In vivo characterization of the human glioblastoma infiltrative edge with label-free intraoperative fluorescence lifetime imaging.

Challenges in identifying a glioblastoma's infiltrative edge during neurosurgical procedures result in rapid recurrence. A label-free fluorescence lifetime imaging (FLIm) device was used to evaluate glioblastoma's infiltrative edge in vivo in 15 patients (89 samples). FLIm data were analyzed according to tumor cell density, infiltrating tissue type (gray and white matter), and diagnosis history (new or recurrent). Infiltrations in white matter from new glioblastomas showed decreasing lifetimes and a spectral red shift with increasing tumor cell density. Areas of high versus low tumor cell density were separated through a linear discriminant analysis with a ROC-AUC=0.74. Current results support the feasibility of intraoperative FLIm for real-time in vivo brain measurements and encourage refinement to predict glioblastoma infiltrative edge, underscoring the ability of FLIm to optimize neurosurgical outcomes.

Intraoperative detection of IDH-mutant glioma using fluorescence lifetime imaging.

Identifying isocitrate dehydrogenase (IDH)-mutation and glioma subtype during surgery instead of days later can aid in modifying tumor resection strategies for better survival outcomes. We report intraoperative identification of IDH-mutant glioma (N = 12 patients) with a clinically compatible fluorescence lifetime imaging (FLIm) device (excitation: 355 nm; emission spectral bands: 390/40 nm, 470/28 nm, 542/50 nm). The fluorescence-derived parameters were analyzed to study the optical contrast between IDH-mutant tumors and surrounding brain tissue. IDH-mutant oligodendrogliomas exhibited shorter lifetimes (3.3 ± 0.1 ns) than IDH-mutant astrocytomas (4.1 ± 0.1 ns). Both IDH-mutant glioma subtypes had shorter lifetimes than white matter (4.6 ± 0.4 ns) but had comparable lifetimes to cortex. Lifetimes also increased with malignancy grade within IDH-mutant oligodendrogliomas (grade 2: 2.96 ± 0.08 ns, grade 3: 3.4 ± 0.3 ns) but not within IDH-mutant astrocytomas. The current results support the feasibility of FLIm as a surgical adjuvant for identifying IDH-mutant glioma tissue.

First in patient assessment of brain tumor infiltrative margins using simultaneous time-resolved measurements of 5-ALA-induced PpIX fluorescence and tissue autofluorescence.

SIGNIFICANCE: 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence is currently used for image-guided glioma resection. Typically, this widefield imaging method highlights the bulk of high-grade gliomas, but it underperforms at the infiltrating edge where PpIX fluorescence is not visible to the eyes. Fluorescence lifetime imaging (FLIm) has the potential to detect PpIX fluorescence below the visible detection threshold. Moreover, simultaneous acquisition of time-resolved nicotinamide adenine (phosphate) dinucleotide [NAD(P)H] fluorescence may provide metabolic information from the tumor environment to further improve overall tumor detection. AIM: We investigate the ability of pulse sampling, fiber-based FLIm to simultaneously image PpIX and NAD(P)H fluorescence of glioma infiltrative margins in patients. APPROACH: A mesoscopic fiber-based point-scanning FLIm device (355 nm pulses) was used to simultaneously resolve the fluorescence decay of PpIX (629/53 nm) and NAD(P)H (470/28 nm). The FLIm device enabled data acquisition at room light and rapid (<33 ms) augmentation of FLIm parameters on the surgical field-of-view. FLIm measurements from superficial tumors and tissue areas around the resection margins were performed on three glioblastoma patients in vivo following inspection of PpIX visible fluorescence with a conventional neurosurgical microscope. Microbiopsies were collected from FLIm imaged areas for histopathological evaluation. RESULTS: The average lifetime from PpIX and NAD(P)H fluorescence distinguished between tumor and surrounding tissue. FLIm measurements of resection margins presented a range of PpIX and NAD(P)H lifetime values (τPpIX   ∼ 3 to 14 ns, τNAD(P)H = 3 to 6 ns) associated with unaffected tissue and areas of low-density tumor infiltration. CONCLUSIONS: Intraoperative FLIm could simultaneously detect the emission of PpIX and NAD(P)H from patients in vivo during craniotomy procedures. This approach doubles as a clinical tool to identify tumor areas while performing tissue resection and as a research tool to study tumor microenvironmental changes in vivo. Intraoperative FLIm of 5-ALA-induced PpIX and tissue autofluorescence makes a promising surgical adjunct to guide tumor resection surgery.

Assessment of Murine Colon Inflammation Using Intraluminal Fluorescence Lifetime Imaging.

Inflammatory bowel disease (IBD) is typically diagnosed by exclusion years after its onset. Current diagnostic methods are indirect, destructive, or target overt disease. Screening strategies that can detect low-grade inflammation in the colon would improve patient prognosis and alleviate associated healthcare costs. Here, we test the feasibility of fluorescence lifetime imaging (FLIm) to detect inflammation from thick tissue in a non-destructive and label-free approach based on tissue autofluorescence. A pulse sampling FLIm instrument with 355 nm excitation was coupled to a rotating side-viewing endoscopic probe for high speed (10 mm/s) intraluminal imaging of the entire mucosal surface (50-80 mm) of freshly excised mice colons. Current results demonstrate that tissue autofluorescence lifetime was sensitive to the colon anatomy and the colonocyte layer. Moreover, mice under DSS-induced colitis and 5-ASA treatments showed changes in lifetime values that were qualitatively related to inflammatory markers consistent with alterations in epithelial bioenergetics (switch between ß-oxidation and aerobic glycolysis) and physical structure (colon length). This study demonstrates the ability of intraluminal FLIm to image mucosal lifetime changes in response to inflammatory treatments and supports the development of FLIm as an in vivo imaging technique for monitoring the onset, progression, and treatment of inflammatory diseases.

Mesoscopic fluorescence lifetime imaging: Fundamental principles, clinical applications and future directions.

Fluorescence lifetime imaging (FLIm) is an optical spectroscopic imaging technique capable of real-time assessments of tissue properties in clinical settings. Label-free FLIm is sensitive to changes in tissue structure and biochemistry resulting from pathological conditions, thus providing optical contrast to identify and monitor the progression of disease. Technical and methodological advances over the last two decades have enabled the development of FLIm instrumentation for real-time, in situ, mesoscopic imaging compatible with standard clinical workflows. Herein, we review the fundamental working principles of mesoscopic FLIm, discuss the technical characteristics of current clinical FLIm instrumentation, highlight the most commonly used analytical methods to interpret fluorescence lifetime data and discuss the recent applications of FLIm in surgical oncology and cardiovascular diagnostics. Finally, we conclude with an outlook on the future directions of clinical FLIm.

Physical, Biomechanical, and Optical Characterization of Collagen and Elastin Blend Hydrogels.

Collagen and elastin proteins are major components of the extracellular matrix of many organs. The presence of collagen and elastin networks, and their associated properties, in different tissues have led scientists to study collagen and elastin composites for use in tissue engineering. In this study, we characterized physical, biochemical, and optical properties of gels composed of collagen and elastin blends. We demonstrated that the addition of varying amounts of elastin to the constructs alters collagen fibrillogenesis, D-banding pattern length, and storage modulus. However, the addition of elastin does not affect collagen fibril diameter. We also evaluated the autofluorescence properties of the different collagen and elastin blends with fluorescence lifetime imaging (FLIm). Autofluorescence emission showed a red shift with the addition of elastin to the hydrogels. The fluorescence lifetime values of the gels increased with the addition of elastin and were strongly correlated with the storage moduli measurements. These results suggest that FLIm can be used to monitor the gels' mechanical properties nondestructively. These collagen and elastin constructs, along with the FLIm capabilities, can be used to develop and study collagen and elastin composites for tissue engineering and regenerative medicine.

FLIm and Raman Spectroscopy for Investigating Biochemical Changes of Bovine Pericardium upon Genipin Cross-Linking.

Biomaterials used in tissue engineering and regenerative medicine applications benefit from longitudinal monitoring in a non-destructive manner. Label-free imaging based on fluorescence lifetime imaging (FLIm) and Raman spectroscopy were used to monitor the degree of genipin (GE) cross-linking of antigen-removed bovine pericardium (ARBP) at three incubation time points (0.5, 1.0, and 2.5 h). Fluorescence lifetime decreased and the emission spectrum redshifted compared to that of uncross-linked ARBP. The Raman signature of GE-ARBP was resonance-enhanced due to the GE cross-linker that generated new Raman bands at 1165, 1326, 1350, 1380, 1402, 1470, 1506, 1535, 1574, 1630, 1728, and 1741 cm-1. These were validated through density functional theory calculations as cross-linker-specific bands. A multivariate multiple regression model was developed to enhance the biochemical specificity of FLIm parameters fluorescence intensity ratio (R2 = 0.92) and lifetime (R2 = 0.94)) with Raman spectral results. FLIm and Raman spectroscopy detected biochemical changes occurring in the collagenous tissue during the cross-linking process that were characterized by the formation of a blue pigment which affected the tissue fluorescence and scattering properties. In conclusion, FLIm parameters and Raman spectroscopy were used to monitor the degree of cross-linking non-destructively.

Investigating Origins of FLIm Contrast in Atherosclerotic Lesions Using Combined FLIm-Raman Spectroscopy.

Background: Fluorescence lifetime imaging (FLIm) is a spectroscopic imaging technique able to characterize the composition of luminal surface of arterial vessels. Studies of human coronary samples demonstrated that distinct atherosclerotic lesion types are characterized by FLIm features associate with distinct tissue molecular makeup. While conventional histology has provided indications about potential sources of molecular contrast, specific information about the origin of FLIm signals is lacking. Here we investigate whether Raman spectroscopy, a technique able to evaluate chemical content of biological samples, can provide additional insight into the origin of FLIm contrast. Methods: Six human coronary artery samples were imaged using FLIm (355 nm excitation)-Raman spectroscopy (785 nm excitation) via a multimodal fiber optic probe. The spatial distribution of molecular contrast in FLIm images was analyzed in relationship with histological findings. Raman data was investigated using an endmember technique and compared with histological findings. A descriptive modeling approach based on multivariate regression was used to identify Raman bands related with changes in lifetime in four spectral channels (violet: 387/35 nm, blue: 443/29 nm, green: 546/38 nm, and red: 628/53 nm). Results: Fluorescence lifetime variations in the violet, blue and green spectral bands were observed for distinct areas of each tissue sample associated with distinct pathologies. Analysis of Raman signals from areas associated with normal, pathological intimal thickening, and fibrocalcific regions demonstrated the presence of hydroxyapatite, collagenous proteins, carotene, cholesterol, and triglycerides. The FLIm and Raman descriptive modeling analysis indicated that lifetime increase in the violet spectral band was associated with increased presence of cholesterol and carotenes, a new finding consistent with LDL accumulation in atherosclerotic lesions, and not with collagen proteins, as expected from earlier studies. Conclusions: The systematic, quantitative analysis of the multimodal FLIm-Raman dataset using a descriptive modeling approach led to the identification of LDL accumulation as the primary source of lifetime contrast in atherosclerotic lesions in the violet spectral range. Earlier FLIm validation studies relying on histopathological findings had associated this contrast to increased collagen content, also present in advanced lesions, thus demonstrating the benefits of alternative validation methods.

FLIm-Guided Raman Imaging to Study Cross-Linking and Calcification of Bovine Pericardium.

Bovine pericardium (BP) is a vascular biomaterial used in cardiovascular surgery that is typically cross-linked for masking antigenicity and enhance stability. There is a need for biochemical evaluation of the tissue properties prior to implantation to ensure that quality and reliability standards are met. Here, engineered antigen removed BP (ARBP) that was cross-linked with 0.2% and 0.6% glutaraldehyde (GA), and further calcified in vitro to simulate graft calcifications upon implantation was characterized nondestructively using fluorescence lifetime imaging (FLIm) to identify regions of interest which were then assessed by Raman spectroscopy. We observed that the tissue fluorescence lifetime shortened, and that Raman bands at 856, 935, 1282, and 1682 cm-1 decreased, and at 1032 and 1627 cm-1 increased with increasing GA cross-linking. Independent classification analysis based on fluorescence lifetime and on Raman spectra discriminated between GA-ARBP and untreated ARBP with an accuracy of 91% and 66%, respectively. Pearson's correlation analysis showed a strong correlation between pyridinium cross-links measured with high-performance liquid chromatography and fluorescence lifetime measured at 380-400 nm (R = -0.76, p = 0.00094), as well as Raman bands at 856 cm-1 for hydroxy-proline (R = -0.68, p = 0.0056) and at 1032 cm-1 for hydroxy-pyridinium (R = 0.74, p = 0.0016). Calcified areas of GA cross-linked tissue showed characteristic hydroxyapatite (959 and 1038 cm-1) bands in the Raman spectrum and fluorescence lifetime shortened by 0.4 ns compared to uncalcified regions. FLIm-guided Raman imaging could rapidly identify degrees of cross-linking and detected calcified regions with high chemical specificity, an ability that can be used to monitor tissue engineering processes for applications in regenerative medicine.

Real-time augmented reality for delineation of surgical margins during neurosurgery using autofluorescence lifetime contrast.

Current clinical brain imaging techniques used for surgical planning of tumor resection lack intraoperative and real-time feedback; hence surgeons ultimately rely on subjective evaluation to identify tumor areas and margins. We report a fluorescence lifetime imaging (FLIm) instrument (excitation: 355 nm; emission spectral bands: 390/40 nm, 470/28 nm, 542/50 nm and 629/53 nm) that integrates with surgical microscopes to provide real-time intraoperative augmentation of the surgical field of view with fluorescent derived parameters encoding diagnostic information. We show the functionality and safety features of this instrument during neurosurgical procedures in patients undergoing craniotomy for the resection of brain tumors and/or tissue with radiation damage. We demonstrate in three case studies the ability of this instrument to resolve distinct tissue types and pathology including cortex, white matter, tumor and radiation-induced necrosis. In particular, two patients with effects of radiation-induced necrosis exhibited longer fluorescence lifetimes and increased optical redox ratio on the necrotic tissue with respect to non-affected cortex, and an oligodendroglioma resected from a third patient reported shorter fluorescence lifetime and a decrease in optical redox ratio than the surrounding white matter. These results encourage the use of FLIm as a label-free and non-invasive intraoperative tool for neurosurgical guidance.

Bovine pericardial extracellular matrix niche modulates human aortic endothelial cell phenotype and function.

Xenogeneic biomaterials contain biologically relevant extracellular matrix (ECM) composition and organization, making them potentially ideal surgical grafts and tissue engineering scaffolds. Defining the effect of ECM niche (e.g., basement membrane vs. non-basement membrane) on repopulating cell phenotype and function has important implications for use of xenogeneic biomaterials, particularly in vascular applications. We aim to understand how serous (i.e., basement membrane) versus fibrous (i.e., non-basement membrane) ECM niche of antigen-removed bovine pericardium (AR-BP) scaffolds influence human aortic endothelial cell (hAEC) adhesion, growth, phenotype, inflammatory response and laminin production. At low and moderate seeding densities hAEC proliferation was significantly increased on the serous side. Similarly, ECM niche modulated cellular morphology, with serous side seeding resulting in a more rounded aspect ratio and intact endothelial layer formation. At moderate seeding densities, hAEC production of human laminin was enhanced following serous seeding. Finally, inflammatory marker and pro-inflammatory cytokine expression decreased following long-term cell growth regardless of seeding side. This work demonstrates that at low and moderate seeding densities AR-BP sidedness significantly impacts endothelial cell growth, morphology, human laminin production, and inflammatory state. These findings suggest that ECM niche has a role in modulating response of repopulating recipient cells toward AR-BP scaffolds for vascular applications.

Simultaneous intraluminal imaging of tissue autofluorescence and eGFP-labeled cells in engineered vascular grafts inside a bioreactor.

The growing demand for tissue engineered vascular grafts (TEVG) motivates the development of optimized fabrication and monitoring procedures. Bioreactors which provide physiologically-relevant conditions are important for improving holistic TEVG properties and performance. Herein we describe a fiber-based intraluminal imaging system that allows for in situ assessment of vascular materials and re-cellularization processes inside a bioreactor by simultaneous and co-registered measurements of endogenous fluorescence lifetime and exogenous marker fluorescence intensity. The lumen of 6 vascular grafts (∼4 mm diameter) were scanned by reciprocally rotating a 41° angle polished multimode optical fiber inside a protective glass tube with outer diameter of 3 mm. Tubular bovine pericardium constructs were recellularized using enhanced Green Fluorescent Protein (eGFP) transfected cells in a custom bioreactor. The imaging system has resolved consistently the cellular autofluorescence from that of tissue matrix in situ based on the lifetime fluorescence properties of endogenous molecular species. The location of the re-cellularized area was validated by the eGFP emission. Current results demonstrate the potential of this system as a valuable tool in tissue engineering for in situ studies of cell-tissue interactions in cylindrical or other 3-dimensional structures.

Fiber-based platform for synchronous imaging of endogenous and exogenous fluorescence of biological tissue.

Endogenous and exogenous fluorescence emission from biological samples encodes complementary information. Here we report, to the best of our knowledge, the first results from an optical imaging platform with interleaved excitation and detection of exogenous and endogenous fluorescence from tissue samples using a single flexible multimode fiber that delivers the excitation beam and collects the emitted light. A custom-built reflective optical chopper wheel with synchronized rotation temporally multiplexes an autofluorescence lifetime imaging apparatus with an intensity-based fluorescence module tailored to imaging green fluorescent protein. We demonstrate the functionality of such platform imaging dyes of varying fluorescence signatures and resolving cellularized areas on bio-engineered tissue constructs.

Multiscale, multispectral fluorescence lifetime imaging using a double-clad fiber.

Fiber-based imaging of tissue autofluorescence using ultraviolet (UV) excitation is a highly flexible tool used to probe structure and composition. In this Letter, we report, to the best of our knowledge, the first results from a single-fiber imaging system employing a custom double-clad fiber to acquire multispectral fluorescence lifetime images at two distinct spatial resolutions. We characterize the lateral point spread function and fluorescent background of the system and show how enhanced resolution can identify features such as trabeculae in ex vivo murine bone samples.

Characterization of expressed human meibum using hyperspectral stimulated Raman scattering microscopy.

PURPOSE: This study examined whether hyperspectral stimulated Raman scattering (hsSRS) microscopy can detect differences in meibum lipid to protein composition of normal and evaporative dry eye subjects with meibomian gland dysfunction. METHODS: Subjects were evaluated for tear breakup time (TBUT), staining, meibum expression and gland dropout. Expressed meibum was analyzed using SRS vibrational signatures in the CH stretching region (2800-3050 cm-1). Vertex component analysis and K-means clustering were used to group the spectral signatures into four fractions containing high lipid (G1) to high protein (G4). RESULTS: Thirty-three subjects could be statistically analyzed using pooled meibum (13 with stable tear films (TBUTs > 10 s) and 20 with unstable tear films (TBUTs ≤ 10 s). Significant differences in meibum from subjects with unstable vs. stable TBUTs were found for the G1 fraction (medians 0.164 and 0.020, respectively; p = 0.012) and the G2 fraction (medians 0.244 and 0.272, respectively; p = 0.045). No differences were observed for the G3 and G4 fractions. Single orifice samples were not significantly different vs. pooled samples from the fellow eye, and eyelid sector samples (nasal, central and temporal) G2:G3 fractional components were not significantly different (p = 0.449). Spearman analysis suggested a significant inverse correlation between G1 fraction and TBUT (R = -0.351; p = 0.045). CONCLUSIONS: hsSRS microscopy allows compositional analysis of expressed meibum from humans which correlated to changes in TBUT. These findings support the hypothesis that hsSRS may be useful in classifying meibum quality and evaluating the effects of therapy.

Label-Free Assessment of Collagenase Digestion on Bovine Pericardium Properties by Fluorescence Lifetime Imaging.

The extracellular matrix architecture of bovine pericardium (BP) has distinct biochemical and biomechanical properties that make it a useful biomaterial in the field of regenerative medicine. Collagen represents the dominant structural protein of BP and is therefore intimately associated with the properties of this biomaterial. Enzymatic degradation of collagen molecules is critical for extracellular matrix turnover, remodeling and ultimately tissue regeneration. We present a quantitative, label-free and non-destructive method for monitoring changes in biochemical and biomechanical properties of BP during tissue degradation, based on multi-spectral fluorescence lifetime imaging (ms-FLIm). Strong correlations of fluorescence intensity ratio and average fluorescence lifetime were identified with collagen content, Young's Modulus and Ultimate tensile strength during collagenase degradation, indicating the potential of optically monitoring collagen degradation using ms-FLIm. The obtained results demonstrate the value of ms-FLIm to assess the quality of biomaterials in situ for applications in regenerative medicine.

Fiber-based fluorescence lifetime imaging of recellularization processes on vascular tissue constructs.

New techniques able to monitor the maturation of tissue engineered constructs over time are needed for a more efficient control of developmental parameters. Here, a label-free fluorescence lifetime imaging (FLIm) approach implemented through a single fiber-optic interface is reported for nondestructive in situ assessment of vascular biomaterials. Recellularization processes of antigen removed bovine pericardium scaffolds with endothelial cells and mesenchymal stem cells were evaluated on the serous and the fibrous sides of the scaffolds, 2 distinct extracellular matrix niches, over the course of a 7 day culture period. Results indicated that fluorescence lifetime successfully report cell presence resolved from extracellular matrix fluorescence. The recellularization process was more rapid on the serous side than on the fibrous side for both cell types, and endothelial cells expanded faster than mesenchymal stem cells on antigen-removed bovine pericardium. Fiber-based FLIm has the potential to become a nondestructive tool for the assessment of tissue maturation by allowing in situ imaging of intraluminal vascular biomaterials.

Pyrroloquinoline quinone prevents developmental programming of microbial dysbiosis and macrophage polarization to attenuate liver fibrosis in offspring of obese mice.

Increasingly, evidence suggests that exposure to maternal obesity creates an inflammatory environment in utero, exerting long-lasting postnatal signatures on the juvenile innate immune system and microbiome that may predispose offspring to development of fatty liver disease. We found that exposure to a maternal Western-style diet (WD) accelerated fibrogenesis in the liver of offspring and was associated with early recruitment of proinflammatory macrophages at 8-12 weeks and microbial dysbiosis as early as 3 weeks of age. We further demonstrated that bone marrow-derived macrophages (BMDMs) were polarized toward an inflammatory state at 8 weeks of age and that a potent antioxidant, pyrroloquinoline quinone (PQQ), reversed BMDM metabolic reprogramming from glycolytic toward oxidative metabolism by restoring trichloroacetic acid cycle function at isocitrate dehydrogenase. This resulted in reduced inflammation and inhibited collagen fibril formation in the liver at 20 weeks of age, even when PQQ was withdrawn at 3 weeks of age. Beginning at 3 weeks of age, WD-fed mice developed a decreased abundance of Parabacteroides and Lactobacillus, together with increased Ruminococcus and decreased tight junction gene expression by 20 weeks, whereas microbiota of mice exposed to PQQ retained compositional stability with age, which was associated with improved liver health. Conclusion: Exposure to a maternal WD induces early gut dysbiosis and disrupts intestinal tight junctions, resulting in BMDM polarization and induction of proinflammatory and profibrotic programs in the offspring that persist into adulthood. Disrupted macrophage and microbiota function can be attenuated by short-term maternal treatment with PQQ prior to weaning, suggesting that reshaping the early gut microbiota in combination with reprogramming macrophages during early weaning may alleviate the sustained proinflammatory environment, preventing the rapid progression of nonalcoholic fatty liver disease to nonalcoholic steatohepatitis in offspring of obese mothers. (Hepatology Communications 2018;2:313-328).

Label-free assessment of carotid artery biochemical composition using fiber-based fluorescence lifetime imaging.

Novel diagnostic tools with the ability to monitor variations in biochemical composition and provide benchmark indicators of vascular tissue maturation are needed to create functional tissue replacements. We investigated the ability of fiber-based, label-free multispectral fluorescent lifetime imaging (FLIm) to quantify the anatomical variations in biochemical composition of native carotid arteries and validated these results against biochemical assays. FLIm-derived parameters in spectral band 415-455 nm correlated with tissue collagen content (R2 = 0.64) and cell number (R2 = 0.61) and in spectral band 465-553 nm strongly correlated with elastin content (R2 = 0.89). These results suggest that FLIm holds great potential for assessing vascular tissue maturation and functional properties based on tissue autofluorescence.

A machine learning framework to analyze hyperspectral stimulated Raman scattering microscopy images of expressed human meibum.

We develop and discuss a methodology for batch-level analysis of hyperspectral stimulated Raman scattering (hsSRS) data sets of human meibum in the CH-stretching vibrational range. The analysis consists of two steps. The first step uses a training set (n=19) to determine chemically meaningful reference spectra that jointly constitute a basis set for the sample. This procedure makes use of batch-level vertex component analysis (VCA), followed by unsupervised k-means clustering to express the data set in terms of spectra that represent lipid and protein mixtures in changing proportions. The second step uses a random forest classifier to rapidly classify hsSRS stacks in terms of the pre-determined basis set. The overall procedure allows a rapid quantitative analysis of large hsSRS data sets, enabling a direct comparison among samples using a single set of reference spectra. We apply this procedure to assess 50 specimens of expressed human meibum, rich in both protein and lipid, and show that the batch-level analysis reveals marked variation among samples that potentially correlate with meibum health quality.