Label-Free Optical Metabolic Imaging in Cells and Tissues.

Over the last half century, the autofluorescence of the metabolic cofactors NADH (reduced nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) has been quantified in a variety of cell types and disease states. With the spread of nonlinear optical microscopy techniques in biomedical research, NADH and FAD imaging has offered an attractive solution to noninvasively monitor cell and tissue status and elucidate dynamic changes in cell or tissue metabolism. Various tools and methods to measure the temporal, spectral, and spatial properties of NADH and FAD autofluorescence have been developed. Specifically, an optical redox ratio of cofactor fluorescence intensities and NADH fluorescence lifetime parameters have been used in numerous applications, but significant work remains to mature this technology for understanding dynamic changes in metabolism. This article describes the current understanding of our optical sensitivity to different metabolic pathways and highlights current challenges in the field. Recent progress in addressing these challenges and acquiring more quantitative information in faster and more metabolically relevant formats is also discussed.

Development of a novel bioengineered 3D brain-like tissue for studying primary blast-induced traumatic brain injury.

Primary blast injury is caused by the direct impact of an overpressurization wave on the body. Due to limitations of current models, we have developed a novel approach to study primary blast-induced traumatic brain injury. Specifically, we employ a bioengineered 3D brain-like human tissue culture system composed of collagen-infused silk protein donut-like hydrogels embedded with human IPSC-derived neurons, human astrocytes, and a human microglial cell line. We have utilized this system within an advanced blast simulator (ABS) to expose the 3D brain cultures to a blast wave that can be precisely controlled. These 3D cultures are enclosed in a 3D-printed surrogate skull-like material containing media which are then placed in a holder apparatus inside the ABS. This allows for exposure to the blast wave alone without any secondary injury occurring. We show that blast induces an increase in lactate dehydrogenase activity and glutamate release from the cultures, indicating cellular injury. Additionally, we observe a significant increase in axonal varicosities after blast. These varicosities can be stained with antibodies recognizing amyloid precursor protein. The presence of amyloid precursor protein deposits may indicate a blast-induced axonal transport deficit. After blast injury, we find a transient release of the known TBI biomarkers, UCHL1 and NF-H at 6 h and a delayed increase in S100B at 24 and 48 h. This in vitro model will enable us to gain a better understanding of clinically relevant pathological changes that occur following primary blast and can also be utilized for discovery and characterization of biomarkers.

Tissue Imaging and Quantification Relying on Endogenous Contrast.

Cell-matrix interactions play an important role in regulating a variety of essential processes in multicellular organisms, and are closely associated with numerous diseases. Modified interactions have major effects upon key features of both cells and extracellular matrix (ECM), and a thorough understanding of changes in these features can lead to critically important insights of diseases as well as the identification of effective therapeutic targets. Here, we summarize recent advances in quantitative, optical imaging of cellular metabolism and ECM spatial organization using endogenous sources of contrast. Specifically, we focus on the two-photon excited fluorescence (TPEF) imaging of autofluorescent cellular coenzymes, NAD(P)H and FAD, for the extraction of metabolic information described by optical biomarkers including cellular redox state, NAD(P)H fluorescence lifetime, and mitochondrial clustering. We show representative applications in assessing adipose tissue function and detecting malignant lesions in human skin, and further demonstrate that a combination of these optical metrics can provide complementary insights into the underlying biological mechanisms. In addition, we review the development of quantitative analysis methods to extract spatial orientation and organization metrics of collagen fibers, a major ECM component, and demonstrate applications of these approaches in two and three dimensions in several diseases, including would healing, osteoarthritis and cancer, as well as assessments of matrix remodeling in hormone-regulated engineered breast tissues. Finally, we summarize this chapter and discuss important research directions that we expect will evolve in the near future.

An essential role for IGF2 in cartilage development and glucose metabolism during postnatal long bone growth.

Postnatal bone growth involves a dramatic increase in length and girth. Intriguingly, this period of growth is independent of growth hormone and the underlying mechanism is poorly understood. Recently, an IGF2 mutation was identified in humans with early postnatal growth restriction. Here, we show that IGF2 is essential for longitudinal and appositional murine postnatal bone development, which involves proper timing of chondrocyte maturation and perichondrial cell differentiation and survival. Importantly, the Igf2 null mouse model does not represent a simple delay of growth but instead uncoordinated growth plate development. Furthermore, biochemical and two-photon imaging analyses identified elevated and imbalanced glucose metabolism in the Igf2 null mouse. Attenuation of glycolysis rescued the mutant phenotype of premature cartilage maturation, thereby indicating that IGF2 controls bone growth by regulating glucose metabolism in chondrocytes. This work links glucose metabolism with cartilage development and provides insight into the fundamental understanding of human growth abnormalities.

Lysyl oxidase enzymes mediate TGF-ß1-induced fibrotic phenotypes in human skin-like tissues.

Cutaneous fibrosis is a common complication seen in mixed connective tissue diseases. It often occurs as a result of TGF-ß-induced deposition of excessive amounts of collagen in the skin. Lysyl oxidases (LOXs), a family of extracellular matrix (ECM)-modifying enzymes responsible for collagen cross-linking, are known to be increased in dermal fibroblasts from patients with fibrotic diseases, denoting a possible role of LOXs in fibrosis. To directly study this, we have developed two bioengineered, in vitro skin-like models: human skin equivalents (hSEs), and self-assembled stromal tissues (SASs) that contain either normal or systemic sclerosis (SSc; scleroderma) patient-derived fibroblasts. These tissues provide an organ-level structure that could be combined with non-invasive, label-free, multiphoton microscopy (SHG/TPEF) to reveal alterations in the organization and cross-linking levels of collagen fibers during the development of cutaneous fibrosis, which demonstrated increased stromal rigidity and activation of dermal fibroblasts in response to TGF-ß1. Specifically, inhibition of specific LOXs isoforms, LOX and LOXL4, in foreskin fibroblasts (HFFs) resulted in antagonistic effects on TGF-ß1-induced fibrogenic hallmarks in both hSEs and SASs. In addition, a translational relevance of these models was seen as similar antifibrogenic phenotypes were achieved upon knocking down LOXL4 in tissues containing SSc patient-derived-dermal fibroblasts (SScDFs). These findings point to a pivotal role of LOXs in TGF-ß1-induced cutaneous fibrosis through impaired ECM homeostasis in skin-like tissues, and show the value of these tissue platforms in accelerating the discovery of antifibrosis therapeutics.

Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles.

A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives the electrostatic assembly of nanoparticles that mediate potent protein delivery and genome editing. These bioreducible lipids efficiently deliver protein cargo into cells, facilitate the escape of protein from endosomes in response to the reductive intracellular environment, and direct protein to its intracellular target sites. The delivery of supercharged Cre protein and Cas9:sgRNA complexed with bioreducible lipids into cultured human cells enables gene recombination and genome editing with efficiencies greater than 70%. In addition, we demonstrate that these lipids are effective for functional protein delivery into mouse brain for gene recombination in vivo. Therefore, the integration of this bioreducible lipid platform with protein engineering has the potential to advance the therapeutic relevance of protein-based genome editing.

Bioluminescence and second harmonic generation imaging reveal dynamic changes in the inflammatory and collagen landscape in early osteoarthritis.

Osteoarthritis (OA) is a leading cause of chronic disability whose mechanism of pathogenesis is largely elusive. Local inflammation is thought to play a key role in OA progression, especially in injury-associated OA. While multiple inflammatory cytokines are detected, the timing and extent of overall inflammatory activities in early OA and the manner by which joint inflammation correlates with cartilage structural damage are still unclear. We induced OA via destabilization of the medial meniscus (DMM) in NFκB luciferase reporter mice, whose bioluminescent signal reflects the activity of NFκB, a central mediator of inflammation. Bioluminescence imaging data showed that DMM and sham control joints had a similar surge of inflammation at 1-week post-surgery, but the DMM joint exhibited a delay in resolution of inflammation in subsequent weeks. A similar trend was observed with synovitis, which we found to be mainly driven by synovial cell density and inflammatory infiltration rather than synovial lining thickness. Interestingly, an association between synovitis and collagen structural damage was observed in early OA. Using Second Harmonic Generation (SHG) imaging, we analyzed collagen fiber organization in articular cartilage. Zonal differences in collagen fiber thickness and organization were observed as soon as OA initiated after DMM surgery, and persisted over time. Even at 1-week post-surgery, the DMM joint showed a decrease in collagen fiber thickness in the deep zone and an increase in collagen fiber disorganization in the superficial zone. Since we were able detect and quantify collagen structural changes very early in OA development by SHG imaging, we concluded that SHG imaging is a highly sensitive tool to evaluate pathological changes in OA. In summary, this study uncovered a dynamic profile of inflammation and joint cartilage damage during OA initiation and development, providing novel insights into OA pathology.

UV and NIR-Responsive Layer-by-Layer Films Containing 6-Bromo-7-hydroxycoumarin Photolabile Groups.

This paper describes polyelectrolyte multilayer films prepared by the layer-by-layer (LbL) technique capable of undergoing dissolution upon exposure to either ultraviolet or near-infrared light. Film dissolution is driven by photochemical deprotection of a random methacrylic copolymer with two types of side chains: (i) 6-bromo-7-hydroxycoumarinyl esters, photocleavable groups that are known to have substantial two-photon photolysis cross sections, and (ii) cationic residues from the commercially available monomer N,N-dimethylaminoethyl methacrylate (DMAEMA). In addition, the dependence of stability of both unirradiated and irradiated films on pH provides experimental evidence for the necessity of disrupting both ion-pairing and hydrophobic interactions between polyelectrolytes to realize film dissolution. This work therefore provides both new fundamental insight regarding photolabile LbL films and expands their applied capabilities to nonlinear photochemical processes.

Laparoscopic narrow band imaging for detection of occult cancer metastases: a randomized feasibility trial.

BACKGROUND: Selection of cancer treatment fundamentally relies on staging of the underlying malignancy. The aim of this study was to evaluate the feasibility and effectiveness of laparoscopic narrow band imaging (NBI) for operative staging and detection of occult peritoneal cancer metastases. METHODS: A randomized, controlled feasibility trial with crossover design evaluating adult patients with gastrointestinal or gynecologic malignancies who have a clinical indication for diagnostic laparoscopy was conducted. Twenty-three patients were randomized to white-light followed by NBI laparoscopy (n = 11) or NBI followed by white-light laparoscopy (n = 12) using the Olympus Evis Exera II system. Three patients were excluded from analysis. RESULTS: In all 20 study patients, the abdominal cavity was sufficiently illuminated. An enhanced contrast of microvasculature and organ surface pattern was appreciated. Eight of the 20 patients (40%) were found to have metastases of the peritoneal surface. While NBI did not show any additional peritoneal lesions, 2 of the 63 suspicious-appearing nodules seen on white-light imaging were not visible on NBI (p = 0.50). The median diameter of all the nodules identified was 2 mm (range 1-50 mm) and was identical with each method. CONCLUSIONS: The information from this feasibility study demonstrated that NBI provides adequate illumination of the abdominal cavity and a unique contrast that enhances microvasculature and architectural surface pattern. The results suggest that NBI laparoscopy is not superior in detecting peritoneal metastases compared to standard white-light laparoscopy, but might provide a technology that could be applied for other abdominal pathologies.

Characterization of mechanical and biochemical properties of developing embryonic tendon.

Tendons have uniquely high tensile strength, critical to their function to transfer force from muscle to bone. When injured, their innate healing response results in aberrant matrix organization and functional properties. Efforts to regenerate tendon are challenged by limited understanding of its normal development. Consequently, there are few known markers to assess tendon formation and parameters to design tissue engineering scaffolds. We profiled mechanical and biological properties of embryonic tendon and demonstrated functional properties of developing tendon are not wholly reflected by protein expression and tissue morphology. Using force volume-atomic force microscopy, we found that nano- and microscale tendon elastic moduli increase nonlinearly and become increasingly spatially heterogeneous during embryonic development. When we analyzed potential biochemical contributors to modulus, we found statistically significant but weak correlation between elastic modulus and collagen content, and no correlation with DNA or glycosaminoglycan content, indicating there are additional contributors to mechanical properties. To investigate collagen cross-linking as a potential contributor, we inhibited lysyl oxidase-mediated collagen cross-linking, which significantly reduced tendon elastic modulus without affecting collagen morphology or DNA, glycosaminoglycan, and collagen content. This suggests that lysyl oxidase-mediated cross-linking plays a significant role in the development of embryonic tendon functional properties and demonstrates that changes in cross-links alter mechanical properties without affecting matrix content and organization. Taken together, these data demonstrate the importance of functional markers to assess tendon development and provide a profile of tenogenic mechanical properties that may be implemented in tissue engineering scaffold design to mechanoregulate new tendon regeneration.

Noninvasive assessment of mitochondrial organization in three-dimensional tissues reveals changes associated with cancer development.

Mitochondrial organization is often altered to accommodate cellular bioenergetic and biosynthetic demands. Changes in metabolism are a hallmark of a number of diseases, including cancer; however, the interdependence between mitochondrial metabolic function and organization is not well understood. Here, we present a noninvasive, automated and quantitative method to assess mitochondrial organization in three-dimensional (3D) tissues using exclusively endogenous two-photon excited fluorescence (TPEF) and show that mitochondrial organization reflects alterations in metabolic activities. Specifically, we examine the organization of mitochondria within live, engineered epithelial tissue equivalents that mimic normal and precancerous human squamous epithelial tissues. We identify unique patterns of mitochondrial organization in the different tissue models we examine, and we attribute these to differences in the metabolic profiles of these tissues. We find that mitochondria are clustered in tissues with high levels of glycolysis and are more highly networked in tissues where oxidative phosphorylation is more dominant. The most highly networked organization is observed within cells with high levels of glutamine consumption. Furthermore, we demonstrate that mitochondrial organization provides complementary information to traditional morphological hallmarks of cancer development, including variations in nuclear size. Finally, we present evidence that this automated quantitative analysis of endogenous TPEF images can identify differences in the mitochondrial organization of freshly excised normal and pre-cancerous human cervical tissue specimens. Thus, this method could be a promising new modality to assess the role of mitochondrial organization in the metabolic activity of 3D tissues and could be further developed to serve as an early cancer clinical diagnostic biomarker.

An automated image processing method to quantify collagen fibre organization within cutaneous scar tissue.

Standard approaches to evaluate scar formation within histological sections rely on qualitative evaluations and scoring, which limits our understanding of the remodelling process. We have recently developed an image analysis technique for the rapid quantification of fibre alignment at each pixel location. The goal of this study was to evaluate its application for quantitatively mapping scar formation in histological sections of cutaneous burns. To this end, we utilized directional statistics to define maps of fibre density and directional variance from Masson's trichrome-stained sections for quantifying changes in collagen organization during scar remodelling. Significant increases in collagen fibre density are detectable soon after burn injury in a rat model. Decreased fibre directional variance in the scar was also detectable between 3 weeks and 6 months after injury, indicating increasing fibre alignment. This automated analysis of fibre organization can provide objective surrogate endpoints for evaluating cutaneous wound repair and regeneration.

From shadow to light: visualization of extrahepatic bile ducts using image-enhanced laparoscopy.

BACKGROUND: Correct recognition of the extrahepatic bile ducts is thought to be crucial to reduce the risk of bile duct injuries during various laparoscopic procedures. Image-enhanced laparoscopy techniques, utilizing various optical modalities other than white light, may help in detecting structures "hidden" underneath connective tissue. METHODS: A systematic literature search was conducted of studies describing image-enhanced laparoscopy techniques for visualization of the extrahepatic bile ducts. RESULTS: In all, 29 articles met inclusion criteria. They describe various techniques in the animal or human setting, including autofluorescence imaging, drug-enhanced fluorescence imaging, infrared thermography, and spectral imaging. This review describes these various techniques and their results. CONCLUSION: Image-enhanced laparoscopy techniques for real-time visualization of extrahepatic bile ducts are still in its infancy. Out of the techniques currently described, indocyanine green-enhanced near-infrared fluorescence laparoscopy has the most mature results, but other techniques also appear promising. It can be expected that in the future, image-enhanced laparoscopy might become a routine adjunct to any white-light laparoscopic operation near the hepatic hilum.

Cell-tethered ligands modulate bone remodeling by osteoblasts and osteoclasts.

The goals of the present study are to establish an in vitro co-culture model of osteoblast and osteoclast function and to quantify the resulting bone remodeling. The bone is tissue engineered using well-defined silk protein biomaterials in 2D and 3D formats in combination with human cells expressing tethered agonists for selected G protein-coupled receptors (GPCRs). The tethered constructs are introduced with the objective of triggering sustained and localized GPCR signaling. The cell-modified biomaterial surfaces are reconstructed from SEM images into 3D models using image processing for quantitative measurement of surface characteristics. Parathyroid hormone (PTH) and glucose-dependent insulinotropic peptide (GIP) are selected because of their roles in bone remodeling for expression in tethered format on bone marrow derived human mesenchymal stem cells (hMSCs). Increased calcium deposition and increased surface roughness are found in 3D digital surface models constructed from SEM images of silk protein films remodeled by the co-cultures containing the tethered PTH, and decreased surface roughness is found for the films remodeled by the tethered GIP co-cultures. Increased surface roughness is not found in monocultures of hMSCs expressing tethered PTH, suggesting that osteoclast-osteoblast interactions in the presence of PTH signaling are responsible for the increased mineralization. These data point towards the design of in vitro bone models in which osteoblast-osteoclast interactions are mimicked for a better understanding of bone remodeling.

Deep learning-enabled detection of rare circulating tumor cell clusters in whole blood using label-free, flow cytometry.

Metastatic tumors have poor prognoses for progression-free and overall survival for all cancer patients. Rare circulating tumor cells (CTCs) and rarer circulating tumor cell clusters (CTCCs) are potential biomarkers of metastatic growth, with CTCCs representing an increased risk factor for metastasis. Current detection platforms are optimized for ex vivo detection of CTCs only. Microfluidic chips and size exclusion methods have been proposed for CTCC detection; however, they lack in vivo utility and real-time monitoring capability. Confocal backscatter and fluorescence flow cytometry (BSFC) has been used for label-free detection of CTCCs in whole blood based on machine learning (ML) enabled peak classification. Here, we expand to a deep-learning (DL)-based, peak detection and classification model to detect CTCCs in whole blood data. We demonstrate that DL-based BSFC has a low false alarm rate of 0.78 events per min with a high Pearson correlation coefficient of 0.943 between detected events and expected events. DL-based BSFC of whole blood maintains a detection purity of 72% and a sensitivity of 35.3% for both homotypic and heterotypic CTCCs starting at a minimum size of two cells. We also demonstrate through artificial spiking studies that DL-based BSFC is sensitive to changes in the number of CTCCs present in the samples and does not add variability in detection beyond the expected variability from Poisson statistics. The performance established by DL-based BSFC motivates its use for in vivo detection of CTCCs. Using transfer learning, we additionally validate DL-based BSFC on blood samples from different species and cancer cell types. Further developments of label-free BSFC to enhance throughput could lead to critical applications in the clinical detection of CTCCs and ex vivo isolation of CTCC from whole blood with minimal disruption and processing steps.

Two photon imaging probe with highly efficient autofluorescence collection at high scattering and deep imaging conditions.

In this paper, we present a 2-photon imaging probe system featuring a novel fluorescence collection method with improved and reliable efficiency. The system aims to miniaturize the potential of 2-photon imaging in the metabolic and morphological characterization of cervical tissue at sub-micron resolution over large imaging depths into a flexible and clinically viable platform towards the early detection of cancers. Clinical implementation of such a probe system is challenging due to inherently low levels of autofluorescence, particularly when imaging deep in highly scattering tissues. For an efficient collection of fluorescence signals, our probe employs 12 0.5 NA collection fibers arranged around a miniaturized excitation objective. By bending and terminating a multitude of collection fibers at a specific angle, we increase collection area and directivity significantly. Positioning of these fibers allows the collection of fluorescence photons scattered away from their ballistic trajectory multiple times, which offers a system collection efficiency of 4%, which is 55% of what our bench-top microscope with 0.75 NA objective achieves. We demonstrate that the collection efficiency is largely maintained even at high scattering conditions and high imaging depths. Radial symmetry of arrangement maintains uniformity of collection efficiency across the whole FOV. Additionally, our probe can image at different tissue depths via axial actuation by a dc servo motor, allowing depth dependent tissue characterization. We designed our probe to perform imaging at 775 nm, targeting 2-photon autofluorescence from NAD(P)H and FAD molecules, which are often used in metabolic tissue characterization. An air core photonic bandgap fiber delivers laser pulses of 100 fs duration to the sample. A miniaturized objective designed with commercially available lenses of 3 mm diameter focuses the laser beam on tissue, attaining lateral and axial imaging resolutions of 0.66 µm and 4.65 µm, respectively. Characterization results verify that our probe achieves collection efficiency comparable to our optimized bench-top 2-photon imaging microscope, minimally affected by imaging depth and radial positioning. We validate autofluorescence imaging capability with excised porcine vocal fold tissue samples. Images with 120 µm FOV and 0.33 µm pixel sizes collected at 2 fps confirm that the 300 µm imaging depth was achieved.

Rapid, high-resolution, non-destructive assessments of metabolic and morphological homogeneity uniquely identify high-grade cervical precancerous lesions.

Purpose: Two-photon microscopy (2PM) is an emerging clinical imaging modality with the potential to non-invasively assess tissue metabolism and morphology in high-resolution. This study aimed to assess the translational potential of 2PM for improved detection of high-grade cervical precancerous lesions. Experimental Design: 2P images attributed to reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and oxidized flavoproteins (FP) were acquired from the full epithelial thickness of freshly excised human cervical tissue biopsies (N = 62). Fifteen biopsies harbored high-grade squamous intraepithelial lesions (HSILs), 14 biopsies harbored low-grade SILs (LSILs), and 33 biopsies were benign. Quadratic discriminant analysis (QDA) leveraged morphological and metabolic functional metrics extracted from these images to predict the presence of HSILs. We performed gene set enrichment analysis (GSEA) using datasets available on the Gene Expression Omnibus (GEO) to validate the presence of metabolic reprogramming in HSILs. Results: Integrating metabolic and morphological 2P-derived metrics from finely sampled, full-thickness epithelia achieved a high 90.8 ± 6.1% sensitivity and 72.3 ± 11.3% specificity of HSIL detection. Notably, sensitivity (91.4 ± 12.0%) and specificity (77.5 ± 12.6%) were maintained when utilizing metrics from only two images at 12- and 72-µm from the tissue surface. Upregulation of glycolysis, fatty acid metabolism, and oxidative phosphorylation in HSIL tissues validated the metabolic reprogramming captured by 2P biomarkers. Conclusion: Label-free 2P images from as few as two epithelial depths enable rapid and robust HSIL detection through the quantitative characterization of metabolic and morphological reprogramming, underscoring the potential of this tool for clinical evaluation of cervical precancers.

A mitochondrial NADPH-cholesterol axis regulates extracellular vesicle biogenesis to support hematopoietic stem cell fate.

Mitochondrial fatty acid oxidation (FAO) is essential for hematopoietic stem cell (HSC) self-renewal; however, the mechanism by which mitochondrial metabolism controls HSC fate remains unknown. Here, we show that within the hematopoietic lineage, HSCs have the largest mitochondrial NADPH pools, which are required for proper HSC cell fate and homeostasis. Bioinformatic analysis of the HSC transcriptome, biochemical assays, and genetic inactivation of FAO all indicate that FAO-generated NADPH fuels cholesterol synthesis in HSCs. Interference with FAO disturbs the segregation of mitochondrial NADPH toward corresponding daughter cells upon single HSC division. Importantly, we have found that the FAO-NADPH-cholesterol axis drives extracellular vesicle (EV) biogenesis and release in HSCs, while inhibition of EV signaling impairs HSC self-renewal. These data reveal the existence of a mitochondrial NADPH-cholesterol axis for EV biogenesis that is required for hematopoietic homeostasis and highlight the non-stochastic nature of HSC fate determination.

Optical imaging using endogenous contrast to assess metabolic state.

Optical microscopic imaging offers opportunities to perform noninvasive assessments of numerous parameters associated with the biochemistry, morphology, and functional state of biological samples. For example, it is possible to detect the endogenous fluorescence from a small number of important biomolecules, including NADH and FAD, which are two coenzymes involved in key metabolic pathways such as glycolysis, the Krebs cycle, and oxidative phosphorylation. Here, we review different imaging approaches to isolate the fluorescence from these chromophores in two- and three-dimensional samples and discuss the origins and potential interpretation of the observed signals in terms of cell metabolic status. Finally, we discuss the challenges and limitations of these approaches, as well as important research directions that we expect will evolve in the near future.

Autocorrelation method for fractal analysis in nonrectangular image domains.

We report an autocorrelation-based approach that accurately measures fractal organization within arbitrarily shaped (nonrectangular) regions of interest of gray-scale images. It extends fractal analysis beyond what is possible using fast Fourier transforms and improves on a previous autocorrelation algorithm. We illustrate its use in detecting subtle changes in mitochondrial organization within murine fibroblasts expressing the human papillomavirus E7 oncogene.