Functional Time Domain Diffuse Correlation Spectroscopy.

Time-domain diffuse correlation spectroscopy (TD-DCS) offers a novel approach to high-spatial resolution functional brain imaging based on the direct quantification of cerebral blood flow (CBF) changes in response to neural activity. However, the signal-to-noise ratio (SNR) offered by previous TD-DCS instruments remains a challenge to achieving the high temporal resolution needed to resolve perfusion changes during functional measurements. Here we present a next-generation optimized functional TD-DCS system that combines a custom 1,064 nm pulse-shaped, quasi transform-limited, amplified laser source with a high-resolution time-tagging system and superconducting nanowire single-photon detectors (SNSPDs). System characterization and optimization was conducted on homogenous and two-layer intralipid phantoms before performing functional CBF measurements in six human subjects. By acquiring CBF signals at over 5 Hz for a late gate start time of the temporal point spread function (TPSF) at 15 mm source-detector separation, we demonstrate for the first time the measurement of blood flow responses to breath-holding and functional tasks using TD-DCS.

Temporal Imaging of Live Cells by High-Speed Confocal Raman Microscopy.

Label-free live cell imaging was performed using a custom-built high-speed confocal Raman microscopy system. For various cell types, cell-intrinsic Raman bands were monitored. The high-resolution temporal Raman images clearly delineated the intracellular distribution of biologically important molecules such as protein, lipid, and DNA. Furthermore, optical phase delay measured using quantitative phase microscopy shows similarity with the image reconstructed from the protein Raman peak. This reported work demonstrates that Raman imaging is a powerful label-free technique for studying various biomedical problems in vitro with minimal sample preparation and external perturbation to the cellular system.

Diffuse correlation spectroscopy measurements of blood flow using 1064 nm light.

SIGNIFICANCE: Diffuse correlation spectroscopy (DCS) is an established optical modality that enables noninvasive measurements of blood flow in deep tissue by quantifying the temporal light intensity fluctuations generated by dynamic scattering of moving red blood cells. Compared with near-infrared spectroscopy, DCS is hampered by a limited signal-to-noise ratio (SNR) due to the need to use small detection apertures to preserve speckle contrast. However, DCS is a dynamic light scattering technique and does not rely on hemoglobin contrast; thus, there are significant SNR advantages to using longer wavelengths (>1000 nm) for the DCS measurement due to a variety of biophysical and regulatory factors. AIM: We offer a quantitative assessment of the benefits and challenges of operating DCS at 1064 nm versus the typical 765 to 850 nm wavelength through simulations and experimental demonstrations. APPROACH: We evaluate the photon budget, depth sensitivity, and SNR for detecting blood flow changes using numerical simulations. We discuss continuous wave (CW) and time-domain (TD) DCS hardware considerations for 1064 nm operation. We report proof-of-concept measurements in tissue-like phantoms and healthy adult volunteers. RESULTS: DCS at 1064 nm offers higher intrinsic sensitivity to deep tissue compared with DCS measurements at the typically used wavelength range (765 to 850 nm) due to increased photon counts and a slower autocorrelation decay. These advantages are explored using simulations and are demonstrated using phantom and in vivo measurements. We show the first high-speed (cardiac pulsation-resolved), high-SNR measurements at large source-detector separation (3 cm) for CW-DCS and late temporal gates (1 ns) for TD-DCS. CONCLUSIONS: DCS at 1064 nm offers a leap forward in the ability to monitor deep tissue blood flow and could be especially useful in increasing the reliability of cerebral blood flow monitoring in adults.

Label-free spectrochemical probe for determination of hemoglobin glycation in clinical blood samples.

Glycated hemoglobin, HbA1c, is an important biomarker that reveals the average value of blood glucose over the preceding 3 months. While significant recent attention has been focused on the use of optical and direct molecular spectroscopic methods for determination of HbA1c, a facile test that minimizes sample preparation needs and turnaround time still remains elusive. Here, we report a label-free approach for identifying low, mid and high-HbA1c groups in hemolysate and in whole blood samples featuring resonance Raman (RR) spectroscopy and support vector machine (SVM)-based classification of spectral patterns. The diagnostic power of RR measurements stems from its selective enhancement of hemoglobin-specific features, which simultaneously minimizes the blood matrix spectral interference and permits detection in the native solution. In this pilot study, our spectroscopic observations reveal that glycation of hemoglobin results in subtle but reproducible changes even when detected in the whole blood matrix. Leveraging SVM analysis of the principal component scores determined from the RR spectra, we show high degree of accuracy in classifying clinical specimen. We envisage that the promising findings will pave the way for more extensive clinical specimen investigations with the ultimate goal of translating molecular spectroscopy for routine point-of-care testing.

Large population cell characterization using quantitative phase cytometer.

A major challenge in cellular analysis is the phenotypic characterization of large cell populations within a short period of time. Among various parameters for cell characterization, the cell dry mass is often used to describe cell size but is difficult to be measured directly with traditional techniques. Here, we propose an interferometric approach based on line-focused beam illumination for high-content precision dry mass measurements of adherent cells in a non-invasive fashion-we call it quantitative phase cytometry (QPC). Besides dry mass, abundant cellular morphological features such as projected area, sphericity, and phase skewness can be readily extracted from the QPC interferometric data. To validate the utility of our technique, we demonstrate characterizing a large population of ∼104 HeLa cells. Our reported QPC system is envisioned as a promising quantitative tool for label-free characterization of a large cell count at single cell resolution. © 2017 International Society for Advancement of Cytometry.

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.

Scanning color optical tomography (SCOT).

We have developed an interferometric optical microscope that provides three-dimensional refractive index map of a specimen by scanning the color of three illumination beams. Our design of the interferometer allows for simultaneous measurement of the scattered fields (both amplitude and phase) of such a complex input beam. By obviating the need for mechanical scanning of the illumination beam or detection objective lens; the proposed method can increase the speed of the optical tomography by orders of magnitude. We demonstrate our method using polystyrene beads of known refractive index value and live cells.

Three-Dimensional Holographic Refractive-Index Measurement of Continuously Flowing Cells in a Microfluidic Channel.

Refractive index of biological specimens is a source of intrinsic contrast that can be explored without any concerns of photobleaching or harmful effects caused by extra contrast agents. In addition, RI contains rich information related to the metabolism of cells at the cellular and subcellular levels. Here, we report a no-moving parts approach that provides three-dimensional refractive index maps of biological samples continuously flowing in a microfluidic channel. Specifically, we use line illumination and off-axis digital holography to record the angular spectra of light scattered from flowing samples at high speed. Applying the scalar diffraction theory, we obtain accurate RI maps of the samples from the measured spectra. Using this method, we demonstrate label-free 3-D imaging of live RKO human colon cancer cells and RPMI8226 multiple myeloma cells, and obtain the volume, dry mass and density of these cells from the measured 3-D refractive index maps. Our results show that the reported method, alone or in combination with the existing flow cytometry techniques, promises as a quantitative tool for stain-free characterization of large number of cells.

Measurement of the time-resolved reflection matrix for enhancing light energy delivery into a scattering medium.

Multiple scatterings occurring in a turbid medium attenuate the intensity of propagating waves. Here, we propose a method to efficiently deliver light energy to the desired target depth in a scattering medium. We measure the time-resolved reflection matrix of a scattering medium using coherent time-gated detection. From this matrix, we derive and experimentally implement an incident wave pattern that optimizes the detected signal corresponding to a specific arrival time. This leads to enhanced light delivery at the target depth. The proposed method will lay a foundation for efficient phototherapy and deep-tissue in vivo imaging in the near future.

Stain-free quantification of chromosomes in live cells using regularized tomographic phase microscopy.

Refractive index imaging is a label-free technique that enables long-term monitoring of the internal structures and molecular composition in living cells with minimal perturbation. Existing tomographic methods for the refractive index imaging lack 3-D resolution and result in artifacts that prevent accurate refractive index quantification. To overcome these limitations without compromising the capability to observe a sample in its most native condition, we have developed a regularized tomographic phase microscope (RTPM) enabling accurate refractive index imaging of organelles inside intact cells. With the enhanced accuracy, we quantify the mass of chromosomes in intact living cells, and differentiate two human colon cancer lines, HT-29 and T84 cells, solely based on the non-aqueous (dry) mass of chromosomes. In addition, we demonstrate chromosomal imaging using a dual-wavelength RTPM, which shows its potential to determine the molecular composition of cellular organelles in live cells.

Measuring uptake dynamics of multiple identifiable carbon nanotube species via high-speed confocal Raman imaging of live cells.

Carbon nanotube uptake was measured via high-speed confocal Raman imaging in live cells. Spatial and temporal tracking of two cell-intrinsic and nine nanotube-derived Raman bands was conducted simultaneously in RAW 264.7 macrophages. Movies resolved single (n, m) species, defects, and aggregation states of nanotubes transiently as well as the cell position, denoted by lipid and protein signals. This work portends the real-time molecular imaging of live cells and tissues using Raman spectroscopy, affording multiplexing and complete photostability.

Single-shot quantitative dispersion phase microscopy.

We present an imaging modality capable of providing high-speed optical dispersion measurements of live cells. The technique permits wide-field measurement of the optical phase shifts introduced by a sample for multiple discrete wavelengths in a single image capture. Utilizing spatial modulation and the wavelength dependence of the interference-fringe spacing, average refractive index as a function of wavelength is obtained, yielding optical dispersion measurements of the sample under observation. Because of its simple and low-cost design, the technique can be readily integrated into a standard microscope to collect additional diagnostic information about biological cells.

Portable optical fiber probe-based spectroscopic scanner for rapid cancer diagnosis: a new tool for intraoperative margin assessment.

There continues to be a significant clinical need for rapid and reliable intraoperative margin assessment during cancer surgery. Here we describe a portable, quantitative, optical fiber probe-based, spectroscopic tissue scanner designed for intraoperative diagnostic imaging of surgical margins, which we tested in a proof of concept study in human tissue for breast cancer diagnosis. The tissue scanner combines both diffuse reflectance spectroscopy (DRS) and intrinsic fluorescence spectroscopy (IFS), and has hyperspectral imaging capability, acquiring full DRS and IFS spectra for each scanned image pixel. Modeling of the DRS and IFS spectra yields quantitative parameters that reflect the metabolic, biochemical and morphological state of tissue, which are translated into disease diagnosis. The tissue scanner has high spatial resolution (0.25 mm) over a wide field of view (10 cm × 10 cm), and both high spectral resolution (2 nm) and high spectral contrast, readily distinguishing tissues with widely varying optical properties (bone, skeletal muscle, fat and connective tissue). Tissue-simulating phantom experiments confirm that the tissue scanner can quantitatively measure spectral parameters, such as hemoglobin concentration, in a physiologically relevant range with a high degree of accuracy (<5% error). Finally, studies using human breast tissues showed that the tissue scanner can detect small foci of breast cancer in a background of normal breast tissue. This tissue scanner is simpler in design, images a larger field of view at higher resolution and provides a more physically meaningful tissue diagnosis than other spectroscopic imaging systems currently reported in literatures. We believe this spectroscopic tissue scanner can provide real-time, comprehensive diagnostic imaging of surgical margins in excised tissues, overcoming the sampling limitation in current histopathology margin assessment. As such it is a significant step in the development of a platform technology for intraoperative management of cancer, a clinical problem that has been inadequately addressed to date.

Fluorescence-guided optical coherence tomography imaging for colon cancer screening: a preliminary mouse study.

A new concept for cancer screening has been preliminarily investigated. A cancer targeting agent loaded with a near-infrared (NIR) dye was topically applied on the tissue to highlight cancer-suspect locations and guide optical coherence tomography (OCT) imaging, which was used to further investigate tissue morphology at the micron scale. A pilot study on ApcMin mice has been performed to preliminarily test this new cancer screening approach. As a cancer-targeting agent, poly(epsilon-caprolactone) microparticles (PCLMPs), labeled with a NIR dye and functionalized with an RGD (argenine-glycine-aspartic acid) peptide, were used. This agent recognizes the α(ν)ß(3) integrin receptor (ABIR), which is over-expressed by epithelial cancer cells. The contrast agent was administered topically in vivo in mouse colon. After incubation, the animals were sacrificed and fluorescence-guided high resolution optical coherence tomography (OCT) imaging was used to visualize colon morphology. The preliminary results show preferential staining of the abnormal tissue, as indicated by both microscopy and laser-induced fluorescence imaging, and OCT's capability to differentiate between normal mucosal areas, early dysplasia, and adenocarcinoma. Although very preliminary, the results of this study suggest that fluorescence-guided OCT imaging might be a suitable approach for cancer screening. If successful, this approach could be used by clinicians to more reliably diagnose early stage cancers in vivo.

Near-Common-Path Self-Reference Quantitative Phase Microscopy.

We report a near-common-path self-reference quantitative phase microscope, wherein a quantitative phase image is formed through the off-axis interference of the sample wave with a 180-degree rotated version of itself. A pair of dove prisms, oriented in different directions, is used to effect the relative transformation between the beams. Our technique features a simple optical design that requires no maintenance, rendering it accessible to nonspecialists in the field of optics. Additionally, its variable magnification and low-noise phase measurement capabilities make it suitable for high-accuracy imaging of biological samples of different sizes.

Combined confocal Raman and quantitative phase microscopy system for biomedical diagnosis.

We have developed a novel multimodal microscopy system that incorporates confocal Raman, confocal reflectance, and quantitative phase microscopy (QPM) into a single imaging entity. Confocal Raman microscopy provides detailed chemical information from the sample, while confocal reflectance and quantitative phase microscopy show detailed morphology. Combining these intrinsic contrast imaging modalities makes it possible to obtain quantitative morphological and chemical information without exogenous staining. For validation and characterization, we have used this multi-modal system to investigate healthy and diseased blood samples. We first show that the thickness of a healthy red blood cell (RBC) shows good correlation with its hemoglobin distribution. Further, in malaria infected RBCs, we successfully image the distribution of hemozoin (malaria pigment) inside the cell. Our observations lead us to propose morphological screening by QPM and subsequent chemical imaging by Raman for investigating blood disorders. This new approach allows monitoring cell development and cell-drug interactions with minimal perturbation of the biological system of interest.

Preliminary evaluation of a nanotechnology-based approach for the more effective diagnosis of colon cancers.

AIM: The goal of this research was to develop and preliminarily test a novel technology and instrumentation that could help to significantly increase the diagnostic yield of current colon cancer screening procedures. This technology is based on a combined fluorescence-optical coherence tomography (OCT) imaging, and topical delivery of a cancer-targeting agent. MATERIALS & METHODS: Gold colloid-adsorbed poly(ε-caprolactone) microparticles were labeled with a near-infrared dye, and functionalized with argentine-glycine-aspartic acid (RGD peptide) to effectively target cancer tissue, and enhance fluorescence-imaging contrast. The RGD peptide recognizes the α(v)ß(3)-integrin receptor, which is overexpressed by epithelial cancer cells. OCT was used under fluorescence guidance to visualize tissue morphology and, thus, to serve as a confirmatory tool for cancer presence. RESULTS: A preliminary testing of this technology on human colon cancer cell lines, a mouse model of colon cancer, as well as human colon tissue specimens, was performed. Strong binding of microparticles to cancer cells and no binding to cells that do not significantly express integrins, such as mouse fibroblasts, was observed. Preferential binding to cancer tissue was also observed. Strong fluorescence signals were obtained from cancer tissue, owing to the efficient binding of the contrast agent. OCT imaging was capable of revealing clear differences between normal and cancer tissue. CONCLUSION: A dual-modality imaging approach combined with topical delivery of a cancer-targeting contrast agent has been preliminarily tested for colon cancer diagnosis. Preferential binding of the contrast agent to cancer tissue allowed the cancer-suspicious locations to be highlighted and, thus, guided OCT imaging to visualize tissue morphology and determine tissue type. If successful, this multimodal approach might help to increase the sensitivity and the specificity of current colon cancer-screening procedures in the future.

High resolution multimodal clinical ophthalmic imaging system.

We developed a multimodal adaptive optics (AO) retinal imager which is the first to combine high performance AO-corrected scanning laser ophthalmoscopy (SLO) and swept source Fourier domain optical coherence tomography (SSOCT) imaging modes in a single compact clinical prototype platform. Such systems are becoming ever more essential to vision research and are expected to prove their clinical value for diagnosis of retinal diseases, including glaucoma, diabetic retinopathy (DR), age-related macular degeneration (AMD), and retinitis pigmentosa. The SSOCT channel operates at a wavelength of 1 microm for increased penetration and visualization of the choriocapillaris and choroid, sites of major disease activity for DR and wet AMD. This AO system is designed for use in clinical populations; a dual deformable mirror (DM) configuration allows simultaneous low- and high-order aberration correction over a large range of refractions and ocular media quality. The system also includes a wide field (33 deg.) line scanning ophthalmoscope (LSO) for initial screening, target identification, and global orientation, an integrated retinal tracker (RT) to stabilize the SLO, OCT, and LSO imaging fields in the presence of lateral eye motion, and a high-resolution LCD-based fixation target for presentation of visual cues. The system was tested in human subjects without retinal disease for performance optimization and validation. We were able to resolve and quantify cone photoreceptors across the macula to within approximately 0.5 deg (approximately 100-150 microm) of the fovea, image and delineate ten retinal layers, and penetrate to resolve features deep into the choroid. The prototype presented here is the first of a new class of powerful flexible imaging platforms that will provide clinicians and researchers with high-resolution, high performance adaptive optics imaging to help guide therapies, develop new drugs, and improve patient outcomes.

Live cell refractometry using Hilbert phase microscopy and confocal reflectance microscopy.

Quantitative chemical analysis has served as a useful tool for understanding cellular metabolisms in biology. Among many physical properties used in chemical analysis, refractive index in particular has provided molecular concentration that is an important indicator for biological activities. In this report, we present a method of extracting full-field refractive index maps of live cells in their native states. We first record full-field optical thickness maps of living cells by Hilbert phase microscopy and then acquire physical thickness maps of the same cells using a custom-built confocal reflectance microscope. Full-field and axially averaged refractive index maps are acquired from the ratio of optical thickness to physical thickness. The accuracy of the axially averaged index measurement is 0.002. This approach can provide novel biological assays of label-free living cells in situ.

Improved phase sensitivity in spectral domain phase microscopy using line-field illumination and self phase-referencing.

We report a quantitative phase microscope based on spectral domain optical coherence tomography and line-field illumination. The line illumination allows self phase-referencing method to reject common-mode phase noise. The quantitative phase microscope also features a separate reference arm, permitting the use of high numerical aperture (NA > 1) microscope objectives for high resolution phase measurement at multiple points along the line of illumination. We demonstrate that the path-length sensitivity of the instrument can be as good as 41 pm/square root of Hz, which makes it suitable for nanometer scale study of cell motility. We present the detection of natural motions of cell surface and two-dimensional surface profiling of a HeLa cell.