Hyperspectral dark-field microscopy of human breast lumpectomy samples for tumor margin detection in breast-conserving surgery.

Significance: Hyperspectral dark-field microscopy (HSDFM) and data cube analysis algorithms demonstrate successful detection and classification of various tissue types, including carcinoma regions in human post-lumpectomy breast tissues excised during breast-conserving surgeries. Aim: We expand the application of HSDFM to the classification of tissue types and tumor subtypes in pre-histopathology human breast lumpectomy samples. Approach: Breast tissues excised during breast-conserving surgeries were imaged by the HSDFM and analyzed. The performance of the HSDFM is evaluated by comparing the backscattering intensity spectra of polystyrene microbead solutions with the Monte Carlo simulation of the experimental data. For classification algorithms, two analysis approaches, a supervised technique based on the spectral angle mapper (SAM) algorithm and an unsupervised technique based on the K-means algorithm are applied to classify various tissue types including carcinoma subtypes. In the supervised technique, the SAM algorithm with manually extracted endmembers guided by H&E annotations is used as reference spectra, allowing for segmentation maps with classified tissue types including carcinoma subtypes. Results: The manually extracted endmembers of known tissue types and their corresponding threshold spectral correlation angles for classification make a good reference library that validates endmembers computed by the unsupervised K-means algorithm. The unsupervised K-means algorithm, with no a priori information, produces abundance maps with dominant endmembers of various tissue types, including carcinoma subtypes of invasive ductal carcinoma and invasive mucinous carcinoma. The two carcinomas' unique endmembers produced by the two methods agree with each other within <2% residual error margin. Conclusions: Our report demonstrates a robust procedure for the validation of an unsupervised algorithm with the essential set of parameters based on the ground truth, histopathological information. We have demonstrated that a trained library of the histopathology-guided endmembers and associated threshold spectral correlation angles computed against well-defined reference data cubes serve such parameters. Two classification algorithms, supervised and unsupervised algorithms, are employed to identify regions with carcinoma subtypes of invasive ductal carcinoma and invasive mucinous carcinoma present in the tissues. The two carcinomas' unique endmembers used by the two methods agree to <2% residual error margin. This library of high quality and collected under an environment with no ambient background may be instrumental to develop or validate more advanced unsupervised data cube analysis algorithms, such as effective neural networks for efficient subtype classification.

Polydimethylsiloxane tissue-mimicking phantoms with tunable optical properties.

SIGNIFICANCE: The polymer, polydimethylsiloxane (PDMS), has been increasingly used to make tissue simulating phantoms due to its excellent processability, durability, flexibility, and limited tunability of optical, mechanical, and thermal properties. We report on a robust technique to fabricate PDMS-based tissue-mimicking phantoms where the broad range of scattering and absorption properties are independently adjustable in the visible- to near-infrared wavelength range from 500 to 850 nm. We also report on an analysis method to concisely quantify the phantoms' broadband characteristics with four parameters. AIM: We report on techniques to manufacture and characterize solid tissue-mimicking phantoms of PDMS polymers. Tunability of the absorption (µa ( λ ) ) and reduced scattering coefficient spectra (µs'(λ)) in the wavelength range of 500 to 850 nm is demonstrated by adjusting the concentrations of light absorbing carbon black powder (CBP) and light scattering titanium dioxide powder (TDP) added into the PDMS base material. APPROACH: The µa ( λ ) and µs'(λ) of the phantoms were obtained through measurements with a broadband integrating sphere system and by applying an inverse adding doubling algorithm. Analyses of µa ( λ ) and µs'(λ) of the phantoms, by fitting them to linear and power law functions, respectively, demonstrate that independent control of µa ( λ ) and µs'(λ) is possible by systematically varying the concentrations of CBP and TDP. RESULTS: Our technique quantifies the phantoms with four simple fitting parameters enabling a concise tabulation of their broadband optical properties as well as comparisons to the optical properties of biological tissues. We demonstrate that, to a limited extent, the scattering properties of our phantoms mimic those of human tissues of various types. A possible way to overcome this limitation is demonstrated with phantoms that incorporate polystyrene microbead scatterers. CONCLUSIONS: Our manufacturing and analysis techniques may further promote the application of PDMS-based tissue-mimicking phantoms and may enable robust quality control and quality checks of the phantoms.

Optical phase contrast imaging for absolute, quantitative measurements of ultrasonic fields with frequencies up to 20 MHz.

The technique of phase contrast imaging, combined with tomographic reconstructions, can rapidly measure ultrasonic fields propagating in water, including ultrasonic fields with complex wavefront shapes, which are difficult to characterize with standard hydrophone measurements. Furthermore, the technique can measure the absolute pressure amplitudes of ultrasonic fields without requiring a pressure calibration. Absolute pressure measurements have been previously demonstrated using optical imaging methods for ultrasonic frequencies below 2.5 MHz. The present work demonstrates that phase contrast imaging can accurately measure ultrasonic fields with frequencies up to 20 MHz and pressure amplitudes near 10 kPa. Accurate measurements at high ultrasonic frequencies are performed by tailoring the measurement conditions to limit optical diffraction as guided by a simple dimensionless parameter. In some situations, differences between high frequency measurements made with the phase contrast method and a calibrated hydrophone become apparent, and the reasons for these differences are discussed. Extending optical imaging measurements to high ultrasonic frequencies could facilitate quantitative applications of ultrasound measurements in nondestructive testing and medical therapeutics and diagnostics such as photoacoustic imaging.

Dual-comb photoacoustic spectroscopy.

Spectrally resolved photoacoustic imaging is promising for label-free imaging in optically scattering materials. However, this technique often requires acquisition of a separate image at each wavelength of interest. This reduces imaging speeds and causes errors if the sample changes in time between images acquired at different wavelengths. We demonstrate a solution to this problem by using dual-comb spectroscopy for photoacoustic measurements. This approach enables a photoacoustic measurement at thousands of wavelengths simultaneously. In this technique, two optical-frequency combs are interfered on a sample and the resulting pressure wave is measured with an ultrasound transducer. This acoustic signal is processed in the frequency-domain to obtain an optical absorption spectrum. For a proof-of-concept demonstration, we measure photoacoustic signals from polymer films. The absorption spectra obtained from these measurements agree with those measured using a spectrophotometer. Improving the signal-to-noise ratio of the dual-comb photoacoustic spectrometer could enable high-speed spectrally resolved photoacoustic imaging.

Scattering based hyperspectral imaging of plasmonic nanoplate clusters towards biomedical applications.

A new optical scattering contrast-agent based on polymer-nanoparticle encapsulated silver nanoplates (PESNs) is presented. Silver nanoplates were chosen due to the flexibility of tuning their plasmon frequencies. The polymer coating preserves their physical and optical properties and confers other advantages such as controlled contrast agent delivery. Finite difference time domain (FDTD) simulations model the interaction of light with the nanoplates in different orientations in the cluster. Hyperspectral dark field microscopy (HYDFM) observes the scattering spectra of the PESNs. An unsupervised sequential maximum angle convex cone (SMACC) image analysis resolves spectral endmembers corresponding to different stacking orientations of the nanoplates. The orientation-dependent endmembers qualitatively agree with the FDTD results. For contrast enhancement, the uptake and spatial distribution of PESNs are demonstrated by an HYDFM study of single melanoma cells to result in an enhanced contrast of up to 400%. A supervised spatial mapping of the endmembers obtained by the unsupervised SMACC algorithm reveals spatial distributions of PESNs with various clustering orientations of encapsulated nanoplates. Our study demonstrates tunability in plasmonics properties in clustered metal nanoparticles and its utility for the development of scatter-based imaging contrast agents for a broad range of applications, including studies of single cells and other biomedical systems.

Single molecule confocal fluorescence lifetime correlation spectroscopy for accurate nanoparticle size determination.

We report on an experimental procedure in confocal single molecule fluorescence lifetime correlation spectroscopy (FLCS) to determine the range of excitation power and molecular or particulate concentration in solution under which the application of an unmodified model autocorrelation function is justified. This procedure enables fitting of the autocorrelation to an accurate model to measure diffusion length (r) and diffusion time (τD) of single molecules in solution. We also report on the pinhole size dependency of r and τD in a confocal FLCS platform. This procedure determines a set of experimental parameters with which the Stokes-Einstein (S-E) equation accurately measures the hydrodynamic radii of spherical nanoparticles, enabling the determination of the particle size range for which the hydrodynamic radius by the S-E equation measures the real particle radius.

Immobilization of proteins on carboxylic acid functionalized nanopatterns.

The immobilization of proteins on nanopatterned surfaces was investigated using in situ atomic force microscopy (AFM) and ex situ infrared reflectance-absorption spectroscopy (IRAS). The AFM-based lithography technique of nanografting provided control of the size, geometry, and spatial placement of nanopatterns within self-assembled monolayers (SAMs). Square nanopatterns of carboxylate-terminated SAMs were inscribed within methyl-terminated octadecanethiolate SAMs and activated using carbodiimide/succinimide coupling chemistry. Staphylococcal protein A was immobilized on the activated nanopatterns before exposure to rabbit immunoglobulin G. In situ AFM was used to monitor changes in the topography and friction of the nanopatterns in solution upon protein immobilization. Complementary studies with ex situ IRAS confirmed the surface chemistry that occurred during the steps of SAM activation and subsequent protein immobilization on unpatterned samples. Since carbodiimide/succinimide coupling chemistry can be used for surface attachment of different biomolecules, this protocol shows promise for development of other aqueous-based studies for nanopatterned protein immobilization.

Cholesterol/phospholipid interactions in hybrid bilayer membranes.

The interactions between cholesterol and saturated phospholipids in hybrid bilayer membranes (HBMs) were investigated using the interface-sensitive technique of vibrational sum frequency spectroscopy (VSFS). The unique sensitivity of VSFS to order/disorder transitions of the lipid acyl chains was used to determine the main gel to liquid crystal phase transition temperature, Tm, for HBMs of binary cholesterol/phospholipid mixtures on octadecanethiolate self-assembled monolayers. The phase transition temperature and the breadth of the transition were shown to increase with cholesterol content, and the phase boundaries observed in the cholesterol/phospholipid HBMs were comparable to the published phase diagrams of binary cholesterol/phospholipid vesicles. A thermodynamic assessment of the cooperative units of the HBM phase transitions revealed the presence of <10 nm diameter domains that were independent of the cholesterol composition.

Characterization and control of lipid layer fluidity in hybrid bilayer membranes.

The main gel-to-liquid-crystal (LC) phase transition temperature, T(m), of the distal lipid layer in hybrid bilayer membranes (HBMs) under water was investigated using vibrational sum frequency spectroscopy (VSFS). VSFS has unique sensitivity to order/disorder transitions in the lipid acyl chains and can determine T(m) for the lipid monolayers in HBMs. We recently reported the observation that T(m) is raised and the transition width is broadened for the overlying phospholipid monolayer in HBM systems formed on densely packed crystalline self-assembled monolayers (SAMs) as compared to that of vesicles in solution. In this report, we establish that T(m) for the lipid layer of HBMs can be controlled by proper choice of the SAM underlayer. The SAM underlayer of the HBM was systematically altered by using an alkane thiol, a saturated thiolipid, a mixed SAM of a saturated lipid-pyridine disulfide, and finally a mixed SAM of an unsaturated lipid-pyridine disulfide. T(m) was measured for two different chain length saturated phosphatidylcholine lipid overlayers on these SAMs. The values obtained show that Tm of the lipid layer of HBMs is sensitive to the composition and/or packing density of molecules in the underlying SAM.

Determination of lipid phase transition temperatures in hybrid bilayer membranes.

The main gel-to-liquid-crystal (LC) phase transition temperature, T(m), of the lipid monolayer in hybrid bilayer membranes (HBMs) was investigated using vibrational sum frequency spectroscopy (VSFS). In the gel phase, the acyl chains of the lipid molecules assume an ordered, all-trans configuration, whereas in the LC phase, the acyl chains exhibit a significant number of disordered gauche conformers. VSFS has unique sensitivity to the order/disorder transitions in the acyl chains and was used to determine T(m) for a series of saturated phosphatidylcholine lipids on octadecanethiolate self-assembled monolayers (SAMs). The values obtained for T(m) for all lipids studied are significantly higher than for the corresponding lipids in vesicles in solution. Additionally, the transition widths are broader for the lipids in HBMs. The underlying SAM clearly influences the phase behavior of the overlying lipid monolayer.

Infrared detection of a phenylboronic acid terminated alkane thiol monolayer on gold surfaces.

Polarization modulation infrared reflectance absorption spectroscopy (PM-IRRAS) and infrared reflectance absorption spectroscopy (IRRAS) have been used to characterize the formation of a self-assembled monolayer of N-(3-dihydroxyborylphenyl)-11-mercaptoundecanamide) (abbreviated PBA) on a gold surface and the subsequent binding of various sugars to the PBA adlayer through the phenylboronic acid moiety to form a phenylboronate ester. Vibrationally resonant sum frequency generation (VR-SFG) spectroscopy confirmed the ordering of the substituted phenyl groups of the PBA adlayer on the gold surface. Solution FTIR spectra and density functional theory were used to confirm the identity of the observed vibrational modes on the gold surface of PBA with and without bound sugar. The detection of the binding of glucose on the gold surface was confirmed in part by the presence of a C-O stretching mode of glucose and the observed O-H stretching mode of glucose that is shifted in position relative to the O-H stretching mode of boronic acid. An IR marker mode was also observed at 1734 cm(-1) upon the binding of glucose. Additionally, changes in the peak profile of the B-O stretching band were observed upon binding, confirming formation of a phenylboronate ester on the gold surface. The binding of mannose and lactose were also detected primarily through the IR marker mode at approximately 1736 to 1742 cm(-1) depending on the identity of the bound sugar.