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Shah VS, Hou J, Vinarsky V, Xu J, Surve MV, Lin CP, Rajagopal J. Autofluorescence imaging permits label-free cell type assignment and reveals the dynamic formation of airway secretory cell associated antigen passages (SAPs). eLife 2023; 12:e84375. [PMID: 36994985 PMCID: PMC10154029 DOI: 10.7554/elife.84375] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2022] [Accepted: 03/29/2023] [Indexed: 03/31/2023] Open
Abstract
The specific functional properties of a tissue are distributed amongst its component cell types. The various cells act coherently, as an ensemble, in order to execute a physiologic response. Modern approaches for identifying and dissecting novel physiologic mechanisms would benefit from an ability to identify specific cell types in live tissues that could then be imaged in real time. Current techniques require the use of fluorescent genetic reporters that are not only cumbersome, but which only allow the study of three or four cell types at a time. We report a non-invasive imaging modality that capitalizes on the endogenous autofluorescence signatures of the metabolic cofactors NAD(P)H and FAD. By marrying morphological characteristics with autofluorescence signatures, all seven of the airway epithelial cell types can be distinguished simultaneously in mouse tracheal explants in real time. Furthermore, we find that this methodology for direct cell type-specific identification avoids pitfalls associated with the use of ostensibly cell type-specific markers that are, in fact, altered by clinically relevant physiologic stimuli. Finally, we utilize this methodology to interrogate real-time physiology and identify dynamic secretory cell associated antigen passages (SAPs) that form in response to cholinergic stimulus. The identical process has been well documented in the intestine where the dynamic formation of SAPs and goblet cell associated antigen passages (GAPs) enable luminal antigen sampling. Airway secretory cells with SAPs are frequently juxtaposed to antigen presenting cells, suggesting that airway SAPs, like their intestinal counterparts, not only sample antigen but convey their cargo for immune cell processing.
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Affiliation(s)
- Viral S Shah
- Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, Massachusetts General HospitalBostonUnited States
- Center for Regenerative Medicine, Massachusetts General HospitalBostonUnited States
| | - Jue Hou
- Advanced Microscopy Program, Center for Systems Biology and Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical SchoolBostonUnited States
| | - Vladimir Vinarsky
- Center for Regenerative Medicine, Massachusetts General HospitalBostonUnited States
| | - Jiajie Xu
- Center for Regenerative Medicine, Massachusetts General HospitalBostonUnited States
| | - Manalee V Surve
- Center for Regenerative Medicine, Massachusetts General HospitalBostonUnited States
| | - Charles P Lin
- Advanced Microscopy Program, Center for Systems Biology and Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical SchoolBostonUnited States
| | - Jayaraj Rajagopal
- Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, Massachusetts General HospitalBostonUnited States
- Center for Regenerative Medicine, Massachusetts General HospitalBostonUnited States
- Harvard Stem Cell InstituteCambridgeUnited States
- Klarman Cell Observatory, Broad InstituteCambridgeUnited States
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O'Sullivan JDB, Bullen A, Mann ZF. Mitochondrial form and function in hair cells. Hear Res 2023; 428:108660. [PMID: 36525891 DOI: 10.1016/j.heares.2022.108660] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/30/2022] [Revised: 11/07/2022] [Accepted: 11/23/2022] [Indexed: 11/27/2022]
Abstract
Hair cells (HCs) are specialised sensory receptors residing in the neurosensory epithelia of inner ear sense organs. The precise morphological and physiological properties of HCs allow us to perceive sound and interact with the world around us. Mitochondria play a significant role in normal HC function and are also intricately involved in HC death. They generate ATP essential for sustaining the activity of ion pumps, Ca2+ transporters and the integrity of the stereociliary bundle during transduction as well as regulating cytosolic calcium homoeostasis during synaptic transmission. Advances in imaging techniques have allowed us to study mitochondrial populations throughout the HC, and how they interact with other organelles. These analyses have identified distinct mitochondrial populations between the apical and basolateral portions of the HC, in which mitochondrial morphology appears determined by the physiological processes in the different cellular compartments. Studies in HCs across species show that ototoxic agents, ageing and noise damage directly impact mitochondrial structure and function resulting in HC death. Deciphering the molecular mechanisms underlying this mitochondrial sensitivity, and how their morphology relates to their function during HC death, requires that we first understand this relationship in the context of normal HC function.
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Affiliation(s)
- James D B O'Sullivan
- Centre for Craniofacial and Regenerative Biology, Faculty of Dentistry, Oral, Craniofacial Sciences, King's College London, London SE1 9RT, U.K
| | - Anwen Bullen
- UCL Ear Institute, University College London, London WC1×8EE, U.K.
| | - Zoë F Mann
- Centre for Craniofacial and Regenerative Biology, Faculty of Dentistry, Oral, Craniofacial Sciences, King's College London, London SE1 9RT, U.K.
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Toward Personalized Diagnosis and Therapy for Hearing Loss: Insights From Cochlear Implants. Otol Neurotol 2022; 43:e903-e909. [PMID: 35970169 DOI: 10.1097/mao.0000000000003624] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
ABSTRACT Sensorineural hearing loss (SNHL) is the most common sensory deficit, disabling nearly half a billion people worldwide. The cochlear implant (CI) has transformed the treatment of patients with SNHL, having restored hearing to more than 800,000 people. The success of CIs has inspired multidisciplinary efforts to address the unmet need for personalized, cellular-level diagnosis, and treatment of patients with SNHL. Current limitations include an inability to safely and accurately image at high resolution and biopsy the inner ear, precluding the use of key structural and molecular information during diagnostic and treatment decisions. Furthermore, there remains a lack of pharmacological therapies for hearing loss, which can partially be attributed to challenges associated with new drug development. We highlight advances in diagnostic and therapeutic strategies for SNHL that will help accelerate the push toward precision medicine. In addition, we discuss technological improvements for the CI that will further enhance its functionality for future patients. This report highlights work that was originally presented by Dr. Stankovic as part of the Dr. John Niparko Memorial Lecture during the 2021 American Cochlear Implant Alliance annual meeting.
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Spankovich C, Walters BJ. Mild Therapeutic Hypothermia and Putative Mechanisms of Hair Cell Survival in the Cochlea. Antioxid Redox Signal 2021; 36:1203-1214. [PMID: 34619988 PMCID: PMC9221161 DOI: 10.1089/ars.2021.0184] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/27/2021] [Accepted: 09/29/2021] [Indexed: 12/20/2022]
Abstract
Significance: Sensorineural hearing loss has significant implications for quality of life and risk for comorbidities such as cognitive decline. Noise and ototoxic drugs represent two common risk factors for acquired hearing loss that are potentially preventable. Recent Advances: Numerous otoprotection strategies have been postulated over the past four decades with primary targets of upstream redox pathways. More recently, the application of mild therapeutic hypothermia (TH) has shown promise for otoprotection for multiple forms of acquired hearing loss. Critical Issues: Systemic antioxidant therapy may have limited application for certain ototoxic drugs with a therapeutic effect on redox pathways and diminished efficacy of the primary drug's therapeutic function (e.g., cisplatin for tumors). Future Directions: Mild TH likely targets multiple mechanisms, contributing to otoprotection, including slowed metabolics, reduced oxidative stress, and involvement of cold shock proteins. Further work is needed to identify the mechanisms of mild TH at play for various forms of acquired hearing loss.
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Affiliation(s)
- Christopher Spankovich
- Department of Otolaryngology-Head and Neck Surgery and University of Mississippi Medical Center, Jackson, Mississippi, USA
| | - Bradley J. Walters
- Department of Otolaryngology-Head and Neck Surgery and University of Mississippi Medical Center, Jackson, Mississippi, USA
- Department of Neurobiology and Anatomical Sciences, University of Mississippi Medical Center, Jackson, Mississippi, USA
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Pham DL, Miller CR, Myers MS, Myers DM, Hansen LA, Nichols MG. Development and characterization of phasor-based analysis for FLIM to evaluate the metabolic and epigenetic impact of HER2 inhibition on squamous cell carcinoma cultures. JOURNAL OF BIOMEDICAL OPTICS 2021; 26:JBO-210187R. [PMID: 34628733 PMCID: PMC8501457 DOI: 10.1117/1.jbo.26.10.106501] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 06/18/2021] [Accepted: 09/14/2021] [Indexed: 06/13/2023]
Abstract
SIGNIFICANCE Deranged metabolism and dysregulated growth factor signaling are closely associated with abnormal levels of proliferation, a recognized hallmark in tumorigenesis. Fluorescence lifetime imaging microscopy (FLIM) of endogenous nicotinamide adenine dinucleotide (NADH), a key metabolic coenzyme, offers a non-invasive, diagnostic indicator of disease progression, and treatment response. The model-independent phasor analysis approach leverages FLIM to rapidly evaluate cancer metabolism in response to targeted therapy. AIM We combined lifetime and phasor FLIM analysis to evaluate the influence of human epidermal growth factor receptor 2 (HER2) inhibition, a prevalent cancer biomarker, on both nuclear and cytoplasmic NAD(P)H of two squamous cell carcinoma (SCC) cultures. While better established, the standard lifetime analysis approach is relatively slow and potentially subject to intrinsic fitting errors and model assumptions. Phasor FLIM analysis offers a rapid, model-independent alternative, but the sensitivity of the bound NAD(P)H fraction to growth factor signaling must also be firmly established. APPROACH Two SCC cultures with low- and high-HER2 expression, were imaged using multiphoton-excited NAD(P)H FLIM, with and without treatment of the HER2 inhibitor AG825. Cells were challenged with mitochondrial inhibition and uncoupling to investigate AG825's impact on the overall metabolic capacity. Phasor FLIM and lifetime fitting analyses were compared within nuclear and cytoplasmic compartments to investigate epigenetic and metabolic impacts of HER2 inhibition. RESULTS NAD(P)H fluorescence lifetime and bound fraction consistently decreased following HER2 inhibition in both cell lines. High-HER2 SCC74B cells displayed a more significant response than low-HER2 SCC74A in both techniques. HER2 inhibition induced greater changes in nuclear than cytoplasmic compartments, leading to an increase in NAD(P)H intensity and concentration. CONCLUSIONS The use of both, complementary FLIM analysis techniques together with quantitative fluorescence intensity revealed consistent, quantitative changes in NAD(P)H metabolism associated with inhibition of growth factor signaling in SCC cell lines. HER2 inhibition promoted increased reliance on oxidative phosphorylation in both cell lines.
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Affiliation(s)
- Dan L. Pham
- Creighton University, Department of Physics, Omaha, Nebraska, United States
| | | | - Molly S. Myers
- Creighton University, Department of Physics, Omaha, Nebraska, United States
| | - Dominick M. Myers
- Creighton University, Department of Biomedical Sciences, Omaha, Nebraska, United States
| | - Laura A. Hansen
- Creighton University, Department of Biomedical Sciences, Omaha, Nebraska, United States
| | - Michael G. Nichols
- Creighton University, Department of Physics, Omaha, Nebraska, United States
- Creighton University, Department of Biomedical Sciences, Omaha, Nebraska, United States
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Romito M, Pu Y, Stankovic KM, Psaltis D. Imaging hair cells through laser-ablated cochlear bone. BIOMEDICAL OPTICS EXPRESS 2019; 10:5974-5988. [PMID: 31799058 PMCID: PMC6865115 DOI: 10.1364/boe.10.005974] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/06/2019] [Revised: 09/29/2019] [Accepted: 10/24/2019] [Indexed: 05/04/2023]
Abstract
We report an innovative technique for the visualization of cells through an overlying scattering medium by combining femtosecond laser bone ablation and two-photon excitation fluorescence (TPEF) microscopy. We demonstrate the technique by imaging hair cells in an intact mouse cochlea ex vivo. Intracochlear imaging is important for the assessment of hearing disorders. However, the small size of the cochlea and its encasement in the densest bone in the body present challenging obstacles, preventing the visualization of the intracochlear microanatomy using standard clinical imaging modalities. The controlled laser ablation reduces the optical scattering of the cochlear bone while the TPEF allows visualization of individual cells behind the bone. We implemented optical coherence tomography (OCT) simultaneously with the laser ablation to enhance the precision of the ablation and prevent inadvertent damage to the cells behind the bone.
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Affiliation(s)
- Marilisa Romito
- Optics Laboratory, School of Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
| | - Ye Pu
- Optics Laboratory, School of Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
| | - Konstantina M. Stankovic
- Department of Otolaryngology – Head and Neck Surgery, Harvard Medical School and Massachusetts Eye and Ear, Boston, MA 02114, USA
| | - Demetri Psaltis
- Optics Laboratory, School of Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
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Abstract
Over the past two decades there have been unprecedented advances in the capabilities for live cell imaging using light and confocal microscopy. Together with the discovery of green fluorescent protein and its derivatives and the development of a vast array of fluorescent imaging probes and conjugates, it is now possible to image virtually any intracellular or extracellular protein or structure. Traditional static imaging of fixed bone cells and tissues takes a snapshot view of events at a specific time point, but can often miss the dynamic aspects of the events being investigated. This chapter provides an overview of the application of live cell imaging approaches for the study of bone cells and bone organ cultures. Rather than emphasizing technical aspects of the imaging equipment, which may vary in different laboratories, we focus on what we consider to be the important principles that are of most practical use for an investigator setting up these techniques in their own laboratory. We also provide detailed protocols that our laboratory has used for live imaging of bone cell and organ cultures.
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Affiliation(s)
- Sarah L Dallas
- Department of Oral and Craniofacial Sciences, School of Dentistry, University of Missouri, Kansas City, Kansas City, MO, USA.
| | - Patricia A Veno
- Department of Oral and Craniofacial Sciences, School of Dentistry, University of Missouri, Kansas City, Kansas City, MO, USA
| | - LeAnn M Tiede-Lewis
- Department of Oral and Craniofacial Sciences, School of Dentistry, University of Missouri, Kansas City, Kansas City, MO, USA
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Desa DE, Nichols MG, Smith HJ. Aminoglycosides rapidly inhibit NAD(P)H metabolism increasing reactive oxygen species and cochlear cell demise. JOURNAL OF BIOMEDICAL OPTICS 2018; 24:1-14. [PMID: 30411553 PMCID: PMC6225535 DOI: 10.1117/1.jbo.24.5.051403] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/10/2018] [Accepted: 09/21/2018] [Indexed: 06/04/2023]
Abstract
Despite causing permanent hearing loss by damaging inner ear sensory cells, aminoglycosides (AGs) remain one of the most widely used classes of antibiotics in the world. Although the mechanisms of cochlear sensory cell damage are not fully known, reactive oxygen species (ROS) are clearly implicated. Mitochondrial-specific ROS formation was evaluated in acutely cultured murine cochlear explants exposed to gentamicin (GM), a representative ototoxic AG antibiotic. Superoxide (O2·-) and hydrogen peroxide (H2O2) were measured using MitoSOX Red and Dihydrorhodamine 123, respectively, in sensory and supporting cells. A 1-h GM exposure significantly increased O2·- formation in IHCs and increased H2O2 formation in all cell types. At the same time point, GM significantly increased manganese superoxide dismutase (MnSOD) levels while significantly decreasing copper/zinc superoxide dismutase (CuZnSOD) in cochlear sensory cells. This suggests (1) a rapid conversion of highly reactive O2·- to H2O2 during the acute stage of ototoxic antibiotic exposure and (2) that the endogenous antioxidant system is significantly altered by AGs. Fluorescence intensity-based measurements of reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and mitochondrial membrane potential were measured to determine if increases in GM-induced ROS production were correlated with changes in mitochondrial metabolism. This project provides a basis for understanding the mechanisms of mitochondrial ROS production in cochlear cells exposed to ototoxic antibiotics. Understanding the nature of ototoxic antibiotic-induced changes in mitochondrial metabolism is critical for developing hearing loss treatment and prevention strategies.
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Affiliation(s)
- Danielle E. Desa
- University of Rochester, Department of Biomedical Engineering, Rochester, New York, United States
| | - Michael G. Nichols
- Creighton University, Department of Physics, Omaha, Nebraska, United States
- Creighton University, Department of Biomedical Sciences, Omaha, Nebraska, United States
| | - Heather Jensen Smith
- University of Nebraska Medical Center, The Eppley Institute for Cancer and Allied Diseases, Omaha, Nebraska, United States
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Schaefer PM, Kalinina S, Rueck A, von Arnim CAF, von Einem B. NADH Autofluorescence-A Marker on its Way to Boost Bioenergetic Research. Cytometry A 2018; 95:34-46. [PMID: 30211978 DOI: 10.1002/cyto.a.23597] [Citation(s) in RCA: 128] [Impact Index Per Article: 18.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2018] [Revised: 07/20/2018] [Accepted: 08/04/2018] [Indexed: 12/20/2022]
Abstract
More than 60 years ago, the idea was introduced that NADH autofluorescence could be used as a marker of cellular redox state and indirectly also of cellular energy metabolism. Fluorescence lifetime imaging microscopy of NADH autofluorescence offers a marker-free readout of the mitochondrial function of cells in their natural microenvironment and allows different pools of NADH to be distinguished within a cell. Despite its many advantages in terms of spatial resolution and in vivo applicability, this technique still requires improvement in order to be fully useful in bioenergetics research. In the present review, we give a summary of technical and biological challenges that have so far limited the spread of this powerful technology. To help overcome these challenges, we provide a comprehensible overview of biological applications of NADH imaging, along with a detailed summary of valid imaging approaches that may be used to tackle many biological questions. This review is meant to provide all scientists interested in bioenergetics with support on how to embed successfully NADH imaging in their research. © 2018 International Society for Advancement of Cytometry.
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Affiliation(s)
| | - Sviatlana Kalinina
- Core Facility Confocal and Multiphoton Microscopy, Ulm University, Ulm, Germany
| | - Angelika Rueck
- Core Facility Confocal and Multiphoton Microscopy, Ulm University, Ulm, Germany
| | - Christine A F von Arnim
- Department of Neurology, Ulm University, Ulm, Germany.,Clinic for Neurogeriatrics and Neurological Rehabilitation, University- and Rehabilitation Hospital Ulm, Ulm, Germany
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Maniwa K, Yamashita H, Tsukano H, Hishida R, Endo N, Shibata M, Shibuki K. Tomographic optical imaging of cortical responses after crossing nerve transfer in mice. PLoS One 2018; 13:e0193017. [PMID: 29444175 PMCID: PMC5812646 DOI: 10.1371/journal.pone.0193017] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2017] [Accepted: 02/03/2018] [Indexed: 11/23/2022] Open
Abstract
To understand the neural mechanisms underlying the therapeutic effects of crossing nerve transfer for brachial plexus injuries in human patients, we investigated the cortical responses after crossing nerve transfer in mice using conventional and tomographic optical imaging. The distal cut ends of the left median and ulnar nerves were connected to the central cut ends of the right median and ulnar nerves with a sciatic nerve graft at 8 weeks of age. Eight weeks after the operation, the responses in the primary somatosensory cortex (S1) elicited by vibratory stimulation applied to the left forepaw were visualized based on activity-dependent flavoprotein fluorescence changes. In untreated mice, the cortical responses to left forepaw stimulation were mainly observed in the right S1. In mice with nerve crossing transfer, cortical responses to left forepaw stimulation were observed in the left S1 together with clear cortical responses in the right S1. We expected that the right S1 responses in the untreated mice were produced by thalamic inputs to layer IV, whereas those in the operated mice were mediated by callosal inputs from the left S1 to layer II/III of the right S1. To confirm this hypothesis, we performed tomographic imaging of flavoprotein fluorescence responses by macroconfocal microscopy. Flavoprotein fluorescence responses in layer IV were dominant compared to those in layer II/III in untreated mice. In contrast, responses in layer II/III were dominant compared to those in layer IV in operated mice. The peak latency of the cortical responses in the operated mice was longer than that in the untreated mice. These results confirmed our expectation that drastic reorganization in the cortical circuits was induced after crossing nerve transfer in mice.
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Affiliation(s)
- Keiichi Maniwa
- Department of Neurophysiology, Brain Research Institute, Niigata University, Niigata, Japan
- Department of Orthopedic Surgery, Faculty of Medicine, Niigata University, Niigata, Japan
| | - Haruyoshi Yamashita
- Department of Orthopedic Surgery, Faculty of Medicine, Niigata University, Niigata, Japan
| | - Hiroaki Tsukano
- Department of Neurophysiology, Brain Research Institute, Niigata University, Niigata, Japan
| | - Ryuichi Hishida
- Department of Neurophysiology, Brain Research Institute, Niigata University, Niigata, Japan
| | - Naoto Endo
- Department of Orthopedic Surgery, Faculty of Medicine, Niigata University, Niigata, Japan
| | - Minoru Shibata
- Department of Plastic Surgery, Faculty of Medicine, Niigata University, Niigata, Japan
| | - Katsuei Shibuki
- Department of Neurophysiology, Brain Research Institute, Niigata University, Niigata, Japan
- * E-mail:
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Zholudeva LV, Ward KG, Nichols MG, Smith HJ. Gentamicin differentially alters cellular metabolism of cochlear hair cells as revealed by NAD(P)H fluorescence lifetime imaging. JOURNAL OF BIOMEDICAL OPTICS 2015; 20:051032. [PMID: 25688541 PMCID: PMC4405084 DOI: 10.1117/1.jbo.20.5.051032] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/23/2014] [Accepted: 01/15/2015] [Indexed: 06/04/2023]
Abstract
Aminoglycoside antibiotics are implicated as culprits of hearing loss in more than 120,000 individuals annually. Research has shown that the sensory cells, but not supporting cells, of the cochlea are readily damaged and/or lost after use of such antibiotics. High-frequency outer hair cells (OHCs) show a greater sensitivity to antibiotics than high- and low-frequency inner hair cells (IHCs). We hypothesize that variations in mitochondrial metabolism account for differences in susceptibility. Fluorescence lifetime microscopy was used to quantify changes in NAD(P)H in sensory and supporting cells from explanted murine cochleae exposed to mitochondrial uncouplers, inhibitors, and an ototoxic antibiotic, gentamicin (GM). Changes in metabolic state resulted in a redistribution of NAD(P)H between subcellular fluorescence lifetime pools. Supporting cells had a significantly longer lifetime than sensory cells. Pretreatment with GM increased NAD(P)H intensity in high-frequency sensory cells, as well as the NAD(P)H lifetime within IHCs. GM specifically increased NAD(P)H concentration in high-frequency OHCs, but not in IHCs or pillar cells. Variations in NAD(P)H intensity in response to mitochondrial toxins and GM were greatest in high-frequency OHCs. These results demonstrate that GM rapidly alters mitochondrial metabolism, differentially modulates cell metabolism, and provides evidence that GM-induced changes in metabolism are significant and greatest in high-frequency OHCs.
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Affiliation(s)
- Lyandysha V. Zholudeva
- Drexel University, Department of Neurobiology and Anatomy, 2900 West Queen Lane, Philadelphia, Pennsylvania 19129, United States
| | - Kristina G. Ward
- Creighton University, Department of Physics, 2500 California Plaza, Omaha, Nebraska 68178, United States
| | - Michael G. Nichols
- Creighton University, Department of Physics, 2500 California Plaza, Omaha, Nebraska 68178, United States
- Creighton University, Department of Biomedical Sciences, 2500 California Plaza, Omaha, Nebraska 68178, United States
| | - Heather Jensen Smith
- Creighton University, Department of Biomedical Sciences, 2500 California Plaza, Omaha, Nebraska 68178, United States
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Li L, Chen Z, Wang X, Zhuo S, Li H, Jiang W, Guan G, Chen J. Assessment of colloid response by nonlinear optical microscopy after preoperative radiochemotherapy for rectal carcinoma. JOURNAL OF BIOMEDICAL OPTICS 2015; 20:051009. [PMID: 25436512 DOI: 10.1117/1.jbo.20.5.051009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/23/2014] [Accepted: 10/27/2014] [Indexed: 06/04/2023]
Abstract
Colloid response is a type of tumor response that occurs after preoperative radiochemotherapy for rectal carcinoma. Given its important influence on survival, the colloid response should be considered when estimating histopathological reactions. Here, multiphoton microscopy (MPM) was applied to evaluate the colloid response ex vivo. This study demonstrated that MPM has the capability to visualize the colloid response in the absence of labels and can, in particular, identify rare residual carcinomatous cells in mucin pools. These results highlight the potential of this nonlinear optical technique as a diagnostic tool for tumor response after neoadjuvant treatment.
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Affiliation(s)
- Lianhuang Li
- Institute of Laser and Optoelectronics Technology, Fujian Provincial Key Laboratory for Photonics Technology, Key Laboratory of OptoElectronic Science and Technology for Medicine of Ministry of Education, Fujian Normal University, Fuzhou 350007, China
| | - Zhifen Chen
- Fujian Medical University, The Affiliated Union Hospital, Department of Colorectal Surgery, Fuzhou 350001, China
| | - Xingfu Wang
- Fujian Medical University, The First Affiliated Hospital, Department of Pathology, Fuzhou 350001, China
| | - Shuangmu Zhuo
- Institute of Laser and Optoelectronics Technology, Fujian Provincial Key Laboratory for Photonics Technology, Key Laboratory of OptoElectronic Science and Technology for Medicine of Ministry of Education, Fujian Normal University, Fuzhou 350007, China
| | - Hongsheng Li
- Institute of Laser and Optoelectronics Technology, Fujian Provincial Key Laboratory for Photonics Technology, Key Laboratory of OptoElectronic Science and Technology for Medicine of Ministry of Education, Fujian Normal University, Fuzhou 350007, China
| | - Weizhong Jiang
- Fujian Medical University, The Affiliated Union Hospital, Department of Colorectal Surgery, Fuzhou 350001, China
| | - Guoxian Guan
- Fujian Medical University, The Affiliated Union Hospital, Department of Colorectal Surgery, Fuzhou 350001, China
| | - Jianxin Chen
- Institute of Laser and Optoelectronics Technology, Fujian Provincial Key Laboratory for Photonics Technology, Key Laboratory of OptoElectronic Science and Technology for Medicine of Ministry of Education, Fujian Normal University, Fuzhou 350007, China
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13
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Majumder P, Duchen MR, Gale JE. Cellular glutathione content in the organ of Corti and its role during ototoxicity. Front Cell Neurosci 2015; 9:143. [PMID: 25972783 PMCID: PMC4412067 DOI: 10.3389/fncel.2015.00143] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2015] [Accepted: 03/26/2015] [Indexed: 12/31/2022] Open
Abstract
Glutathione (GSH) is the major scavenger of reactive oxygen species (ROS) inside cells. We used live confocal imaging in order to clarify the role of GSH in the biology of the organ of Corti, the sensory epithelium of the cochlea, before, during and after the onset of hearing and in ~1 year old mice. GSH content was measured using monochlorobimane (MCB), a non-fluorescent cell permeant bimane that reacts with GSH, forming a fluorescent adduct through a reaction catalyzed by glutathione-S-transferase. GSH content increased significantly in inner hair cells during maturation in young adult animals, whereas there was no significant change in the outer hair cells. However, the GSH content in inner hair cells was significantly reduced in ~1 year old mice. The GSH content of supporting cells was comparatively stable over these ages. To test whether the GSH content played a significant protective role during ototoxicity, GSH synthesis was inhibited by buthionine sulfoximine (BSO) in organotypic cochlear explant cultures from immature mice. BSO treatment alone, which reduced GSH by 65 and 85% in inner hair cells and outer hair cells respectively, did not cause any significant cell death. Surprisingly, GSH depletion had no significant effect on hair cell survival even during exposure to the ototoxic aminoglycoside neomycin. These data suggest that the involvement of ROS during aminoglycoside-induced hair cell death is less clear than previously thought and requires further investigation.
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Affiliation(s)
| | - Michael R Duchen
- Department of Cell and Developmental Biology, University College London London, UK
| | - Jonathan E Gale
- UCL Ear Institute, University College London London, UK ; Department of Cell and Developmental Biology, University College London London, UK
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Li LH, Chen ZF, Wang XF, Zhuo SM, Li HS, Jiang WZ, Guan GX, Chen JX. Multiphoton microscopy for tumor regression grading after neoadjuvant treatment for colorectal carcinoma. World J Gastroenterol 2015; 21:4210-4215. [PMID: 25892870 PMCID: PMC4394081 DOI: 10.3748/wjg.v21.i14.4210] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/04/2014] [Revised: 01/10/2015] [Accepted: 01/21/2015] [Indexed: 02/06/2023] Open
Abstract
AIM: To evaluate the feasibility of using multiphoton microscopy (MPM) to assess a tumor regression grading (TRG) system.
METHODS: Fresh specimens from seven patients with colorectal carcinoma undergoing neoadjuvant radiochemotherapy at the Fujian Medical University Union Hospital were obtained immediately after proctectomy. Specimens were serially sectioned (10 µm thickness) and used for MPM or stained with hematoxylin and eosin for comparison. Sections were imaged by MPM using 810 nm excitation, and images were collected in two wavelength channels corresponding to second-harmonic generation (SHG) and two-photon excited fluorescence (TPEF) signals. The ratio of these signal intensities was used to distinguish fibrosis from normal mucosal and serosal tissues.
RESULTS: TRG of specimens assessed by MPM were in complete agreement with histologic grading performed by a consulting pathologist. SHG and TPEF images clearly revealed collagen fibers and fragmented elastic fibers in the muscularis propria specimens following neoadjuvant radiochemotherapy. Additionally, blood vessel hyperplasia was observed as thickening and fibrosis of the intima and media, which was accompanied by minimal inflammatory cell infiltration. Furthermore, the SHG/TPEF ratio in stromal fibrosis (4.15 ± 0.58) was significantly higher than those in the normal submucosal (2.31 ± 0.52) and serosal (1.47 ± 0.10) tissues (P < 0.001 for both). Analysis of emission spectra from cancerous tumor cells revealed two peaks corresponding to nicotinamide adenine dinucleotide hydrogen and flavin adenine dinucleotide signals; the ratio of these values was 1.19 ± 0.02, which is close to a normal metabolic state.
CONCLUSION: MPM can be used to perform real-time diagnosis of tumor response after neoadjuvant treatment, and can be applied to evaluate TRG.
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Chen Y, Chen J, Chen H, Hong Z, Zhu X, Zhuo S, Chen Y, Chen J. Multiphoton microscopy as a diagnostic imaging modality for pancreatic neoplasms without hematoxylin and eosin stains. JOURNAL OF BIOMEDICAL OPTICS 2014; 19:96008. [PMID: 25216027 DOI: 10.1117/1.jbo.19.9.096008] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/20/2014] [Accepted: 08/11/2014] [Indexed: 05/11/2023]
Abstract
Hematoxylin and eosin (H&E) staining of tissue samples is the standard approach in histopathology for imaging and diagnosing cancer. Recent reports have shown that multiphoton microscopy (MPM) provides better sample interface with single-cell resolution, which enhances traditional H&E staining and offers a powerful diagnostic tool with potential applications in oncology. The purpose of this study was to further expand the versatility of MPM by establishing the optical parameters required for imaging unstained histological sections of pancreatic neoplasms, thereby providing an efficient and environmentally sustainable alternative to H&E staining while improving the accuracy of pancreatic cancer diagnoses. We found that the high-resolution MPM images clearly distinguish between the structure of normal pancreatic tissues compared with pancreatic neoplasms in unstained histological sections, and discernable differences in tissue architecture and cell morphology between normal versus tumorigenic cells led to enhanced optical diagnosis of cancerous tissue. Moreover, quantitative assessment of the cytomorphological features visualized from MPM images showed significant differences in the nuclear–cytoplasmic ratios of pancreatic neoplasms compared with normal pancreas, as well as further distinguished pancreatic malignant tumors from benign tumors. These results indicate that the MPM could potentially serve as an optical tool for the diagnosis of pancreatic neoplasms in unstained histological sections.
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Affiliation(s)
- Youting Chen
- The First Affiliated Hospital of Fujian Medical University, Department of Hepatobiliary Surgery, Fuzhou 350005, China
| | - Jing Chen
- Fujian Normal University, Key Laboratory of OptoElectronic Science and Technology for Medicine of Ministry of Education, Fujian Provincial Key Laboratory for Photonics Technology, Institute of Laser and Optoelectronics Technology, Fuzhou 350007, China
| | - Hong Chen
- The First Affiliated Hospital of Fujian Medical University, Department of Pathology, Fuzhou 350005, China
| | - Zhipeng Hong
- The First Affiliated Hospital of Fujian Medical University, Department of Hepatobiliary Surgery, Fuzhou 350005, China
| | - Xiaoqin Zhu
- Fujian Normal University, Key Laboratory of OptoElectronic Science and Technology for Medicine of Ministry of Education, Fujian Provincial Key Laboratory for Photonics Technology, Institute of Laser and Optoelectronics Technology, Fuzhou 350007, China
| | - Shuangmu Zhuo
- Fujian Normal University, Key Laboratory of OptoElectronic Science and Technology for Medicine of Ministry of Education, Fujian Provincial Key Laboratory for Photonics Technology, Institute of Laser and Optoelectronics Technology, Fuzhou 350007, China
| | - Yanling Chen
- The Affiliated Union Hospital of Fujian Medical University, Department of Hepatobiliary Surgery, Fuzhou 350001, China
| | - Jianxin Chen
- Fujian Normal University, Key Laboratory of OptoElectronic Science and Technology for Medicine of Ministry of Education, Fujian Provincial Key Laboratory for Photonics Technology, Institute of Laser and Optoelectronics Technology, Fuzhou 350007, China
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Integrity and regeneration of mechanotransduction machinery regulate aminoglycoside entry and sensory cell death. PLoS One 2013; 8:e54794. [PMID: 23359017 PMCID: PMC3554584 DOI: 10.1371/journal.pone.0054794] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2012] [Accepted: 12/14/2012] [Indexed: 12/04/2022] Open
Abstract
Sound perception requires functional hair cell mechanotransduction (MET) machinery, including the MET channels and tip-link proteins. Prior work showed that uptake of ototoxic aminoglycosides (AG) into hair cells requires functional MET channels. In this study, we examined whether tip-link proteins, including Cadherin 23 (Cdh23), regulate AG entry into hair cells. Using time-lapse microscopy on cochlear explants, we found rapid uptake of gentamicin-conjugated Texas Red (GTTR) into hair cells from three-day-old Cdh23+/+ and Cdh23v2J/+ mice, but failed to detect GTTR uptake in Cdh23v2J/v2J hair cells. Pre-treatment of wildtype cochleae with the calcium chelator 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) to disrupt tip-links also effectively reduced GTTR uptake into hair cells. Both Cdh23v2J/v2J and BAPTA-treated hair cells were protected from degeneration caused by gentamicin. Six hours after BAPTA treatment, GTTR uptake remained reduced in comparison to controls; by 24 hours, drug uptake was comparable between untreated and BAPTA-treated hair cells, which again became susceptible to cell death induced by gentamicin. Together, these results provide genetic and pharmacologic evidence that tip-links are required for AG uptake and toxicity in hair cells. Because tip-links can spontaneously regenerate, their temporary breakage offers a limited time window when hair cells are protected from AG toxicity.
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Vergen J, Hecht C, Zholudeva LV, Marquardt MM, Hallworth R, Nichols MG. Metabolic imaging using two-photon excited NADH intensity and fluorescence lifetime imaging. MICROSCOPY AND MICROANALYSIS : THE OFFICIAL JOURNAL OF MICROSCOPY SOCIETY OF AMERICA, MICROBEAM ANALYSIS SOCIETY, MICROSCOPICAL SOCIETY OF CANADA 2012; 18:761-70. [PMID: 22832200 PMCID: PMC3842212 DOI: 10.1017/s1431927612000529] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/22/2023]
Abstract
Metabolism and mitochondrial dysfunction are known to be involved in many different disease states. We have employed two-photon fluorescence imaging of intrinsic mitochondrial reduced nicotinamide adenine dinucleotide (NADH) to quantify the metabolic state of several cultured cell lines, multicell tumor spheroids, and the intact mouse organ of Corti. Historically, fluorescence intensity has commonly been used as an indicator of the NADH concentration in cells and tissues. More recently, fluorescence lifetime imaging has revealed that changes in metabolism produce not only changes in fluorescence intensity, but also significant changes in the lifetimes and concentrations of free and enzyme-bound pools of NADH. Since NADH binding changes with metabolic state, this approach presents a new opportunity to track the cellular metabolic state.
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Affiliation(s)
- Jorge Vergen
- Department of Physics, Creighton University, 2500 California Plaza, Omaha, NE 68178
| | - Clifford Hecht
- Department of Physics, Creighton University, 2500 California Plaza, Omaha, NE 68178
| | | | - Meg M. Marquardt
- Department of Physics, Creighton University, 2500 California Plaza, Omaha, NE 68178
| | - Richard Hallworth
- Department of Biomedical Sciences, Creighton University, 2500 California Plaza, Omaha, NE 68178
| | - Michael G. Nichols
- Department of Physics, Creighton University, 2500 California Plaza, Omaha, NE 68178
- Corresponding Author: Phone: 402.280.2159; FAX: 402.280.2140;
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Jensen-Smith HC, Hallworth R, Nichols MG. Gentamicin rapidly inhibits mitochondrial metabolism in high-frequency cochlear outer hair cells. PLoS One 2012; 7:e38471. [PMID: 22715386 PMCID: PMC3371036 DOI: 10.1371/journal.pone.0038471] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2012] [Accepted: 05/10/2012] [Indexed: 01/08/2023] Open
Abstract
Aminoglycosides (AG), including gentamicin (GM), are the most frequently used antibiotics in the world and are proposed to cause irreversible cochlear damage and hearing loss (HL) in 1/4 of the patients receiving these life-saving drugs. Akin to the results of AG ototoxicity studies, high-frequency, basal turn outer hair cells (OHCs) preferentially succumb to multiple HL pathologies while inner hair cells (IHCs) are much more resilient. To determine if endogenous differences in IHC and OHC mitochondrial metabolism dictate differential sensitivities to AG-induced HL, IHC- and OHC-specific changes in mitochondrial reduced nicotinamide adenine dinucleotide (NADH) fluorescence during acute (1 h) GM treatment were compared. GM-mediated decreases in NADH fluorescence and succinate dehydrogenase activity were observed shortly after GM application. High-frequency basal turn OHCs were found to be metabolically biased to rapidly respond to alterations in their microenvironment including GM and elevated glucose exposures. These metabolic biases may predispose high-frequency OHCs to preferentially produce cell-damaging reactive oxygen species during traumatic challenge. Noise-induced and age-related HL pathologies share key characteristics with AG ototoxicity, including preferential OHC loss and reactive oxygen species production. Data from this report highlight the need to address the role of mitochondrial metabolism in regulating AG ototoxicity and the need to illuminate how fundamental differences in IHC and OHC metabolism may dictate differences in HC fate during multiple HL pathologies.
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Affiliation(s)
- Heather C Jensen-Smith
- Department of Biomedical Sciences, Creighton University, Omaha, Nebraska, United States of America.
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Hu W, Zhao G, Wang C, Zhang J, Fu L. Nonlinear optical microscopy for histology of fresh normal and cancerous pancreatic tissues. PLoS One 2012; 7:e37962. [PMID: 22655087 PMCID: PMC3360059 DOI: 10.1371/journal.pone.0037962] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2012] [Accepted: 04/26/2012] [Indexed: 11/19/2022] Open
Abstract
BACKGROUND Pancreatic cancer is a lethal disease with a 5-year survival rate of only 1-5%. The acceleration of intraoperative histological examination would be beneficial for better management of pancreatic cancer, suggesting an improved survival. Nonlinear optical methods based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) of intrinsic optical biomarkers show the ability to visualize the morphology of fresh tissues associated with histology, which is promising for real-time intraoperative evaluation of pancreatic cancer. METHODOLOGY/PRINCIPAL FINDINGS In order to investigate whether the nonlinear optical imaging methods have the ability to characterize pancreatic histology at cellular resolution, we studied different types of pancreatic tissues by using label-free TPEF and SHG. Compared with other routine methods for the preparation of specimens, fresh tissues without processing were found to be most suitable for nonlinear optical imaging of pancreatic tissues. The detailed morphology of the normal rat pancreas was observed and related with the standard histological images. Comparatively speaking, the preliminary images of a small number of chemical-induced pancreatic cancer tissues showed visible neoplastic differences in the morphology of cells and extracellular matrix. The subcutaneous pancreatic tumor xenografts were further observed using the nonlinear optical microscopy, showing that most cells are leucocytes at 5 days after implantation, the tumor cells begin to proliferate at 10 days after implantation, and the extracellular collagen fibers become disordered as the xenografts grow. CONCLUSIONS/SIGNIFICANCE In this study, nonlinear optical imaging was used to characterize the morphological details of fresh pancreatic tissues for the first time. We demonstrate that it is possible to provide real-time histological evaluation of pancreatic cancer by the nonlinear optical methods, which present an opportunity for the characterization of the progress of spontaneous pancreatic cancer and further application in a non-invasive manner.
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Affiliation(s)
- Wenyan Hu
- Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan, People's Republic of China
- Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science and Technology, Wuhan, China
| | - Gang Zhao
- Pancreatic Surgery Center, Union Hospital, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Chunyou Wang
- Pancreatic Surgery Center, Union Hospital, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Jungang Zhang
- Pancreatic Surgery Center, Union Hospital, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Ling Fu
- Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan, People's Republic of China
- Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science and Technology, Wuhan, China
- * E-mail:
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20
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Heikal AA. A Multiparametric Imaging of Cellular Coenzymes for Monitoring Metabolic and Mitochondrial Activities. REVIEWS IN FLUORESCENCE 2010 2012. [DOI: 10.1007/978-1-4419-9828-6_10] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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Dittmar R, Potier E, van Zandvoort M, Ito K. Assessment of cell viability in three-dimensional scaffolds using cellular auto-fluorescence. Tissue Eng Part C Methods 2011; 18:198-204. [PMID: 21981657 DOI: 10.1089/ten.tec.2011.0334] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
After assessing cell viability (CV), tissue-engineered constructs are often discarded, as current CV assays commonly require specific (fluorescent) dyes to stain cells and may need scaffold/tissue digestion before quantifying the live and dead cells. Here, we demonstrate and evaluate how cellular auto-fluorescence can be exploited to facilitate a noninvasive CV estimation in three-dimensional scaffolds using two advanced microscopy methods. Mixtures of live and dead C2C12 myoblasts (0%, 25%, 50%, 75%, and 100% live cells) were prepared, and CV was determined before seeding cells into collagen carriers using the trypan blue (TB) assay. Cell-seeded collagen gels ([CSCGs], n=5/cell mixture) were produced by mixing collagen solution with the live/dead cell mixtures (7×10(6) cells/mL). After polymerization, two-photon microscopy (TPM) and confocal microscopy images of the CSCG were acquired (n=30 images/CSCG). It was found that live and dead cells systematically emit auto-fluorescent light with different spectral characteristics. Viable cells showed predominantly blue fluorescence with a peak emission around 470 nm, whereas dead cells appeared to mainly emit green fluorescent light with a peak intensity around 560 nm. For TPM, live and dead cells were distinguished spectrally. For confocal images, the intensity ratio of images taken with band-pass filters was used to distinguish live from dead cells. CV values obtained with both TPM and confocal imaging did not significantly differ from those acquired with the established TB method. In comparison to TPM, confocal microscopy was found to be less accurate in assessing the exact CV in constructs containing mostly live or dead cells. In summary, monitoring cellular auto-fluorescence using advanced microscopy techniques allows CV assessment requiring no additional dyes and/or scaffold digestion and, thus, may be especially suitable for tissue-engineering studies where CV is measured at multiple time points.
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Affiliation(s)
- Roman Dittmar
- Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands
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Alharazneh A, Luk L, Huth M, Monfared A, Steyger PS, Cheng AG, Ricci AJ. Functional hair cell mechanotransducer channels are required for aminoglycoside ototoxicity. PLoS One 2011; 6:e22347. [PMID: 21818312 PMCID: PMC3144223 DOI: 10.1371/journal.pone.0022347] [Citation(s) in RCA: 196] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2011] [Accepted: 06/19/2011] [Indexed: 12/02/2022] Open
Abstract
Aminoglycosides (AG) are commonly prescribed antibiotics with potent bactericidal activities. One main side effect is permanent sensorineural hearing loss, induced by selective inner ear sensory hair cell death. Much work has focused on AG's initiating cell death processes, however, fewer studies exist defining mechanisms of AG uptake by hair cells. The current study investigated two proposed mechanisms of AG transport in mammalian hair cells: mechanotransducer (MET) channels and endocytosis. To study these two mechanisms, rat cochlear explants were cultured as whole organs in gentamicin-containing media. Two-photon imaging of Texas Red conjugated gentamicin (GTTR) uptake into live hair cells was rapid and selective. Hypocalcemia, which increases the open probability of MET channels, increased AG entry into hair cells. Three blockers of MET channels (curare, quinine, and amiloride) significantly reduced GTTR uptake, whereas the endocytosis inhibitor concanavalin A did not. Dynosore quenched the fluorescence of GTTR and could not be tested. Pharmacologic blockade of MET channels with curare or quinine, but not concanavalin A or dynosore, prevented hair cell loss when challenged with gentamicin for up to 96 hours. Taken together, data indicate that the patency of MET channels mediated AG entry into hair cells and its toxicity. Results suggest that limiting permeation of AGs through MET channel or preventing their entry into endolymph are potential therapeutic targets for preventing hair cell death and hearing loss.
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Affiliation(s)
- Abdelrahman Alharazneh
- Department of Otolaryngology-Head and Neck Surgery, Stanford University, Stanford, California, United States of America
- Department of Special Surgery, Mu'tah University, Alkarak, Jordan
| | - Lauren Luk
- Department of Otolaryngology-Head and Neck Surgery, Stanford University, Stanford, California, United States of America
| | - Markus Huth
- Department of Otolaryngology-Head and Neck Surgery, Stanford University, Stanford, California, United States of America
| | - Ashkan Monfared
- Department of Otolaryngology-Head and Neck Surgery, Stanford University, Stanford, California, United States of America
| | - Peter S. Steyger
- Department of Otolaryngology-Head and Neck Surgery, Oregon Health & Science University, Portland, Oregon, United States of America
| | - Alan G. Cheng
- Department of Otolaryngology-Head and Neck Surgery, Stanford University, Stanford, California, United States of America
- * E-mail: (AGC); (AJR)
| | - Anthony J. Ricci
- Department of Otolaryngology-Head and Neck Surgery, Stanford University, Stanford, California, United States of America
- Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, United States of America
- * E-mail: (AGC); (AJR)
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KRASIEVA TATIANAB, GIEDZINSKI ERICH, TRAN KATHERINE, LAN MARY, LIMOLI CHARLESL, TROMBERG BRUCEJ. PROBING THE IMPACT OF GAMMA-IRRADIATION ON THE METABOLIC STATE OF NEURAL STEM AND PRECURSOR CELLS USING DUAL-WAVELENGTH INTRINSIC SIGNAL TWO-PHOTON EXCITED FLUORESCENCE. JOURNAL OF INNOVATIVE OPTICAL HEALTH SCIENCES 2011; 4:289-300. [PMID: 23869199 PMCID: PMC3712535 DOI: 10.1142/s1793545811001629] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/02/2023]
Abstract
Two-photon excited fluorescence (TPEF) spectroscopy and imaging were used to investigate the effects of gamma-irradiation on neural stem and precursor cells (NSPCs). While the observed signal from reduced nicotinamide adenine dinucleotide (NADH) was localized to the mitochondria, the signal typically associated with oxidized flavoproteins (Fp) was distributed diffusely throughout the cell. The measured TPEF emission and excitation spectra were similar to the established spectra of NAD(P)H and Fp. Fp fluorescence intensity was markedly increased by addition of the electron transport chain (ETC) modulator menadione to the medium, along with a concomitant decrease in the NAD(P)H signal. Three-dimensional (3D) neurospheres were imaged to obtain the cellular metabolic index (CMI), calculated as the ratio of Fp to NAD(P)H fluorescence intensity. Radiation effects were found to differ between low-dose (≤ 50 cGy) and high-dose (≥ 50 cGy) exposures. Low-dose irradiation caused a marked drop in CMI values accompanied by increased cellular proliferation. At higher doses, both NAD(P)H and Fp signals increased, leading to an overall elevation in CMI values. These findings underscore the complex relationship between radiation dose, metabolic state, and proliferation status in NSPCs and highlight the ability of TPEF spectroscopy and imaging to characterize metabolism in 3D spheroids.
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Affiliation(s)
- TATIANA B. KRASIEVA
- Laser Microbeam and Medical Program, Beckman Laser Institute, University of California, Irvine, 1002 Health Sciences Road East, Irvine, CA 92612, USA
| | - ERICH GIEDZINSKI
- Department of Radiation Oncology, University of California, Irvine, California 92697, USA
| | - KATHERINE TRAN
- Department of Radiation Oncology, University of California, Irvine, California 92697, USA
| | - MARY LAN
- Department of Radiation Oncology, University of California, Irvine, California 92697, USA
| | - CHARLES L. LIMOLI
- Department of Radiation Oncology, University of California, Irvine, California 92697, USA
| | - BRUCE J. TROMBERG
- Laser Microbeam and Medical Program, Beckman Laser Institute, University of California, Irvine, 1002 Health Sciences Road East, Irvine, CA 92612, USA
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Abstract
OBJECTIVE Human mutations in the DNA repair genes, Xeroderma pigmentosum (XP)-C and XPA result in hearing loss, which has fueled the hypothesis that there is a significant demand for these genes in protecting cochlear genetic material. Therefore, we quantified the level of XPC and XPA mRNA in the mammalian cochlea. DESIGN XPC and XPA mRNAs were purified from the cochlea of 15 Fischer344 rats and quantified using SYBR Green chemistry. Another 15 Fischer344 rats were sacrificed for immunolocalization of XPC and XPA polypeptides in the cochlea and kidney (control organ). RESULTS XP mRNA levels were up to 95% (XPA) and 69% (XPC) of the respective maximum expression capacity of each gene. In addition, these cochlear levels were up to sixfold (XPC) and threefold (XPA) greater than that of the kidney, which is known to exhibit XP-DNA repair activity that is greater than most organs of the body. Immunohistochemistry revealed that most kidney and cochlear cells were immunopositive. CONCLUSION These data suggest that under normal conditions the cochlea is experiencing persistent genomic stress that helps to explain the hypersensitivity of the cochlea to exogenous stressors (ototoxic xenobiotics and/or acoustic-overexposure) as well as provide a basis to interpret hearing loss among patients with XP.
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Heikal AA. Intracellular coenzymes as natural biomarkers for metabolic activities and mitochondrial anomalies. Biomark Med 2010; 4:241-63. [PMID: 20406068 DOI: 10.2217/bmm.10.1] [Citation(s) in RCA: 305] [Impact Index Per Article: 20.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
Mitochondria play a pivotal role in energy metabolism, programmed cell death and oxidative stress. Mutated mitochondrial DNA in diseased cells compromises the structure of key enzyme complexes and, therefore, mitochondrial function, which leads to a myriad of health-related conditions such as cancer, neurodegenerative diseases, diabetes and aging. Early detection of mitochondrial and metabolic anomalies is an essential step towards effective diagnoses and therapeutic intervention. Reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) play important roles in a wide range of cellular oxidation-reduction reactions. Importantly, NADH and FAD are naturally fluorescent, which allows noninvasive imaging of metabolic activities of living cells and tissues. Furthermore, NADH and FAD autofluorescence, which can be excited using distinct wavelengths for complementary imaging methods and is sensitive to protein binding and local environment. This article highlights recent developments concerning intracellular NADH and FAD as potential biomarkers for metabolic and mitochondrial activities.
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Affiliation(s)
- Ahmed A Heikal
- Department of Chemistry & Biochemistry and Department of Pharmacy Practice & Pharmaceutical Sciences, The University of Minnesota Duluth, 1039 University Drive, Duluth, MN 55812-2496, USA.
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Two-photon microscopy for non-invasive, quantitative monitoring of stem cell differentiation. PLoS One 2010; 5:e10075. [PMID: 20419124 PMCID: PMC2855700 DOI: 10.1371/journal.pone.0010075] [Citation(s) in RCA: 110] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2009] [Accepted: 03/04/2010] [Indexed: 11/24/2022] Open
Abstract
Background The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed. Methodology/Principal Findings Here, we present the quantitative use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs) using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(P)H, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(P)H and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress. Conclusions/Significance In this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool, which enables researchers to monitor engineered tissues and optimize culture conditions in a near real time manner.
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XU HEN, WU BAOHUA, NIOKA SHOKO, CHANCE BRITTON, LI LINZ. QUANTITATIVE REDOX SCANNING OF TISSUE SAMPLES USING A CALIBRATION PROCEDURE. JOURNAL OF INNOVATIVE OPTICAL HEALTH SCIENCES 2009; 2:375-385. [PMID: 31827629 PMCID: PMC6905636 DOI: 10.1142/s1793545809000681] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/04/2023]
Abstract
The fluorescence properties of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp) including flavin adenine dinucleotide (FAD) in the respiratory chain are sensitive indicators of intracellular metabolic states and have been applied to the studies of mitochondrial function with energy-linked processes. The redox scanner, a three-dimensional (3D) low temperature imager previously developed by Chance et al., measures the in vivo metabolic properties of tissue samples by acquiring fluorescence images of NADH and Fp. The redox ratios, i.e. Fp/(Fp+NADH) and NADH/(Fp+NADH), provided a sensitive index of the mitochondrial redox state and were determined based on relative signal intensity ratios. Here we report the further development of the redox scanning technique by using a calibration method to quantify the nominal concentration of the fluorophores in tissues. The redox scanner exhibited very good linear response in the range of NADH concentration between 165-1318μM and Fp between 90-720 μM using snap-frozen solution standards. Tissue samples such as human tumor mouse xenografts and various mouse organs were redox-scanned together with adjacent NADH and Fp standards of known concentration at liquid nitrogen temperature. The nominal NADH and Fp concentrations as well as the redox ratios in the tissue samples were quantified by normalizing the tissue NADH and Fp fluorescence signal to that of the snap-frozen solution standards. This calibration procedure allows comparing redox images obtained at different time, independent of instrument settings. The quantitative multi-slice redox images revealed heterogeneity in mitochondrial redox state in the tissues.
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Affiliation(s)
- HE N. XU
- Molecular Imaging Laboratory, Department of Radiology
University of Pennsylvania, Philadelphia PA 19104, USA
| | - BAOHUA WU
- Molecular Imaging Laboratory, Department of Radiology
University of Pennsylvania, Philadelphia PA 19104, USA
- Johnson Research Foundation Department of
Biochemistry and Biophysics University of Pennsylvania, PA 19104, USA
| | - SHOKO NIOKA
- Johnson Research Foundation Department of
Biochemistry and Biophysics University of Pennsylvania, PA 19104, USA
| | - BRITTON CHANCE
- Johnson Research Foundation Department of
Biochemistry and Biophysics University of Pennsylvania, PA 19104, USA
| | - LIN Z. LI
- Molecular Imaging Laboratory, Department of Radiology
University of Pennsylvania, Philadelphia PA 19104, USA
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LI LINZ, XU HEN, RANJI MAHSA, NIOKA SHOKO, CHANCE BRITTON. MITOCHONDRIAL REDOX IMAGING FOR CANCER DIAGNOSTIC AND THERAPEUTIC STUDIES. JOURNAL OF INNOVATIVE OPTICAL HEALTH SCIENCES 2009; 2:325-341. [PMID: 26015810 PMCID: PMC4442014 DOI: 10.1142/s1793545809000735] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/04/2023]
Abstract
Mitochondrial redox states provide important information about energy-linked biological processes and signaling events in tissues for various disease phenotypes including cancer. The redox scanning method developed at the Chance laboratory about 30 years ago has allowed 3D high-resolution (~ 50 × 50 × 10 μm3) imaging of mitochondrial redox state in tissue on the basis of the fluorescence of NADH (reduced nicotinamide adenine dinucleotide) and Fp (oxidized flavoproteins including flavin adenine dinucleotide, i.e., FAD). In this review, we illustrate its basic principles, recent technical developments, and biomedical applications to cancer diagnostic and therapeutic studies in small animal models. Recently developed calibration procedures for the redox imaging using reference standards allow quantification of nominal NADH and Fp concentrations, and the concentration-based redox ratios, e.g., Fp/(Fp+NADH) and NADH/(Fp+NADH) in tissues. This calibration facilitates the comparison of redox imaging results acquired for different metabolic states at different times and/or with different instrumental settings. A redox imager using a CCD detector has been developed to acquire 3D images faster and with a higher in-plane resolution down to 10 μm. Ex vivo imaging and in vivo imaging of tissue mitochondrial redox status have been demonstrated with the CCD imager. Applications of tissue redox imaging in small animal cancer models include metabolic imaging of glioma and myc-induced mouse mammary tumors, predicting the metastatic potentials of human melanoma and breast cancer mouse xenografts, differentiating precancerous and normal tissues, and monitoring the tumor treatment response to photodynamic therapy. Possible future directions for the development of redox imaging are also discussed.
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Affiliation(s)
- LIN Z. LI
- Department of Radiology, School of Medicine, University of Pennsylvania, B6 Blockley Hall, 423 Guardian Drive, Philadelphia, PA 19104-6069, USA
- The Institute for Translational Medicine and Therapeutics, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - HE N. XU
- Department of Radiology, School of Medicine, University of Pennsylvania, B6 Blockley Hall, 423 Guardian Drive, Philadelphia, PA 19104-6069, USA
| | - MAHSA RANJI
- Biophotonics Laboratory, Department of Electrical Engineering and Computer Science, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA
| | - SHOKO NIOKA
- Johnson Research Foundation, Department of Biochemistry and Molecular Biophysics, School of Medicine, University of Pennsylvania, 250 Anatomy Chemistry Building, Philadelphia, PA 19104, USA
| | - BRITTON CHANCE
- Johnson Research Foundation, Department of Biochemistry and Molecular Biophysics, School of Medicine, University of Pennsylvania, 250 Anatomy Chemistry Building, Philadelphia, PA 19104, USA
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Tiede L, Steyger PS, Nichols MG, Hallworth R. Metabolic imaging of the organ of corti--a window on cochlea bioenergetics. Brain Res 2009; 1277:37-41. [PMID: 19272358 DOI: 10.1016/j.brainres.2009.02.052] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2009] [Revised: 02/16/2009] [Accepted: 02/23/2009] [Indexed: 11/28/2022]
Abstract
Hair cell loss is a major cause of sensorineural hearing loss. We have developed a method to examine metabolic events in hair cells in response to stimuli known to cause hair cell loss, such as acoustic trauma and aminoglycoside administration. The method employs two-photon excitation of the metabolic intermediate, reduced nicotinamide adenine dinucleotide (NADH), in hair cell mitochondria in an explanted mouse cochlea. Using this method, we show evidence that the aminoglycoside gentamicin selectively affects the level of mitochondrial NADH in outer hair cells, but not inner hair cells, within minutes of administration.
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Affiliation(s)
- LeAnn Tiede
- Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE 68178, USA
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Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level. JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY 2008; 95:46-57. [PMID: 19179090 DOI: 10.1016/j.jphotobiol.2008.12.010] [Citation(s) in RCA: 181] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/01/2008] [Revised: 12/17/2008] [Accepted: 12/17/2008] [Indexed: 11/20/2022]
Abstract
Reduced nicotinamide adenine dinucleotide, NADH, is a major electron donor in the oxidative phosphorylation and glycolytic pathways in cells. As a result, there has been recent resurgence in employing intrinsic NADH fluorescence as a natural probe for a range of cellular processes that include apoptosis, cancer pathology, and enzyme kinetics. Here, we report on two-photon fluorescence lifetime and polarization imaging of intrinsic NADH in breast cancer (Hs578T) and normal (Hs578Bst) cells for quantitative analysis of the concentration and conformation (i.e., free-to-enzyme-bound ratios) of this coenzyme. Two-photon fluorescence lifetime imaging of intracellular NADH indicates sensitivity to both cell pathology and inhibition of the respiratory chain activities using potassium cyanide (KCN). Using a newly developed non-invasive assay, we estimate the average NADH concentration in cancer cells (168+/-49 microM) to be approximately 1.8-fold higher than in breast normal cells (99+/-37 microM). Such analyses indicate changes in energy metabolism and redox reactions in normal breast cells upon inhibition of the respiratory chain activity using KCN. In addition, time-resolved associated anisotropy of cellular autofluorescence indicates population fractions of free (0.18+/-0.08) and enzyme-bound (0.82+/-0.08) conformations of intracellular NADH in normal breast cells. These fractions are statistically different from those in breast cancer cells (free: 0.25+/-0.08; bound: 0.75+/-0.08). Comparative studies on the binding kinetics of NADH with mitochondrial malate dehydrogenase and lactate dehydrogenase in solution mimic our findings in living cells. These quantitative studies demonstrate the potential of intracellular NADH dynamics (rather than intensity) imaging for probing mitochondrial anomalies associated with neurodegenerative diseases, cancer, diabetes, and aging. Our approach is also applicable to other metabolic and signaling pathways in living cells, without the need for cell destruction as in conventional biochemical assays.
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Huang Z, Zhuo S, Chen J, Chen R, Jiang X. Multiphoton microscopic imaging of adipose tissue based on second-harmonic generation and two-photon excited fluorescence. SCANNING 2008; 30:452-6. [PMID: 18942106 DOI: 10.1002/sca.20130] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/23/2023]
Abstract
The fresh adipose tissue was investigated by the use of multiphoton microscopy (MPM) based on two-photon excited fluorescence and second-harmonic generation (SHG). Microstructure of collagen and adipose cells in the adipose tissue is clearly imaged at a subcellular level with the excitation light wavelengths of 850 and 730 nm, respectively. The emission spectrum of collagen SHG signal and NADH and FAD fluorescence signal can also be obtained, which can be used to quantify the content of collagen and adipose cells and reflect the degree of pathological changes when comparing normal tissue with abnormal adipose tissue in the same condition. The results indicate that MPM has the potential to be applied to investigate the adipose tissue and can be used in the research field of lipid and connective tissues.
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Affiliation(s)
- Zufang Huang
- Fujian Normal University, Fuzhou, People's Republic of China
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Zhuo S, Chen J, Yu B, Jiang X, Luo T, Liu Q, Chen R, Xie S. Nonlinear optical microscopy of the bronchus. JOURNAL OF BIOMEDICAL OPTICS 2008; 13:054024. [PMID: 19021404 DOI: 10.1117/1.2982534] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/12/2023]
Abstract
Because of frequent exposure to carcinogens, the bronchus is prone to early pathologic alterations. The assessment of these early changes is of key significance in physiological studies and disease diagnosis of the bronchus. We utilize nonlinear optical microscopy (NLOM) to image mouse bronchial tissue based on intrinsic nonlinear optical contrast. Our results show that NLOM is effective for imaging the bronchial intact microstructural components, providing quantitative information about the biomorphology and biochemistry of tissue. Our findings also display that NLOM can provide a two-photon ratiometric redox fluorometry, based on mitochondrial signals and reduced pyridine nucleotide (NADH and NADPH) and oxidized flavoproteins (Fp) signals, to assess the metabolic state of the epithelial cells and chondrocytes. It was found that NLOM can offer a sensitive tool, based on the second-harmonic signal depth-dependent decay, to obtain quantitative information on the optical property of the stroma associated with normal and diseased tissue states. Our results suggest that with the advent of the clinical portability of typical nonlinear optical endoscopy, the NLOM technique has the potential to be applied in vivo to the clinical diagnosis and monitoring of bronchial disease.
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Affiliation(s)
- Shuangmu Zhuo
- Fujian Normal University, Institute of Laser and Optoelectronics Technology, Fujian Provincial Key Laboratory for Photonics Technology, Key Laboratory of Optoelectronic Science and Technology for Medicine, Ministry of Education, Fuzhou 350007, P. R. China
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Zhuo S, Chen J, Luo T, Jiang X, Xie S, Chen R. Two-layered multiphoton microscopic imaging of cervical tissue. Lasers Med Sci 2008; 24:359-63. [PMID: 18536958 DOI: 10.1007/s10103-008-0570-2] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2007] [Accepted: 04/23/2008] [Indexed: 01/17/2023]
Abstract
We demonstrate the application of multiphoton microscopy (MPM) based on two-photon excited fluorescence and second harmonic generation signals in imaging two-layered structures of cervical tissue in mice. It was found that MPM is effective for visualizing two-layered cervical microstructures, monitoring the metabolic activity and the density (nuclear-to-cytoplasmic) of epithelial cells, measuring the size of epithelial cell nuclei and the thickness of the epithelial layer, and quantifying collagen in the stroma. These results suggest that, with the advent of clinical portability of typical multiphoton endoscopes, the MPM technique has the potential to be applied in vivo to the clinical diagnosis and monitoring of cervical cancer.
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Affiliation(s)
- Shuangmu Zhuo
- Key Laboratory of Optoelectronic Science and Technology for Medicine, Fujian Normal University, Ministry of Education, Fuzhou, 350007, China
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Zhuo S, Chen J, Cao N, Jiang X, Xie S, Xiong S. Imaging collagen remodeling and sensing transplanted autologous fibroblast metabolism in mouse dermis using multimode nonlinear optical imaging. Phys Med Biol 2008; 53:3317-25. [DOI: 10.1088/0031-9155/53/12/017] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
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