Journal of Biomedical Optics最新文献

Significance: Although spatial frequency domain imaging (SFDI) has been well characterized under diffuse optical conditions, tissue measurements made outside the diffuse regime can provide new diagnostic information. Before such measurements can become clinically relevant, however, the behavior of sub-diffuse SFDI and its effect on the accuracy of derived tissue parameters must be assessed.

Aim: We aim to characterize the impact that both the assumed scattering phase function (SPF) and the polarization state of the illumination light source have on the accuracy of SFDI-derived optical properties when operating under diffuse or sub-diffuse conditions, respectively.

Approach: Through the use of a set of well-characterized optical phantoms, SFDI accuracy was assessed at four wavelengths (395, 545, 625, and 850 nm) and two different spatial frequencies (0.3 and $1.0{\mathrm{mm}}^{-1}$ ), which provided a broad range of diffuse and sub-diffuse conditions, using three different SPFs. To determine the effects of polarization, the SFDI accuracy was assessed using both unpolarized and cross-polarized illumination.

Results: It was found that the assumed SPF has a direct and significant impact on the accuracy of the SFDI-derived optical properties, with the best choice of SPF being dictated by the polarization state. As unpolarized SFDI retains the sub-diffuse portion of the signal, optical properties were found to be more accurate when using the full SPF that includes forward and backscattering components. By contrast, cross-polarized SFDI yielded accurate optical properties when using a forward-scattering SPF, matching the behavior of cross-polarization to attenuate the immediate backscattering of sub-diffuse reflectance. Using the correct pairings of SPF and polarization enabled using a reflectance standard, instead of a more subjective phantom, as the reference measurement.

Conclusions: These results provide the foundation for a more thorough understanding of SFDI and enable new applications of this technology in which sub-diffuse conditions dominate (e.g., ${\mu }_a\nless {\mu }_s^{\text{'}}$ ) or high spatial frequencies are required.

Significance: Although the depth detection limit of fluorescence objects in tissue has been studied, reports with a model including noise statistics for designing the optimum measurement configuration are missing. We demonstrate a variance analysis of the depth detection limit toward clinical applications such as noninvasively assessing the risk of aspiration.

Aim: It is essential to analyze how the depth detection limit of the fluorescence object in a strong scattering medium depends on the measurement configuration to optimize the configuration. We aim to evaluate the depth detection limit from theoretical analysis and phantom experiments and discuss the source-detector distance that maximizes this limit.

Approach: Experiments for detecting a fluorescent object in a biological tissue-mimicking phantom of ground beef with background emission were conducted using continuous wave fluorescence measurements with a point source-detector scheme. The results were analyzed using a model based on the photon diffusion equations. Then, variance analysis of the signal fluctuation was introduced.

Results: The model explained the measured fluorescence intensities and their fluctuations well. The variance analysis showed that the depth detection limit in the presence of ambient light increased with the decrease in the source-detector distance, and the optimum distance was in the range of 10 to 15 mm. The depth detection limit was found to be $\sim 30\mathrm{mm}$ with this optimum distance for the phantom.

Conclusions: The presented analysis provides a guide for the optimum design of the measurement configuration for detecting fluorescence objects in clinical applications.

Significance: Minimally invasive surgery (MIS) has shown vast improvement over open surgery by reducing post-operative stays, intraoperative blood loss, and infection rates. However, in spite of these improvements, there are still prevalent issues surrounding MIS that may be addressed through hyperspectral imaging (HSI). We present a laparoscopic HSI system to further advance the field of MIS.

Aim: We present an imaging system that integrates high-speed HSI technology with a clinical laparoscopic setup and validate the system's accuracy and functionality. Different configurations that cover the visible (VIS) to near-infrared (NIR) range of electromagnetism are assessed by gauging the spectral fidelity and spatial resolution of each hyperspectral camera.

Approach: Standard Spectralon reflectance tiles were used to provide ground truth spectral footprints to compare with those acquired by our system using the root mean squared error (RMSE). Demosaicing techniques were investigated and used to measure and improve spatial resolution, which was assessed with a USAF resolution test target. A perception-based image quality evaluator was used to assess the demosaicing techniques we developed. Two configurations of the system were developed for evaluation. The functionality of the system was investigated in a phantom study and by imaging ex vivo tissues.

Results: Multiple configurations of our system were tested, each covering different spectral ranges, including VIS (460 to 600 nm), red/NIR (RNIR) (610 to 850 nm), and NIR (665 to 950 nm). Each configuration is capable of achieving real-time imaging speeds of up to 20 frames per second. RMSE values of $3.51\pm 2.03\%$ , $3.43\pm 0.84\%$ , and 3.47% were achieved for the VIS, RNIR, and NIR systems, respectively. We obtained sub-millimeter resolution using our demosaicing techniques.

Conclusions: We developed and validated a high-speed hyperspectral laparoscopic imaging system. The HSI system can be used as an intraoperative imaging tool for tissue classification during laparoscopic surgery.

Significance: Diffuse optical modalities such as broadband near-infrared spectroscopy (bNIRS) and hyperspectral imaging (HSI) represent a promising alternative for low-cost, non-invasive, and fast monitoring of living tissue. Particularly, the possibility of extracting the molecular composition of the tissue from the optical spectra deems the spectroscopy techniques as a unique diagnostic tool.

Aim: No established method exists to streamline the inference of the biochemical composition from the optical spectrum for real-time applications such as surgical monitoring. We analyze a machine learning technique for inference of changes in the molecular composition of brain tissue.

Approach: We propose modifications to the existing learnable methodology based on the Beer-Lambert law. We evaluate the method's applicability to linear and nonlinear formulations of this physical law. The approach is tested on data obtained from the bNIRS- and HSI-based monitoring of brain tissue.

Results: The results demonstrate that the proposed method enables real-time molecular composition inference while maintaining the accuracy of traditional methods. Preliminary findings show that Beer-Lambert law-based spectral unmixing allows contrasting brain anatomy semantics such as the vessel tree and tumor area.

Conclusion: We present a data-driven technique for inferring molecular composition change from diffuse spectroscopy of brain tissue, potentially enabling intra-operative monitoring.

Significance: Developing stable, robust, and affordable tissue-mimicking phantoms is a prerequisite for any new clinical application within biomedical optics. To this end, a thorough understanding of the phantom structure and optical properties is paramount.

Aim: We characterized the structural and optical properties of PlatSil SiliGlass phantoms using experimental and numerical approaches to examine the effects of phantom microstructure on their overall optical properties.

Approach: We employed scanning electron microscope (SEM), hyperspectral imaging (HSI), and spectroscopy in combination with Mie theory modeling and inverse Monte Carlo to investigate the relationship between phantom constituent and overall phantom optical properties.

Results: SEM revealed that microspheres had a broad range of sizes with average $(13.47\pm 5.98)\mu m$ and were also aggregated, which may affect overall optical properties and warrants careful preparation to minimize these effects. Spectroscopy was used to measure pigment and SiliGlass absorption coefficient in the VIS-NIR range. Size distribution was used to calculate scattering coefficients and observe the impact of phantom microstructure on scattering properties. The results were surmised in an inverse problem solution that enabled absolute determination of component volume fractions that agree with values obtained during preparation and explained experimentally observed spectral features. HSI microscopy revealed pronounced single-scattering effects that agree with single-scattering events.

Conclusions: We show that knowledge of phantom microstructure enables absolute measurements of phantom constitution without prior calibration. Further, we show a connection across different length scales where knowledge of precise phantom component constitution can help understand macroscopically observable optical properties.

Significance: A better understanding of diffusion reflection (DR) behavior may allow it to be used for more noninvasive applications, including the development of in vivo non-damaging techniques, especially for medical topical diagnosis and treatments.

Aim: For a bilayer opaque substance where the attenuation of the upper layer is larger than the attenuation of the lower layer, the DR crossover point ( $C_p$ ) is location where the photons coming from the bottom layer start affecting the DR. We aim to study the dependency of the $C_p$ on absorption changes in different layers for constant scattering and top layer thickness.

Approach: Monolayer and bilayer optical tissue-like phantoms were prepared and measured using a DR system. The results were compared with Monte Carlo simulations.

Results: There is an agreement between the experiments and the simulations. $C_p$ correlates with the square root of the absorption coefficient ratio of the lower layer to the top layer.

Conclusion: The experimental findings support and validate the theoretical prediction describing the dependency of the $C_p$ on the square root of the ratio of the layers' absorption coefficients. In addition, a secondary breaking point is suggested to be observed experimentally at the entrance to the noise area.

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.