Thick Biological Tissues Research Articles

Slicing Network for Wide-Field Fluorescence Image Based on the Improved U-Net Model.

Fluorescence imaging stands as a pivotal component in biomedical research, requiring the elimination of out-of-focus background noise resulting from wide-field volumetric illumination of the whole field-of-view and scattering within thick biological tissues. Traditional methods struggle to effectively address varying degrees of defocusing in fluorescence images. This study introduces the utilization of upU-Net, 3D U-Net, and 3D upU-Net as defocusing networks tailored for 2D and 3D wide-field fluorescence images, yielding notable enhancements. These advancements facilitate more economically viable confocal microscopy, delivering significant advantages to biologists presently utilizing wide-field fluorescence microscopy.

Construction of vector vortex beams on hybrid-order Poincaré sphere through highly scattering media

Vector vortex beams (VVBs) have attracted extensive attention due to their unique properties and their wide applications in fields such as optical manipulation and optical imaging. However, the wavefronts of the vector vortex beams are highly scrambled when they encounter highly scattering media (HSM), such as thick biological tissues, which greatly prevents the applications of VVBs behind HSM. To address this issue, we propose a scheme to construct VVBs of freewill position on the surface of hybrid-order Poincaré sphere (HyOPS) through HSM. With the measurement of two orthogonal scalar transmission matrices, the conjugated wavefronts for constructing orbital angular momentum beams with arbitrary topological charge in right and left circularly polarized states through HSM can be calculated, respectively. When an input wavefront superimposed by the two conjugated wavefronts with an appropriate ratio and phase delay, impinges on the HSM, the desired VVB can be created through HSM. To demonstrate the viability of our scheme, a series of VVBs on different locations of various HyOPSs have been reconstructed through a ZnO scattering layer experimentally. Furthermore, to characterize the polarization distribution of the generated beams, the polarization maps of these beams are derived by measuring the four Stokes parameters, which agree well with the theoretical distributions. This work will promote the applications of VVBs in highly scattering environments.

Expanding the Palette of SWIR Emitting Nanoparticles Based on Au Nanoclusters for Single-Particle Tracking Microscopy.

Single-molecule localization microscopy has proved promising to unravel the dynamics and molecular architecture of thin biological samples down to nanoscales. For applications in complex, thick biological tissues shifting single-particle emission wavelengths to the shortwave infrared (SWIR also called NIR II) region between 900 to 2100nm, where biological tissues are more transparent is key. To date, mainly single-walled carbon nanotubes (SWCNTs) enable such applications, but they are inherently 1D objects. Here, 0D ultra-small luminescent gold nanoclusters (AuNCs, <3nm) and ≈25nm AuNC-loaded-polymeric particles that can be detected at the single-particle level in the SWIR are presented. Thanks to high brightness and excellent photostability, it is shown that the dynamics of the spherical polymeric particles can be followed at the single-particle level in solution at video rates for minutes. We compared single particle tracking of AuNC-loaded-polymeric particles with that of SWCNT diffusing in agarose gels demonstrating the specificity and complementarity of diffusion properties of these SWIR-emitting nano-objects when exploring a complex environment. This extends the library of photostable SWIR emitting nanomaterials to 0D nano-objects of variable size for single-molecule localization microscopy in the second biological window, opening unprecedented possibilities for mapping the structure and dynamics of complex biological systems.

Time domain diffuse Raman spectroscopy using single pixel detection.

Diffuse Raman spectroscopy (DIRS) extends the high chemical specificity of Raman scattering to in-depth investigation of thick biological tissues. We present here a novel approach for time-domain diffuse Raman spectroscopy (TD-DIRS) based on a single-pixel detector and a digital micromirror device (DMD) within an imaging spectrometer for wavelength encoding. This overcomes the intrinsic complexity and high cost of detection arrays with ps-resolving time capability. Unlike spatially offset Raman spectroscopy (SORS) or frequency offset Raman spectroscopy (FORS), TD-DIRS exploits the time-of-flight distribution of photons to probe the depth of the Raman signal at a single wavelength with a single source-detector separation. We validated the system using a bilayer tissue-bone mimicking phantom composed of a 1 cm thick slab of silicone overlaying a calcium carbonate specimen and demonstrated a high differentiation of the two Raman signals. We reconstructed the Raman spectra of the two layers, offering the potential for improved and quantitative material analysis. Using a bilayer phantom made of porcine muscle and calcium carbonate, we proved that our system can retrieve Raman peaks even in the presence of autofluorescence typical of biomedical tissues. Overall, our novel TD-DIRS setup proposes a cost-effective and high-performance approach for in-depth Raman spectroscopy in diffusive media.

Locating Three-Dimensional Position of Deep-Seated SERS Phantom Lesions in Thick Tissues Using Tomographic Transmission Raman Spectroscopy.

Locating distinct objects within a thick scattering medium remains a long-standing challenge in the fields of materials science, health, and engineering. Transmission Raman spectroscopy (TRS) with the use of surface-enhanced Raman scattering (SERS) nanoparticles has proven to be an effective approach to detect deep-seated lesions inside thick biological tissues. However, it has not yet been proven to spatially locate deep lesions in three dimensions using optical modalities. Herein, we present the concept of tomographic TRS and report its successful use for accurately locating SERS nanoparticles in elongated rod-like thick tissues. Our work starts with theoretical simulations of Raman photon propagation in tissues. We discovered a linear relationship between the Raman spectral peak ratio and propagation distance of Raman photons in tissues, allowing us to predict the location of lesions tagged by SERS NPs. Based on this, we propose a two-step tomographic TRS strategy, which includes axial scanning and ring scanning. We demonstrate the robustness of our approach using ex vivo thick tissue (4.5 cm in thickness) and locate an embedded SERS phantom lesion, with a ring scanning step of 10-30°. We successfully locate multiple SERS phantom lesions in the ex vivo porcine muscle stack with high accuracy (absolute error of <2 mm). Our method is rapid, efficient, and of low cost compared to current tomographic medical imaging techniques. This work advances Raman techniques for three-dimensional positioning and offers new insights toward practical diagnosis applications.

Expandable ELAST for super-resolution imaging of thick tissue slices using a hydrogel containing charged monomers

Hydrogels have been utilized extensively as a material for retaining position information in tissue imaging procedures, such as tissue clearing and super-resolution imaging. Immunostaining thick biological tissues, however, poses a bottleneck that restricts sample size. The recently developed technique known as entangled link-augmented stretchable tissue-hydrogel (ELAST) accelerates the immunostaining process by embedding specimens in long-chain polymers and stretching them. A more advanced version of ELAST, magnifiable entangled link-augmented stretchable tissue-hydrogel (mELAST), achieves rapid immunostaining and tissue expansion by embedding specimens in long-chain neutral polymers and subsequently hydrolyzing them. Building on these techniques, we introduce a variant of mELAST called ExELAST. This approach uses charged monomers to stretch and expand tissue slices. Using ExELAST, we first tested two hydrogel compositions that could permit uniform expansion of biological specimens. Then, we apply the tailored hydrogel to the 500-μm-thick mouse brain slices and demonstrated that they can be stained within two days and imaged with a resolution below the diffraction limit of light.

A rapid and universal method for depth estimation of lesions in heterogeneous tissues via photosafe ratiometric transmission Raman spectroscopy

AbstractNoninvasive detection of deep lesions remains a long‐standing goal for clinical applications, with depth estimation of a single lesion in heterogeneous tissues being a key challenge. Currently, optical techniques are widely applied for lesion detection, but they face difficulties in achieving rapid and precise lesion depth estimation, particularly when the lesions are buried in thick heterogeneous tissues and the applied irradiance is below clinically maximum permissible exposure. Herein, we theoretically and experimentally demonstrate a universal method for depth prediction of phantom lesions labeled with surface‐enhanced Raman scattering nanotags in thick biological tissues using ratiometric transmission Raman spectroscopy (TRS). We begin by highlighting the linear relationship between the natural logarithm of Raman peak‐to‐peak ratio and the lesion depth and establishing a home‐built TRS system with the clinically safe irradiance. We achieve an accurate depth prediction for phantom lesions hidden in 6‐cm‐thick ex vivo homogeneous tissue with a root mean squared error (RMSE) as low as 2.42%. Additionally, we predict the depth of phantom lesions buried in 5‐cm‐thick ex vivo heterogeneous tissues with an RMSE of down to 8.35%. We also demonstrate the applicability of this method theoretically for highly heterogeneous tissues such as complex in vivo environments. This work provides a rapid, robust, and universal method to estimate the depth of lesions in complex biological samples, demonstrating the potential of ratiometric Raman spectroscopy for lesion localization in clinical applications.

Probing the deep brain: Enhanced multi-photon imaging by aggregation-induced emission luminogens via nanocrystallization

The latest requirement of optical imaging is to visualize vital, thick, and complex biological tissues in real-time and minimum-invasive mode. Among diverse available diagnostic tools, multi-photon imaging (MPI) with long wavelength excitation and exceptional lateral resolution has been a perfect option. However, the existing palette of MPI contrast agents either suffers from sub-optimal imaging performance or non-negligible risk in biosafety, exploration of suitable probes is thus of great significance. To address these concerns, in this work, pure organic nanocrystals (NCs) were developed based on aggregation-induced emission luminogens (AIEgens) (DPTA-BT) with an ultrasound-assisted method. Compared to the amorphous DPTA-BT nanoparticles (NPs), NCs with orderly arranged molecules exhibited highly enhanced nonlinear optical signals, including up to 15.35 folds of two-photon action cross-section (2PACS) and up to 4.11 folds of three-photon action cross-section (3PACS). The combination of MPI and DPTA-BT NCs has contributed to a 1900 μm imaging depth of vasculature after craniotomy and a 765 μm imaging depth through the intact skull. The safe use of DPTA-BT NCs was verified by the in vitro and in vivo biosafety assessment. This work is thus expected to encourage more innovation in the development of organic contrast agents for MPI.

Volumetric light sheet imaging with adaptive optics correction.

Light sheet microscopy has developed quickly over the past decades and become a popular method for imaging live model organisms and other thick biological tissues. For rapid volumetric imaging, an electrically tunable lens can be used to rapidly change the imaging plane in the sample. For larger fields of view and higher NA objectives, the electrically tunable lens introduces aberrations in the system, particularly away from the nominal focus and off-axis. Here, we describe a system that employs an electrically tunable lens and adaptive optics to image over a volume of 499 × 499 × 192 μm3 with close to diffraction-limited resolution. Compared to the system without adaptive optics, the performance shows an increase in signal to background ratio by a factor of 3.5. While the system currently requires 7s/volume, it should be straightforward to increase the imaging speed to under 1s per volume.

Reflectional quantitative differential phase microscopy using polarized wavefront phase modulation.

Quantitative phase microscopy (QPM), as a label-free and nondestructive technique, has been playing an indispensable tool in biomedical imaging and industrial inspection. Herein, we introduce a reflectional quantitative differential phase microscopy (termed RQDPM) based on polarized wavefront phase modulation and partially coherent full-aperture illumination, which has high spatial resolution and spatio-temporal phase sensitivity and is applicable to opaque surfaces and turbid biological specimens. RQDPM does not require additional polarized devices and can be easily switched from reflectional mode to transmission mode. In addition, RQDPM inherits the characteristic of high axial resolution of differential interference contrast microscope, thereby providing topography for opaque surfaces. We experimentally demonstrate the reflectional phase imaging ability of RQDPM with several samples: semiconductor wafer, thick biological tissues, red blood cells, and Hela cells. Furthermore, we dynamically monitor the flow state of microspheres in a self-built microfluidic channel by using RQDPM converted into the transmission mode.

Digging Deeper through Biological Specimens Using Adaptive Optics-Based Optical Microscopy

Optical microscopy is a vital tool for visualizing the cellular and sub-cellular structures of biological specimens. However, due to its limited penetration depth, its biological applicability has been hindered. The scattering and absorption of light by a wide array of biomolecules causes signal attenuation and restricted imaging depth in tissues. Researchers have put forth various approaches to address this, including designing novel probes for imaging applications and introducing adaptive optics (AO) technology. Various techniques, such as direct wavefront sensing to quickly detect and fix wavefront deformation and indirect wavefront sensing using modal and zonal methods to rectify complex aberrations, have been developed through AO paradigms. In addition, algorithmic post-processing without mechanical feedback has been utilized to correct the optical patterns using the matrix-based method. Hence, reliable optical imaging through thick biological tissue is made possible by sensorless AO. This review highlights the latest advancements in various AO-based optical microscopy techniques for depth-resolved imaging and briefly discusses their potential in various biomedical applications.

Confocal rescan structured illumination microscopy for real-time deep tissue imaging with superresolution

Structured illumination microscopy (SIM) is an established optical superresolution imaging technique. However, conventional SIM based on wide-field image acquisition is generally limited to visualizing thin cellular samples. We propose combining one-dimensional image rescan and structured illumination in the orthogonal direction to achieve superresolution without the need to rotate the illumination pattern. The image acquisition speed is consequently improved threefold, which is also beneficial for minimizing photobleaching and phototoxicity. Optical sectioning in thick biological tissue is enhanced by including a confocal slit in the system to significantly suppress the out-of-focus background and the associated noise. With all the technical improvements, our method captures three-dimensional superresolved image stacks of neuronal structures in mouse brain tissue samples for a depth range of more than 200 μm.

Creation of cylindrical vector beams through highly anisotropic scattering media with a single scalar transmission matrix calibration

Cylindrical vector (CV) beams have attracted increasing interest due to their particular properties and their applications in optical imaging, optical manipulation, and light–matter interactions. However, it is challenging to construct CV beams through highly anisotropic scattering media (HASM), such as thick biological tissue, posing a barrier to the applications of CV beams that involve HASM. Here, we present a scheme to construct CV beams beyond high scattering that only requires a single scalar transmission matrix (TM) calibration and manipulation of the spatial degrees of freedom of the scalar input field. Assisted by a radial polarization converter (S-waveplate) and a polarizer, the scheme enables one to obtain the correct incident wavefront for the creation of CV beams through HASM with only one single scalar TM calibration. Compared to the existing method, this user-friendly approach is fast and simple in terms of the optical implements and computations. Both radially and azimuthally polarized beams are experimentally constructed through a ZnO scattering layer to demonstrate the viability of the method. Arbitrarily generalized CV beams and arrays of CV beams are also created through the HASM to further prove the flexibility of the method. We believe this work may pave the way for applications of CV beams that involve a highly anisotropic scattering environment.

Accurate optical information transmission through thick tissues using zero-frequency modulation and single-pixel detection

Delivering accurate information through thick biological tissues is critical in many biomedical applications. However, it is challenging to realize accurate optical information transmission and achieve large penetration depth due to complex structures and compositions of thick biological tissues. The absorption and scattering through biotissues are inter-related, and could cause light attenuation dramatically. In this paper, a new method based on zero-frequency modulation (ZFM) is proposed to generate a series of 2D random amplitude-only patterns for accurate optical information transmission through thick tissues at low light intensities. Light source modulated by the generated 2D patterns propagates through biological tissues, and a single-pixel bucket detector is used to record light intensity with a differential detection technique. Different sample thicknesses and the light source with different wavelengths are used to experimentally verify the proposed method. The proposed method can realize accurate optical information transmission through biological tissues with a thickness of 16.0 mm, when a low laser power of only 0.08 mW/cm2 is used to illuminate the sample at wavelength of 658.0 nm. The proposed method realizes accurate optical information transmission through thick biological tissues, and is able to overcome the challenges, e.g., penetration through small-thickness tissues and low quality of the retrieved signals. The proposed method provides a promising means for optical data transmission through deep tissues.

Comparison of fluorescence lifetime and multispectral imaging for quantitative multiplexing in biological tissue.

Fluorescence lifetime (FLT) multiplexing and multispectral imaging (MSI) are both frequently employed for in vitro and ex vivo biological studies. In vivo applications of MSI for deep seated fluorophores require consideration of diffusive light propagation in biological tissue. We have previously shown that a well-known redshift of fluorescence spectra in diffusive medium induces a fluorophore cross-talk, which cannot be accounted for even with known optical properties of the medium. In contrast, FLT measurements remain largely unaffected by light propagation in tissue, enabling zero cross-talk and accurate relative quantification. While a fully quantitative estimation of fluorophore concentrations requires depth resolved tomographic imaging, this is often not possible due to the difficulty of estimating tissue optical properties and modelling light propagation in complex tissue geometries. Here, we experimentally investigate the performance of planar (non-tomographic) MSI and FLT multiplexing for the quantitative recovery of multiple near-infrared fluorophores embedded in 4-8 mm thick tissue. We show that FLT multiplexing provides a superior quantification accuracy (error < 10%) compared to MSI (error = 20-107%) in tissue. The error rates for MSI increased with tissue thickness and can be directly attributed to the spectral redshift induced cross-talk between emission spectra. Our data indicate that planar FLT multiplexing can provide high quantification accuracy in thick biological tissue without a need for optical property estimation, thereby offering an important validation tool for rapid quantification of fluorophore concentrations in bulk tissue.

Multi-Color Two-Photon Microscopic Imaging Based on a Single-Wavelength Excitation.

Two-photon probes with broad absorption spectra are beneficial for multi-color two-photon microscopy imaging, which is one of the most powerful tools to study the dynamic processes of living cells. To achieve multi-color two-photon imaging, multiple lasers and detectors are usually required for excitation and signal collection, respectively. However, one makes the imaging system more complicated and costly. Here, we demonstrate a multi-color two-photon imaging method with a single-wavelength excitation by using a signal separation strategy. The method can effectively solve the problem of spectral crosstalk by selecting a suitable filter combination and applying image subtraction. The experimental results show that the two-color and three-color two-photon imaging are achieved with a single femtosecond laser. Furthermore, this method can also be combined with multi-photon imaging technology to reveal more information and interaction in thick biological tissues.

First Clinical Results of Fluorescence Lifetime-enhanced Tumor Imaging Using Receptor-targeted Fluorescent Probes.

Fluorescence molecular imaging, using cancer-targeted near infrared (NIR) fluorescent probes, offers the promise of accurate tumor delineation during surgeries and the detection of cancer specific molecular expression in vivo. However, nonspecific probe accumulation in normal tissue results in poor tumor fluorescence contrast, precluding widespread clinical adoption of novel imaging agents. Here we present the first clinical evidence that fluorescence lifetime (FLT) imaging can provide tumor specificity at the cellular level in patients systemically injected with panitumumab-IRDye800CW, an EGFR-targeted NIR fluorescent probe. We performed wide-field and microscopic FLT imaging of resection specimens from patients injected with panitumumab-IRDye800CW under an FDA directed clinical trial. We show that the FLT within EGFR-overexpressing cancer cells is significantly longer than the FLT of normal tissue, providing high sensitivity (>98%) and specificity (>98%) for tumor versus normal tissue classification, despite the presence of significant nonspecific probe accumulation. We further show microscopic evidence that the mean tissue FLT is spatially correlated (r > 0.85) with tumor-specific EGFR expression in tissue and is consistent across multiple patients. These tumor cell-specific FLT changes can be detected through thick biological tissue, allowing highly specific tumor detection and noninvasive monitoring of tumor EFGR expression in vivo. Our data indicate that FLT imaging is a promising approach for enhancing tumor contrast using an antibody-targeted NIR probe with a proven safety profile in humans, suggesting a strong potential for clinical applications in image guided surgery, cancer diagnostics, and staging.

Analysis of the photoacoustic spectral dispersion in dielectric colloids

The foremost advanced photoacoustic (PA) transport theory, dealing with image formation, relates to target volumes of shallow depths. It assumes the medium as homogeneous with negligible PA amplitude attenuation. Causal changes in the velocity distribution spectrum, as described in Debye’s theory, and related to propagation distance and sample's density, are also neglected. However, these are relevant for imaging targets at larger depths and improving image resolution of PA images of thick biological tissues. Here we introduce some concepts for extending the PA transport model. These are theoretical and experimental considerations for analysing PA attenuation and the significance of spectral dispersion; and in consequence, disclose those conditions at which they should be included as part of the PA transport theory. Departing from the PA Heaviside telegraph equation and causality conditions, we obtain analytic expressions for associated attenuation and dispersion coefficients. As part of the analysis, we propose expressions for the PA group and PA phase velocities, and for the group velocity dispersion parameter; those in analogy with optical fields. In this way we get a refined description for the spectral dispersion. As proof of consistency, the introduced expressions are tested with experimental data extracted from homogeneous colloid samples. The observed performance is compared against already known general acoustic dispersion theory.

Witnessing the survival of time-energy entanglement through biological tissue and scattering media.

We demonstrate the preservation of the time-energy entanglement of near-IR photons through thick biological media (≤1.55 mm) and tissue (≤ 235 μm) at room temperature. Using a Franson-type interferometer, we demonstrate interferometric contrast of over 0.9 in skim milk, 2% milk, and chicken tissue. This work supports the many proposed opportunities for nonclassical light in biological imaging and analyses from sub-shot noise measurements to entanglement-enhanced fluorescence imaging, clearly indicating that the entanglement characteristics of photons can be maintained even after propagation through thick, turbid biological samples.

Erbium-doped tungsten selenide nanosheets with near-infrared II emission and photothermal conversion

Two-dimensional transition metal dichalcogenides (2D TMDs) have drawn considerable attention due to their promising applications in biomedical and optoelectronic areas. However, the emission of 2D TMDs materials and devices is mostly limited from visible to the edge of near-infrared (NIR), which restricts their wide applications. Herein, we develop Er doped WSe2 nanosheets through cation exchange and ultrasonication-assisted liquid-phase exfoliation. Thanks to the Er ions, the luminescence of 2D nanosheets can be extended to NIR II window. The emission at NIR II range can effectively penetrate several centimeters thick biological tissue. Furthermore, a series of photothermal experiments is also performed with 808 nm laser. The photothermal conversion efficiency of as-produced nanosheets is determined to be as high as 35.2%, which is greatly higher than many previous reports. It suggests that as-prepared nanosheets combining NIR II emission and highly effective photothermal conversion property may be promising as a bifunctional agent for bioimaging and photothermal therapy.