Accurate detection of brain tumor boundaries is crucial for successful tumor removal and better patient outcomes. A novel method using label-free Fluorescence Lifetime Imaging (iFLIm) is presented in this study. The approach involved developing an optimized classification model based on tumor enhancement status, utilizing multispectral FLIm. The method was evaluated on 52 patients with adult-type diffuse glioma, demonstrating promising results with 87% sensitivity, 92% specificity, and an AUC of 0.90. This FLIm-based model has the potential to offer a non-invasive and real-time technique to assist neurosurgeons in accurately identifying tumor infiltrates, potentially improving tumor resection and patient outcomes.
This study assesses the sensitivity of label-free fluorescence lifetime imaging (FLIm) for detecting low-grade gastrointestinal inflammation in mice. A FLIm probe was developed for in vivo colon imaging, enabling complete colon scanning. Using the probe, low-grade inflammation induced by streptomycin was imaged, showing decreased fluorescence lifetime at wavelengths related to metabolic activity, indicating a shift to glycolytic metabolism in inflamed tissue. The potential of validating FLIm maps with spatial transcriptomics was explored. These methodologies provide a basis for further experimentation to establish FLIm as a tool to quantify colon epithelial metabolism over time and its relevance to monitor colorectal inflammatory disease.
Stereotactic needle biopsy is a time-consuming and invasive procedure that often cannot accurately distinguish recurrent tumors from treatment effect in gliomas. We report an intraoperative multispectral fluorescence lifetime imaging (FLIm) system coupled with a custom-made fiber optic probe integrated with the stealth biopsy needle as an optical biopsy tool. FLIm parameters collected from 3 suspected recurrent glioma patients changed over the biopsy trajectory as the needle passed different brain areas. An SVM classifier validated using a leave-one-patient-out validation scheme could identify the lesions from the normal surrounding tissue with sensitivity=0.99, specificity=0.91, and accuracy=0.95.
Label-free biomedical imaging represents a range of powerful technologies used to visualize natural sources of biological contrast. Label-free techniques such as autofluorescence and fluorescence lifetime imaging measure contrast produced by various cellular products and provide high sensitivity for detecting tissue changes that occur with disease onset. However, a major limitation of these modalities, and many label-free modalities broadly, is the lack of robust validation methods that confirm signal specificity. Moreover, existing approaches are limited to assessing correlations and fail to provide mechanistic information into pathological events. Spatially resolved gene sequencing methods (e.g., spatial transcriptomics) are a powerful tool to gain detailed biological insight into tissue properties by creating 2-D maps of variations in gene expression that influence tissue properties. Thus, these techniques represent an avenue for validation of label-free imaging markers through the examination of how label-free image features correspond to gene expression. Toward this aim, we performed autofluorescence and fluorescence lifetime imaging on tissue specimens from four patients presenting with pancreatic neuroendocrine tumors. We then performed spatial transcriptome sequencing on serial tissue sections to measure transcriptome-wide signatures. We assessed imaging biomarkers related to cellular metabolism, vasculature, and extracellular matrix properties. After registering the label-free images to the transcriptomic signatures, we performed k-means clustering, and assessed the correlation between imaging markers and differentially expressed genes associated with tissue properties of interest. Specifically, we aimed to examine correlations between gene expression and established optical biomarkers (e.g., optical redox ratio), along with identifying other potential connections between label-free optics and cellular genetics. The results show that spatial transcriptomics can be used as an effective validation tool for label-free imaging markers, while simultaneously providing additional biological insight to improve the specificity of imaging studies.
In-situ identification of glioma subtype can enable modifications of clinical and surgical strategies. Particularly, astrocytoma benefit from more aggressive resection than oligodendroglioma, which have a more favorable response to post-surgical chemotherapy. Preoperative MRI and intraoperative histology cannot accurately determine glioma subtype. There is a need for real-time identification of adult-type diffuse glioma subtypes to aid the neurosurgeon’s decision-making during resection surgery. Fluorescence lifetime imaging (FLIm) where tissue autofluorescence can be used as an indicator to distinguish among brain tumor tissue types in real-time could aid this process. Here, we report the use of label-free FLIm in distinguishing IDH-mutant glioma subtypes (astrocytoma and oligodendroglioma). The FLIm system (excitation: 355 nm; emission bands: 390/40 nm, 470/28 nm, 542/50 nm) was used to scan brain tissue from the resection margins of glioma patients during tumor resection. Fluorescence lifetimes were extracted and analyzed by constrained least-squares deconvolution with the Laguerre expansion method. FLIm data was validated with histopathology of collected biopsies. Current results show that FLIm provides optical contrast between tumor and healthy white matter, and between IDH-mutant astrocytoma (N=7 patients) and oligodendroglioma (N=5 patients). Tumors showed shorter lifetime values (470-nm: 3.6±0.6ns; 542-nm: 3.3±0.7ns) than healthy white matter (470-nm: 4.6±0.4ns; 542-nm: 4.3±0.5ns, p<0.01). Oligodendroglioma had shorter lifetimes in the 470-nm (3.3±0.1ns) and 542-nm (2.8±0.2ns) channels, which are associated with NAD(P)H and FAD fluorescence respectively, when compared with IDH-mutant astrocytoma (470-nm: 4.1±0.1ns; 542-nm: 3.9±0.2ns, p<0.01). Together, these results demonstrate the feasibility of using FLIm as an intraoperative tool in glioma diagnosis.
The standard treatment for infiltrative gliomas is surgery to remove as much tumor tissue as possible without compromising neurological functions. Thus, real-time identification of infiltrative tumor tissue is necessary. Here a fluorescence lifetime imaging (FLIm) was used to distinguish between healthy brain and areas with different degrees of tumor cellularity as defined by histopathology. We conducted FLIm measurements and collected microbiopsies from tumor resection margins to identify the FLIm characteristics of tumor edges in cortex and white matter of low- and high-grade gliomas. Results from a 13-patient cohort indicate that FLIm identifies infiltrations of up to moderate tumor cellularity.
5-ALA induced PpIX is increasingly used for guiding brain tumor resection surgery. The current intensity-based approach fails at detecting lower concentrations of PpIX found in low-grade gliomas or the infiltrative edge of glioblastomas. Here, we report the first results in patients of real-time, PpIX fluorescence lifetime measurements using a hand-held fiber probe. Fluorescence from different spectral channels (390/40 nm (Collagen), 470/28 nm (NADH), 629/53 nm (PPIX)), is augmented onto a video stream of the surgical field-of-view to provide intraoperative tumor visualization in real-time. In-vivo data reveals strong contrast between regions of high PpIX accumulation associated with tumor (>8 ns) and healthy brain tissue (<4 ns).
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.
Gastrointestinal disorders such as colorectal cancer or inflammatory bowel disease are linked to gut dysbiosis, an unbalanced gut microbiota. This early manifestation of the disease alters colon epithelial metabolism influencing the gut autofluorescence emission, which is susceptible to carry diagnostic value. We analyzed the fluorescence properties of healthy and dysbiotic ex vivo murine colons with an intraluminal fiber-based fluorescence lifetime imaging (FLIm) instrument. The results indicate that fluorescence lifetime reacts to inflammation in a spectrally dependant manner, and the full-length colon images allow to localize specific areas of activity. Imaging results were correlated to biochemical metabolic readouts (i.e. intracellular NADH, lactate) to establish the diagnostic potential of intraluminal FLIm.
We demonstrated the ability of fiber-based Fluorescence Lifetime Imaging (FLIm) guided Raman spectroscopy to monitor the quality of engineered vascular grafts with high speed and specificity. We report FLIm guided Raman imaging as an effective multimodal technique to evaluate scaffold cross-linking and localized calcification. Current results indicate that the lifetime of AR-BP shortens upon GA cross-linking,and Raman spectroscopy reveals secondary structural changes occurring in the Amide I region of cross-linked pericardia. GA fixed vascular grafts are prone to calcification, an effect linked to graft failure. The calcified regions exhibited shorter lifetimes in fluorescence spectral bands ranging from 380 to 455 nm and Raman spectra exhibited the specific hydroxyapatite signature at 960 cm-1 co-localized with these lower lifetime regions. We conclude that FLIm guided Raman imaging can detect cross-linking signatures and areas of calcification in tissue with biochemical specificity.
Endogenous and exogenous fluorescence imaging have shown great values for monitoring and studying all kind of biological processes including vascular tissue regeneration and disease progression. In this study, we present a fiber-based optical imaging instrument that is able to simultaneously acquire endogenous and exogenous fluorescence images from tissue samples using a reflective optical chopper wheel to temporally interleave the two modalities. The functionality of the system was demonstrated by imaging native tissue constructs seeded with cells labeled with different dyes. The cellular regions were clearly resolved in both modalities, providing an exogenous and in-situ validation for endogenous fluorescence lifetime images.
Genipin cross-linked engineered tissues are 10000 times less toxic than glutaraldehyde cross-linked tissues. Hence, genipin is a better fixative to support the recellularization of tissue-engineered constructs such as vascular grafts. Here, we demonstrate the ability of fiber-based Fluorescence Lifetime Imaging (FLIm) guided Raman spectroscopy to monitor the quality of genipin cross-linked vascular grafts with high speed and specificity.
Current results indicate that the fluorescence lifetime of AR-BP shortens upon GE cross-linking. Raman spectroscopy reveals secondary structural changes occurring in the extracellular matrix of pericardia that correspond to Amide I, Amide III and C-C stretch vibrations.
We conclude that FLIm guided Raman imaging can detect cross-linking signatures with biochemical specificity and that this imaging modality provides a non-destructive and label-free method to assess the quality of vascular grafts
Tissue engineered vascular graft (TEVG) are used when native vessels are not available to repair vascular damage. At the time of implantation in human body, these constructs present poor cellularity. To understand the cellularization kinetics under physiological conditions in a setting suitable for experimentation, bioreactors are often used in laboratory setting because of its controllable culture parameters including seeding conditions, flow type, pressure and temperature. Therefore, a non-destructive, label-free imaging modality that is capable of evaluating cell migration on luminal surfaces of TEVGs inside bioreactors is valuable for studying cellularization kinetics and providing a potential quality control method for manufacturing mature TEVGs. A multispectral Fluorescence Lifetime Imaging (ms-FLIm) using 355 nm excitation was configured to accommodate a rotating side-firing scanning probe for intraluminal imaging of tubular-shaped bovine pericardium (BP) scaffolds. The scanning was realized by reciprocal rotation and pullback of the fiber probe. Mesenchymal stem cells were seeded on BP-based TEVGs and cultured in the prototype bioreactor for up to one week. Distinct experimental conditions including the seeding side (i.e. BP serious and fibrous side) and media flow (i.e. static and dynamic pulsatile flow) were evaluated. Using ms-FLIm, the migration of cells on antigen removed BP TEVGs was periodically examined over a week; and the migration rates under different conditions were analyzed. Current results suggest helical ms-FLIm has potential to monitor in situ tissue recellularization process in bioreactors.
A fiber-based, label-free multispectral fluorescence lifetime imaging and intravascular ultrasound (FLIm/IVUS) system was evaluated as a new tool for monitoring variations in biochemical and structural composition of vascular biomaterials, including native arteries and engineered vascular grafts both in vitro and in vivo. Fiber-based FLIm was adapted to assess the hollow geometry of vasculature, allowing for imaging of the luminal surface of vessels. The capacity of FLIm to resolve tissue cellular location (i.e. scaffold reendothelialization) and collagen to elastin ratio on the vessel wall was investigated. Quantitative imaging parameters derived from spectrally- and temporally-resolved autofluorescence (i.e. intensity ratios and fluorescence lifetime) provide benchmark indicators to identify areas of recellularized tissue, and to distinguish wall matrix compositions within and across biomaterials. In addition, fiber-based FLIm was complemented with intravascular ultrasound (IVUS) for simultaneous in vivo evaluation of biochemical and structural tissue properties. Here, we performed an in vitro evaluation of pig carotid arteries and show correlations between FLIm parameters and biochemical composition in different anatomical locations. We discuss the spectral and lifetime differences between native pig carotid artery, acellular antigen removed bovine pericardium grafts, and reendotheliarized grafts. Finally, we translate the findings to an in vivo clinical FLIm/IVUS imaging study with antigen removed bovine pericardium grafted on healthy pig native carotid artery. Upon implantation, the graft is expected to repopulate with cells, and change composition as cells remodel it. These experiments demonstrate the feasibility of fiber-based FLIm/IVUS to examine vascular engineered tissue in research and clinical settings.
KEYWORDS: Collagen, Fluorescence lifetime imaging, In vivo imaging, Luminescence, Proteins, Molecules, Tissue engineering, Nondestructive evaluation, In vitro testing, Imaging systems
Bovine pericardium (BP) exhibits distinct biochemical and biomechanical properties that are dominant by the structural protein collagen. The enzymatic degradation of collagen molecules is critical for in vivo incorporation and remodeling of BP in tissue engineering applications. A non-destructive method for monitoring BP during degradation would provide a valuable tool for quantifying functional changes initially in vitro and ultimately in vivo. In this study, we demonstrated the sensitivity of multi-spectral fluorescence lifetime imaging system (ms-FLIm) developed by our group to collagen content and compressive modulus of BP during collagenase degradation. A pairwise study was performed using bacterial collagenase to partially digest BP. We measured the biomaterials properties with ms-FLIm and destructive conventional measurements including collagen assay, compressive test and histology. A single factor study design was utilized. Test group samples were digested by bacterial collagenase for 0, 8, 16 and 24 hours, while control group samples were prepared in the Hank’s balanced salt solution to control for time in solution. Statistical analysis was performed using the Kendall τB correlation test. The results demonstrate that fluorescence parameters measured by ms-FLIm are significantly correlated with collagen content and compressive modulus (|τB| > 0.45, p < 0.05). Based on these findings, we aim to predict BP’s collagen content and mechanical properties using fluorescence metrics, and ultimately apply ms-FLIm for non-destructively monitoring of in vivo remodeling of BP.
Techniques that dynamically assess the maturation of tissue engineered constructs allow more efficient longitudinal control of developmental parameters than traditional destructive analyses, enhancing the likelihood of successful outcomes. We present a non-destructive and minimally invasive imaging method to monitor the growth of engineered vascular tissue based on label-free fluorescence lifetime imaging (FLIm) using a single fiber optic interface. We demonstrate the potential of the fiber-based FLIm system on vascular grafts composed of antigen removed bovine pericardium extracellular scaffolds seeded with human endothelial or mesenchymal stem cells. Tissue constructs are illuminated with 355 nm pulsed laser light that excites tissue autofluorescence, stemming from scaffold proteins (e.g., collagen), and cellular metabolic co-factors (i.e., NADH and FAD). Fluorescence lifetime images are acquired by scanning the distal tip of a multimode fiber across the sample surface, to deliver fluorescence excitation and collect fluorescence emission. A wavelength selection module is used to spectrally separate autofluorescence into four spectral bands that were selected to match the emission peaks of the main tissue fluorophores. By examining the relative intensity and mean fluorescence lifetime in each spectral band we identify the composition of engineered tissues, and evaluate the progression of recellularization. The fiber-based apparatus is compatible with imaging a range of sample geometries including planar and tubular constructs, and imaging in regions of restricted space such as inside tissue bioreactors, or in vivo. Future applications for the system include longitudinal monitoring of the luminal surface of engineered vascular tissues, or intravascular imaging in vivo to monitor viability of vascular implants.
Surface enhanced Raman scattering (SERS) is a popular technique for detecting and analyzing molecules at very low concentrations. The sensitivity of SERS is high enough to detect single molecules. It has proven difficult, however, to perform similar measurements in the so-called nonlinear optical regime, a regime in which the molecule is responding to multiple light pulses. Nonetheless, recent experiments indicate that after careful optimization, it is possible to generate signals derived from nonlinear analogs of SERS. Such measurements make it possible to view molecular vibrations in real time, which amounts to the femto- to pico-second range. In this contribution, we discuss in detail under which conditions detectable surface-enhanced coherent Raman signals can be expected, provide experimental evidence of coherent Raman scattering of single molecules, and highlight the unique information that can be attained from such measurements.
We discuss the implementation of wavelength-swept coherent Raman scattering (CRS) microscopy for the rapid acquisition of hyperspectral datacubes. We highlight two multivariate analysis approaches for efficiently generating spectroscopic maps from the acquired data: principal component analysis (PCA), which is a popular method for extracting information from multidimensional datasets, and vertex component analysis (VCA), which has previously been successfully used for the analysis of spontaneous Raman microscopy data. Through several biomedical imaging examples, we discuss the advantages and disadvantages of these approaches for CRS microscopy.
Macrophages adopt a variety of phenotypes that are a reflection of the many functions they perform as part of the immune system. In particular, metabolism is a phenotypic trait that differs between classically activated, proinflammatory macrophages, and alternatively activated, prohealing macrophages. Inflammatory macrophages have a metabolism based on glycolysis while alternatively activated macrophages generally rely on oxidative phosphorylation to generate chemical energy. We employ this shift in metabolism as an endogenous marker to identify the phenotype of individual macrophages via live-cell fluorescence lifetime imaging microscopy (FLIM). We demonstrate that polarized macrophages can be readily discriminated with the aid of a phasor approach to FLIM, which provides a fast and model-free method for analyzing fluorescence lifetime images.
We generated a highly deuterated cholesterol analog (D38-cholesterol) and demonstrated its use for selective vibrational imaging of cholesterol storage in mammalian cells. D38-cholesterol produces detectable signals in stimulated Raman scattering (SRS) imaging, is rapidly taken up by cells, and is efficiently metabolized by acyl-CoA cholesterol acyltransferase to form cholesteryl esters. Using hyperspectral SRS imaging of D38-cholesterol, we visualized cholesterol storage in lipid droplets. We found that some lipid droplets accumulated preferentially unesterified D38-cholesterol, whereas others stored D38-cholesteryl esters. In steroidogenic cells, D38-cholesteryl esters and triacylglycerols were partitioned into distinct sets of lipid droplets. Thus, hyperspectral SRS imaging of D38-cholesterol demonstrates a heterogeneous incorporation of neutral lipid species, i.e., free cholesterol, cholesteryl esters, and triacylglycerols, between individual lipid droplets in a cell.
Coherent Raman scattering (CRS) microscopy is gaining acceptance as a valuable addition to the imaging toolset of biological researchers. Optimal use of this label-free imaging technique benefits from a basic understanding of the physical principles and technical merits of the CRS microscope. This tutorial offers qualitative explanations of the principles behind CRS microscopy and provides information about the applicability of this nonlinear optical imaging approach for biological research.
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