Two-site microendoscopic imaging probe for simultaneous three-dimensional imaging at two anatomic locations in tissues.

Systems that can image in three dimensions at cellular resolution and across different locations within an organism may enable insights into complex biological processes, such as immune responses, for which a single location measurement may be insufficient. In this Letter, we describe an in vivo two-site imaging probe (TIP) that can simultaneously image two anatomic sites with a maximum separation of a few centimeters. The TIP consists of two identical bendable graded index (GRIN) lenses and is demonstrated by a two-photon two-color fluorescence imaging system. Each GRIN lens has a field of view of 162 × 162 × 170 µm3, a nominal numerical aperture of 0.5, a magnification of 0.7, and working distances of 0.2 mm in air for both ends. A blind linear unmixing algorithm is applied to suppress bleedthrough between channels. We use this system to successfully demonstrate two-site two-photon two-color imaging of two biomedically relevant samples, i.e., (1) a mixture of two autofluorescent anti-cancer drugs and (2) a live hybrid tumor consisting of two spectrally distinct fluorescent cell lines.

Stomach tissue classification using autofluorescence spectroscopy and machine learning.

BACKGROUND AND OBJECTIVES: Determination of stomach tumor location and invasion depth requires delineation of gastric histological structure, which has hitherto been widely accomplished by histochemical staining. In recent years, alternative histochemical evaluation methods have been pursued to accelerate intraoperative diagnosis, often by bypassing the time-consuming step of dyeing. Owing to strong endogenous signals from coenzymes, metabolites, and proteins, autofluorescence spectroscopy is a favorable candidate technique to achieve this aim. MATERIALS AND METHODS: We investigated stomach tissue slices and block specimens using a fast fluorescence imaging scanner. To obtain histological information from broad and structureless fluorescence spectra, we analyzed tens of thousands of spectra with multiple machine-learning algorithms and built a tissue classification model trained with dissected gastric tissues. RESULTS: A machine-learning-based spectro-histological model was built based on the autofluorescence spectra measured from stomach tissue samples with delineated and validated histological structures. The scores from a principal components analysis were employed as input features, and prediction accuracy was confirmed to be 92.0%, 90.1%, and 91.4% for mucosa, submucosa, and muscularis propria, respectively. We investigated the tissue samples in both sliced and block forms using a fast fluorescence imaging scanner. CONCLUSION: We successfully demonstrated differentiation of multiple tissue layers of well-defined specimens with the guidance of a histologist. Our spectro-histology classification model is applicable to histological prediction for both tissue blocks and slices, even though only sliced samples were trained.

Bendable long graded index lens microendoscopy.

Graded index (GRIN) lens endoscopy has broadly benefited biomedical microscopic imaging by enabling accessibility to sites not reachable by traditional benchtop microscopes. It is a long-held notion that GRIN lenses can only be used as rigid probes, which may limit their potential for certain applications. Here, we describe bendable and long-range GRIN microimaging probes for a variety of potential micro-endoscopic biomedical applications. Using a two-photon fluorescence imaging system, we have experimentally demonstrated the feasibility of three-dimensional imaging through a 500-µm-diameter and ∼11 cm long GRIN lens subject to a cantilever beam-like deflection with a minimum bend radius of ∼25 cm. Bend-induced perturbation to the field of view and resolution has also been investigated quantitatively. Our development alters the conventional notion of GRIN lenses and enables a range of innovative applications. For example, the demonstrated flexibility is highly desirable for implementation into current and emerging minimally invasive clinical procedures, including a pioneering microdevice for high-throughput cancer drug selection.

Compact Three-Dimensional Digital Microfluidic Platforms with Programmable Contact Charge Electrophoresis Actuation.

Digital microfluidics (DMF) has garnered considerable interest as a straightforward, rapid, and programmable technique for controlling microdroplets in various biological, chemical, and medicinal research disciplines. This study details the construction of compact and low-cost 3D DMF platforms with programmable contact charge electrophoresis (CCEP) actuations by employing electrode arrays composed of a small commercial pin socket and a 3D-printed housing. We demonstrate basic 3D droplet manipulation on the platform, including horizontal and vertical transport via lifting and climbing techniques, and droplet merging. Furthermore, phenolphthalein reaction and precipitation process are evaluated using the proposed 3D DMF manipulations as a proof of concept for chemical reaction-based analysis and synthesis. The threshold voltage (or electrical field) and maximum vertical transport velocity are quantified as a function of applied voltage and electrode distance to determine the CCEP actuation conditions for 3D droplet manipulations. The ease of manufacturing and flexibility of the proposed 3D DMF platform may provide an effective technique for programmable 3D manipulation of droplets in biochemical and medical applications, such as biochemical analysis and medical diagnostics.

Dual modal spectroscopic tissue scanner for colorectal cancer diagnosis.

BACKGROUND: Margin status is an important prognostic factor for treating colorectal cancer. This study aimed to investigate the usefulness of a multimodal spectroscopic tissue scanner for real-time cancer diagnosis without tissue staining. PATIENTS AND METHODS: Diffuse reflectance spectra (DRS) and fluorescence spectra (FS) of < 1-mm-sized paired cancer and normal mucosa tissue were acquired using custom-built spectroscopic tissue scanners. For FS, we analyzed wavelengths and intensities at peaks and highest intensities near (± 1.25 nm) the known fluorescence spectral peaks of collagen (380 nm), reduced nicotinamide adenine dinucleotide (NADH, 460 nm), and flavin adenine dinucleotide (FAD, 550 nm). For DRS, we performed a similar analysis near the peaks of strong absorbers, oxyhemoglobin (oxyHb; 414 nm, 540 nm, and 576 nm) and deoxyhemoglobin (deoxyHb; 432 nm and 556 nm). Logistic regression analysis for these parameters was performed in the testing set. RESULTS: We acquired 17,735 spectra of cancer tissues and 9438 of normal tissues from 30 patients. Intensity peaks of representative normal spectra for FS and DRS were higher than those of representative cancer spectra. Logistic regression analysis showed wavelength and intensity at peaks, and the intensities of the peak wavelength of NADH, FAD, deoxyHb, and oxyHb had significant coefficients. The area under the receiver operating characteristic curve was 0.927. The scanner had 100%, 64.3%, and 85.3% sensitivity, specificity, and accuracy, respectively. CONCLUSIONS: The spectroscopic tissue scanner has high sensitivity and accuracy and provides real-time intraoperative resection margin assessments and should be further investigated as an alternative to frozen section.

A Two-Photon Microimaging-Microdevice System for Four-Dimensional Imaging of Local Drug Delivery in Tissues.

Advances in the intratumor measurement of drug responses have included a pioneering biomedical microdevice for high throughput drug screening in vivo, which was further advanced by integrating a graded-index lens based two-dimensional fluorescence micro-endoscope to monitor tissue responses in situ across time. While the previous system provided a bulk measurement of both drug delivery and tissue response from a given region of the tumor, it was incapable of visualizing drug distribution and tissue responses in a three-dimensional (3D) way, thus missing the critical relationship between drug concentration and effect. Here we demonstrate a next-generation system that couples multiplexed intratumor drug release with continuous 3D spatial imaging of the tumor microenvironment via the integration of a miniaturized two-photon micro-endoscope. This enables optical sectioning within the live tissue microenvironment to effectively profile the entire tumor region adjacent to the microdevice across time. Using this novel microimaging-microdevice (MI-MD) system, we successfully demonstrated the four-dimensional imaging (3 spatial dimensions plus time) of local drug delivery in tissue phantom and tumors. Future studies include the use of the MI-MD system for monitoring of localized intra-tissue drug release and concurrent measurement of tissue responses in live organisms, with applications to study drug resistance due to nonuniform drug distribution in tumors, or immune cell responses to anti-cancer agents.

Spectrochemical Probing of MicroRNA Duplex Using Spontaneous Raman Spectroscopy for Biosensing Applications.

MicroRNAs are emerging as both diagnostic and therapeutic targets in different human pathologies. An accurate understanding of the structural dependency of microRNAs for their biological functions is essential for designing synthetic oligos with various base and linkage modifications that can transform into highly sensitive diagnostic devices and therapeutic molecules. In this proof-of-principle study, we have utilized label-free spontaneous Raman spectroscopy to understand the structural differences in sense and antisense microRNA-21 by hybridizing them with complementary RNA and DNA oligos. Overall, the results suggest that the changes in the Raman band at 785 cm-1 originating from the phosphodiester bond of the nucleic acid backbone, linking 5' phosphate of the nucleic acid with 3' OH of the other nucleotide, can serve as a marker to identify these structural variations. Our results support the application of Raman spectroscopy in discerning intramolecular (ssRNA and ssDNA) and intermolecular (RNA-RNA, RNA-DNA, and DNA-DNA hybrids) interactions of nucleic acids. This is potentially useful for developing biosensors to quantify microRNAs in clinical samples and to design therapeutic microRNAs with robust functionality.

Analysis of subcutaneous swine fat via deep Raman spectroscopy using a fiber-optic probe.

Since the fat content of pork is a deciding factor in meat quality grading, the use of a noninvasive subcutaneous probe for real-time in situ monitoring of the fat components is of importance to vendors and other interested parties. In this work, we developed a spectroscopic method using a fiber-optic probe for subcutaneous fat analysis that utilizes spatially offset Raman spectroscopy (SORS). Here, normalized Raman spectra were acquired as a function of spatial offset, and the relative composition of fat-to-skin was determined. We found that the Raman intensity ratio varied disproportionately depending on the fat content and that the variations of the slope were correlated to the thickness of the fat layer. Furthermore, ordinary least square (OLS) regression using two components indicated that the depth-resolved SORS spectra reflected the relative thickness of the fat layer. We concluded that the local distribution of subcutaneous fat could be measured noninvasively using a pair of fiber-optic probes.

Correction to: Evaluation of accuracy dependence of Raman spectroscopic models on the ratio of calibration and validation points for non-invasive glucose sensing.

The authors would like to bring to the reader's attention that the Clarke error grid plot presented in Fig. 3 was generated using codes adapted from following reference.

In vivo detection of drug-induced apoptosis in tumors using Raman spectroscopy.

We describe a label-free approach based on Raman spectroscopy, to study drug-induced apoptosis in vivo. Spectral-shifts at wavenumbers associated with DNA, proteins, lipids, and collagen have been identified on breast and melanoma tumor tissues. These findings may enable a new analytical method for rapid readout of drug-therapy with miniaturized probes.

Evaluation of accuracy dependence of Raman spectroscopic models on the ratio of calibration and validation points for non-invasive glucose sensing.

Optical monitoring of blood glucose levels for non-invasive diagnosis is a growing area of research. Recent efforts in this direction have been inclined towards reducing the requirement of calibration framework. Here, we are presenting a systematic investigation on the influence of variation in the ratio of calibration and validation points on the prospective predictive accuracy of spectral models. A fiber-optic probe coupled Raman system has been employed for transcutaneous measurements. Limit of agreement analysis between serum and partial least square regression predicted spectroscopic glucose values has been performed for accurate comparison. Findings are suggestive of strong predictive accuracy of spectroscopic models without requiring substantive calibration measurements. Graphical abstract.

Raman Spectroscopy Differentiates Each Tissue from the Skin to the Spinal Cord: A Novel Method for Epidural Needle Placement?

BACKGROUND: Neuraxial anesthesia and epidural steroid injection techniques require precise anatomical targeting to ensure successful and safe analgesia. Previous studies suggest that only some of the tissues encountered during these procedures can be identified by spectroscopic methods, and no previous study has investigated the use of Raman, diffuse reflectance, and fluorescence spectroscopies. The authors hypothesized that real-time needle-tip spectroscopy may aid epidural needle placement and tested the ability of spectroscopy to distinguish each of the tissues in the path of neuraxial needles. METHODS: For comparison of detection methods, the spectra of individual, dissected ex vivo paravertebral and neuraxial porcine tissues were collected using Raman spectroscopy (RS), diffuse reflectance spectroscopy, and fluorescence spectroscopy. Real-time spectral guidance was tested using a 2-mm inner-diameter fiber-optic probe-in-needle device. Raman spectra were collected during the needle's passage through intact paravertebral and neuraxial porcine tissue and analyzed afterward. The RS tissue signatures were verified as mapping to individual tissue layers using histochemical staining and widefield microscopy. RESULTS: RS revealed a unique spectrum for all ex vivo paravertebral and neuraxial tissue layers; diffuse reflectance spectroscopy and fluorescence spectroscopy were not distinct for all tissues. Moreover, when accounting for the expected order of tissues, real-time Raman spectra recorded during needle insertion also permitted identification of each paravertebral and neuraxial porcine tissue. CONCLUSIONS: This study demonstrates that RS can distinguish the tissues encountered during epidural needle insertion. This technology may prove useful during needle placement by providing evidence of its anatomical localization.

A vector-free microfluidic platform for intracellular delivery.

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations, including their reliance on exogenous materials or electrical fields, which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30-80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material, such as carbon nanotubes, proteins, and siRNA, to 11 cell types, including embryonic stem cells and immune cells. When used for the delivery of transcription factors, the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed, its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications.

Investigating Effects of Proteasome Inhibitor on Multiple Myeloma Cells Using Confocal Raman Microscopy.

Due to its label-free and non-destructive nature, applications of Raman spectroscopic imaging in monitoring therapeutic responses at the cellular level are growing. We have recently developed a high-speed confocal Raman microscopy system to image living biological specimens with high spatial resolution and sensitivity. In the present study, we have applied this system to monitor the effects of Bortezomib, a proteasome inhibitor drug, on multiple myeloma cells. Cluster imaging followed by spectral profiling suggest major differences in the nuclear and cytoplasmic contents of cells due to drug treatment that can be monitored with Raman spectroscopy. Spectra were also acquired from group of cells and feasibility of discrimination among treated and untreated cells using principal component analysis (PCA) was accessed. Findings support the feasibility of Raman technologies as an alternate, novel method for monitoring live cell dynamics with minimal external perturbation.

High resolution live cell Raman imaging using subcellular organelle-targeting SERS-sensitive gold nanoparticles with highly narrow intra-nanogap.

We report a method to achieve high speed and high resolution live cell Raman images using small spherical gold nanoparticles with highly narrow intra-nanogap structures responding to NIR excitation (785 nm) and high-speed confocal Raman microscopy. The three different Raman-active molecules placed in the narrow intra-nanogap showed a strong and uniform Raman intensity in solution even under transient exposure time (10 ms) and low input power of incident laser (200 µW), which lead to obtain high-resolution single cell image within 30 s without inducing significant cell damage. The high resolution Raman image showed the distributions of gold nanoparticles for their targeted sites such as cytoplasm, mitochondria, or nucleus. The high speed Raman-based live cell imaging allowed us to monitor rapidly changing cell morphologies during cell death induced by the addition of highly toxic KCN solution to cells. These results strongly suggest that the use of SERS-active nanoparticle can greatly improve the current temporal resolution and image quality of Raman-based cell images enough to obtain the detailed cell dynamics and/or the responses of cells to potential drug molecules.

Diffraction optical tomography using a quantitative phase imaging unit.

A simple and practical method to measure three-dimensional (3-D) refractive index (RI) distributions of biological cells is presented. A common-path self-reference interferometry consisting of a compact set of polarizers is attached to a conventional inverted microscope equipped with a beam scanning unit, which can precisely measure multiple 2-D holograms of a sample with high phase stability for various illumination angles, from which accurate 3-D optical diffraction tomograms of the sample can be reconstructed. 3-D RI tomograms of nonbiological samples such as polystyrene microspheres, as well as biological samples including human red blood cells and breast cancer cells, are presented.

Depth-selective microscopic observation of a photomobile liquid crystal polymer under UV illumination.

By using the depth selective imaging method, we studied the UV induced change in a photomobile liquid crystalline polymer film. With 1 µm depth resolution, each slice inside the film was selectively observed. A network-like structure mixed with the ordered and disordered regions of molecules in the middle of the film, and a rubbed polymer layer at the bottom of the film were observed. In each slice of the film, the phase change induced by UV light was observed strongly dependent on the director direction, which indicates the ordering change of the liquid crystalline molecules in the director direction. It took several tens of seconds for the ordering change caused by the collaborative interaction between the molecules. Furthermore, it was suggested that the UV induced change travelled from the bottom layer to the middle layer on the micron order.

Shedding light on the extinction-enhancement duality in gold nanostar-enhanced Raman spectroscopy.

Surface-enhanced Raman spectroscopy (SERS) has evolved from an esoteric physical phenomenon to a robust and effective analytical method recently. The need of addressing both the field enhancement and the extinction of nanoparticle suspensions, however, has been underappreciated despite its substantive impact on the sensing performance. A systematic experimental investigation of SERS enhancement and attenuation is performed in suspensions of gold nanostars, which exhibit a markedly different behavior in relation to conventional nanoparticles. The relationship is elucidated between the SERS enhancement and the localized surface plasmon resonance band, and the effect of the concentration of the gold nanostars on the signal propagation is investigated. It is shown that an optimal concentration of gold nanostars exists to maximize the enhancement factor (EF), and the maximum EF occurs when the LSPR band is blue-shifted from the excitation wavelength rather than at the on-resonance position.

Swept-source Raman spectroscopy of chemical and biological materials.

Significance: Raman spectroscopy has been used as a powerful tool for chemical analysis, enabling the noninvasive acquisition of molecular fingerprints from various samples. Raman spectroscopy has proven to be valuable in numerous fields, including pharmaceutical, materials science, and biomedicine. Active research and development efforts are currently underway to bring this analytical instrument into the field, enabling in situ Raman measurements for a wider range of applications. Dispersive Raman spectroscopy using a fixed, narrowband source is a common method for acquiring Raman spectra. However, dispersive Raman spectroscopy requires a bulky spectrometer, which limits its field applicability. Therefore, there has been a tremendous need to develop a portable and sensitive Raman system. Aim: We developed a compact swept-source Raman (SS-Raman) spectroscopy system and proposed a signal processing method to mitigate hardware limitations. We demonstrated the capabilities of the SS-Raman spectroscopy by acquiring Raman spectra from both chemical and biological samples. These spectra were then compared with Raman spectra obtained using a conventional dispersive Raman spectroscopy system. Approach: The SS-Raman spectroscopy system used a wavelength-swept source laser (822 to 842 nm), a bandpass filter with a bandwidth of 1.5 nm, and a low-noise silicon photoreceiver. Raman spectra were acquired from various chemical samples, including phenylalanine, hydroxyapatite, glucose, and acetaminophen. A comparative analysis with the conventional dispersive Raman spectroscopy was conducted by calculating the correlation coefficients between the spectra from the SS-Raman spectroscopy and those from the conventional system. Furthermore, Raman mapping was obtained from cross-sections of swine tissue, demonstrating the applicability of the SS-Raman spectroscopy in biological samples. Results: We developed a compact SS-Raman system and validated its performance by acquiring Raman spectra from both chemical and biological materials. Our straightforward signal processing method enhanced the quality of the Raman spectra without incurring high costs. Raman spectra in the range of 900 to 1200 cm-1 were observed for phenylalanine, hydroxyapatite, glucose, and acetaminophen. The results were validated with correlation coefficients of 0.88, 0.84, 0.87, and 0.73, respectively, compared with those obtained from dispersive Raman spectroscopy. Furthermore, we performed scans across the cross-section of swine tissue to generate a biological tissue mapping plot, providing information about the composition of swine tissue. Conclusions: We demonstrate the capabilities of the proposed compact SS-Raman spectroscopy system by obtaining Raman spectra of chemical and biological materials, utilizing straightforward signal processing. We anticipate that the SS-Raman spectroscopy will be utilized in various fields, including biomedical and chemical applications.

Multiphoton imaging of the monosachharide induced formation of fluorescent advanced glycation end products in tissues.

We studied the in vitro rate of fluorescent advanced glycation end products (fAGEs) formation with multiphoton microscopy in different porcine tissues (aorta, cornea, kidney, dermis, and tendon). These tissues were treated with d-glucose, d-galactose, and d-fructose, three primary monosaccharides found in human diets. We found that the use of d-fructose resulted in the highest glycation rate, followed by d-galactose and then d-glucose. Moreover, compared to non-collagen tissue constituents such as elastic fibers and cells, the rate of tissue glycation was consistently higher in collagen, suggesting that collagen is a more sensitive target for fAGE formation. However, we also found that collagen in different tissues exhibits different rates of fAGE formation, with slower rates observed in tightly packed tissues such as cornea and tendon. Our study suggests that for fAGE to be developed into a long-term glycemic biomarker, loosely organized collagen tissues located in the proximity of vasculature may be the best targets.