material properties, (5) material characterization methods, and (6) phantom design to (7) reproducibility efforts.

Conclusions: The tutorial offers a comprehensive framework for the development of tissue-mimicking phantoms in PAI to streamline efforts in system testing and push forward the advancement and translation of the technology.

Significance: Azimuth-resolved optical scattering signals obtained from cell nuclei are sensitive to changes in their internal refractive index profile. These two-dimensional signals can therefore offer significant insights into chromatin organization.

Aim: We aim to determine whether two-dimensional scattering signals can be used in an inverse scheme to extract the spatial correlation length ${\ell }_c$ and extent $\delta n$ of subnuclear refractive index fluctuations to provide quantitative information on chromatin distribution.

Approach: Since an analytical formulation that links azimuth-resolved signals to ${\ell }_c$ and $\delta n$ is not feasible, we set out to assess the potential of machine learning to predict these parameters via a data-driven approach. We carry out a convolutional neural network (CNN)-based regression analysis on 198 numerically computed signals for nuclear models constructed with ${\ell }_c$ varying in steps of $0.1\mu m$ between 0.4 and $1.0\mu m$ , and $\delta n$ varying in steps of 0.005 between 0.005 and 0.035. We quantify the performance of our analysis using a five-fold cross-validation technique.

Results: The results show agreement between the true and predicted values for both ${\ell }_c$ and $\delta n$ , with mean absolute percent errors of 8.5% and 13.5%, respectively. These errors are smaller than the minimum percent increment between successive values for respective parameters characterizing the constructed models and thus signify an extremely good prediction performance over the range of interest.

Conclusions: Our results reveal that CNN-based regression can be a powerful approach for exploiting the information content of two-dimensional optical scattering signals and hence monitoring chromatin organization in a quantitative manner.