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PubMedDate not supplied

Optical imaging in microfluidic bioreactors enables oxygen monitoring for continuous cell culture.

For the first time, a fluorescence lifetime calibration method for an oxygen-sensitive dye ruthenium tris(2,2'-dipyridyl) dichloride hexahydrate (RTDP) is applied to image oxygen levels in poly(dimethyl siloxane) (PDMS) bioreactors containing living C2C12 mouse myoblasts. PDMS microsystems are broadly used in bioengineering applications due to their biocompatibility and ease of handling. For these systems, oxygen concentrations are of significance and are likely to play an important role in cell behavior and gene expression. Fluorescence lifetime imaging microscopy (FLIM) bases image contrast on fluorophore excited state lifetimes, which reflect local biochemistry. Unique attributes of the widefield, time-domain FLIM system include tunable excitation (337.1 to 960 nm), large temporal dynamic range (> or =600 ps), high spatial resolution (1.4 microm), calibrated detection (0 to 300+/-8 microM of oxygen), and rapid data acquisition and processing times (10 s). Oxygen levels decrease with increasing cell densities and are consistent with model outcomes obtained by simulating bioreactor oxygen diffusion and cell proliferation. In single bioreactor loops, FLIM detects spatial heterogeneity in oxygen levels with variations as high as 20%. The fluorescence lifetime-based imaging approach we describe avoids intensity-based artifacts (including photobleaching and concentration variations) and provides a technique with high spatial discrimination for oxygen monitoring in continuous cell culture systems.

Animals↗
PubMed2001

Biological characterization and optical imaging of brain-derived neurotrophic factor-green fluorescent protein suggest an activity-dependent local release of brain-derived neurotrophic factor in neurites of cultured hippocampal neurons.

To visualize the release dynamics of the brain-derived neurotrophic factor (BDNF) involved in neural plasticity, we constructed a plasmid encoding green fluorescent protein (GFP) fused with BDNF. First, several biological studies confirmed that this fusion protein (BDNF-GFP) mimics the biological functions and the release kinetics of unfused (native) BDNF. Second, when BDNF-GFP was expressed in cultured hippocampal neurons, we observed that this protein formed striking clusters in the neurites of mature neurons and colocalized with the PSD-95 immunoreactivity. Such a clustered BDNF-GFP rapidly disappeared in response to depolarization with KCl, as revealed by confocal microscopic studies. These data suggest that BDNF is locally and rapidly released at synaptic sites in an activity-dependent manner. Optical studies using BDNF-GFP may provide important evidence regarding the participation of BDNF in synaptic plasticity.

Animals↗
PubMed2005

Multimodal in vivo optical imaging, including confocal microscopy, facilitates presurgical margin mapping for clinically complex lentigo maligna melanoma.

Knowledge of the accurate margins of a lentigo maligna melanoma (LMM) is crucial in the presurgical evaluation of the patient. Towards this end clinicians have utilized the Wood's lamp and dermoscopy to help delineate the borders of the LMM. However, many LMMs arise on photodamaged skin, making it difficult to determine the border of the LMM and separate it from the background lentiginous skin. We present a case of a patient with a recurrent LMM on the scalp that developed in a background of photodamage with diffuse melanocytic atypia and lentigines, making it virtually impossible to determine the precise margins of the LMM by clinical, Wood's lamp or dermoscopic examination. To avoid subjecting the patient to multiple staged excisions we attempted to determine the margins of the LMM by utilizing in vivo confocal laser scanning reflectance microscopy. Using this, it was apparent that there were increased numbers of atypical/dendritic intraepidermal melanocytes in all layers of the epidermis within the LMM. In contrast, skin not involved with the LMM, as viewed under confocal laser examination, had normal honeycomb architecture and no abnormal melanocytes. The confocally determined border was further confirmed by obtaining multiple punch biopsies that were evaluated by haematoxylin and eosin histology and immunohistochemistry. Based on this information, the presurgical margins were marked and the tumour excised accordingly. The excised tissue was examined with multiple-step sections and the margins were determined to be clear. There has been no evidence of tumour recurrence after 1 year. In conclusion, this case illustrates that confocal reflectance microscopy, in conjunction with other in vivo optical instruments, can be utilized to enhance the accuracy for the presurgical margin mapping of LMM.

Aged↗
PubMed1999

Novel form of spreading acidification and depression in the cerebellar cortex demonstrated by neutral red optical imaging.

A novel form of spreading acidification and depression in the rat cerebellar cortex was imaged in vivo using the pH-sensitive dye, Neutral red. Surface stimulation evoked an initial beam of increased fluorescence (i.e., decreased pH) that spread rostrally and caudally across the folium and into neighboring folia. A transient but marked suppression in the excitability of the parallel fiber-Purkinje cell circuitry accompanied the spread. Characteristics differentiating this phenomenon from the spreading depression of Leao include: high speed of propagation on the surface (average of 450 microm/s), stable extracellular DC potential, no change in blood vessel diameter, and repeatability at short intervals. This propagating acidification constitutes a previously unknown class of neuronal processing in the cerebellar cortex.

Acids↗
PubMed2006

Iteration of high-frequency stimulation enhances long-lasting excitatory responses in the spinal dorsal horn of rats: characterization by optical imaging of signal propagation.

To investigate plastic changes in nociceptive sensitivity of the dorsal horn, slow excitatory responses elicited by iteration of high-frequency stimulation were spatiotemporally observed in spinal cord slices of young-adult rats using membrane excitation imaging techniques. Single-pulse stimulation to the dorsal root elicited membrane excitation in lamina II, and high-frequency pulse-train stimulation evoked long-lasting excitation that expanded widely in the dorsal horn. Iteration of high-frequency stimulation enhanced the strength and extent of the excitatory responses, but such augmentation of the excitatory responses disappeared in the presence of an NMDA receptor antagonist (CPP) and was hindered by an NK1 receptor antagonist (L-703.606). The results suggest that activation of both NMDA and NK1 receptors is involved in the enhancement of slow excitatory responses evoked by iteration of high-frequency stimulation.

Action Potentials↗
PubMed1999

Optical imaging of the ventral medullary surface across sleep-wake states.

We hypothesized that spontaneous activity declines over widespread areas of the cat ventral medullary surface (VMS) during rapid eye movement (REM) sleep. We assessed neural and hemodynamic activity, measured as changes in reflected 660- and 560-nm wavelength light, from the VMS during sleep and waking states in five adult, unrestrained cats and in two control cats. Relative to quiet sleep, overall activity declined, and variability, assessed by standard deviation, increased by 25% during REM sleep. Variability in activity during waking also increased by 45% over quiet sleep, but mean activity was unchanged. REM sleep onset was preceded by a reduction in the hemodynamic signal from 5 to 60 s before neural activity decline. The activity decline during REM sleep, previously noted in the goat rostral VMS, extends to intermediate VMS areas of the cat and differs from most neural sites, such as the cortex, hippocampus, and thalamus, which increase activity during REM sleep. The activity decline during REM sleep has the potential to modify VMS responsiveness to baroreceptor and chemoreceptor challenges during the REM state.

Animals↗
PubMed2005

Optical imaging of pancreatic beta cells in living mice expressing a mouse insulin I promoter-firefly luciferase transgene.

We generated transgenic mice expressing firefly (Photinus pyralis) luciferase (luc) under the control of the mouse insulin I promoter (MIP). The mice have normal glucose tolerance and pancreatic islet architecture. The luciferase-expressing beta cells can be readily visualized in living mice using whole-body bioluminescent imaging. The MIP-luc transgenic mice may be useful for monitoring changes in beta cell function or mass in living animals with normal or altered metabolic states.

Animals↗
PubMed2004

Noninvasive optical imaging in the visual cortex in young infants.

During the developmental stage, the brain undergoes anatomic, functional, and metabolic changes necessary to support the complex adaptive behavior of a mature individual. Estimation of developmental changes occurring in different regions of the brain would provide a means of relating various behavioral phenomena to maturation-specific brain structures, thereby providing useful information on structure-function relationships in both normal and disease states. We used multichannel near-infrared spectroscopy (MNIRS), a new noninvasive imaging technique for revealing the course of neural activity in selected brain regions, to monitor the activities of the visual cortex as mirrored by hemodynamic responses in infants subjected to photostimulation during natural sleep. In the infants, oxyhemoglobin and total hemoglobin decreased and deoxyhemoglobin increased in the visual cortex with photostimulation. This pattern of responses was different from the response pattern in adults reported previously. The different patterns of responses to photostimulation in the visual cortices of infants and adults might reflect developmental and behavioral differences. It may reflect a different functional organization of the visual cortex in infants or ongoing retinal development. Our results demonstrated that regional hemodynamic change could be detected in a small area around the visual cortex. MNIRS offers considerable potential for research and noninvasive clinical applications.

Adult↗
PubMed2003

Optical imaging and tumor angiogenesis.

Tumor angiogenesis is essential for tumor growth and progression. Therefore, targeting tumor blood vessels is a promising approach for cancer therapy. Angiogenesis, the formation of blood vessels, is a multistep process, and strongly influenced by the microenvironment. There are no in vitro assays that can resemble this dynamic process in vivo. For this reason, animal models and imaging technologies are critical for studying tumor angiogenesis, identifying therapeutic targets as well as validating the targets. Non-invasive molecular imaging in animal models presents an unprecedented opportunity and ability for us to perform repetitive observations and analysis of the biological processes underlying tumor angiogenesis and tumor progression in living animals in real time. As we gain a better understanding of the fundamental molecular nature of cancer, these techniques will be an important adjunct in translating the knowledge into clinical practice. This important information may elucidate how the tumor blood vessels behave and respond to certain treatments and therapies.

Angiogenesis Inhibitors↗
PubMed2001

In vivo optical imaging of expression of vascular endothelial growth factor following laser incision in skin.

BACKGROUND AND OBJECTIVE: Laser-tissue interaction studies have focused on laser-induced secondary effects on tissue and the postmortem histological analysis of laser wounds. In this study we addressed wound healing and possible impairment of wound healing due to collateral tissue damage by in vivo imaging of gene expression. STUDY DESIGN/MATERIALS AND METHODS: We used a transgenic mouse model containing a VEGF promoter driving a GFP reporter gene to image VEGF expression in vivo. Twenty-two mice received two full thickness incisions in the dorsal skin: one with the Free Electron Laser (lambda = 6.45 microm, 52.9 mJ/mm(2)) and one with a scalpel. Mice were imaged for GFP expression at 3 days, 1, 2, 3, and 4 weeks. Confocal microscopic imaging was performed at 2 weeks. RESULTS: Peak GFP expression was seen at 2-3 weeks and was localized in fibroblasts. FEL lesions showed more total GFP expression than scalpel lesions but this was only statistically significant (P < 0.05) at 2 and 4 weeks. The full-width half-max (FWHM) of the GFP expression was always larger for the FEL lesion compared to the scalpel lesion but was only statistically significant (P < 0.05) at 2 and 3 weeks. At 2 weeks the extent of the GFP expression in the laser lesion was on average 55 microm beyond that seen in the scalpel lesion but correlated with the number of laser passes. CONCLUSIONS: Feasibility of using transgenic mice carrying photoactive reporter genes for studying cellular process of laser-inflicted wound repair in a noninvasive, in vivo manner was shown. GFP expression mediated by the VEGF promoter in fibroblast showed minimal impairment of wound healing due to the laser.

Animals↗
PubMed2005

Modeling and simulations of the pharmacokinetics of fluorophore conjugated antibodies in tumor vicinity for the optimization of fluorescence-based optical imaging.

BACKGROUND AND OBJECTIVES: One of the methods to detect and localize tumors in tissue is to use fluorophore conjugated specific antibodies as tumor surface markers. The goals of this study are to understand and quantify the pharmacokinetics of fluorophore conjugated antibodies in the vicinity of a tumor. This study concludes another stage of the development of a non-invasive fluorescenated antibody-based technique for imaging and localization of tumors in vivo. STUDY DESIGN/MATERIALS AND METHODS: A mathematical model of the pharmacokinetics of fluorophore conjugated antibodies in the vicinity of a tumor was developed based on histological staining experiments. We present the model equations of concentrations of antibodies and free binding sites. We also present a powerful simulation tool that we developed to simulate the imaging process. We analyzed the model and studied the effects of various independent parameters on the imaging result. These parameters included initial volume of markers (injected volume), total number of binding sites, tumor size, binding and dissociation rate constants, and the diffusion coefficient. We present the relations needed between these parameters in order to optimize the imaging results. RESULTS AND CONCLUSIONS: A powerful and accurate tool was developed which may assist in optimizing the imaging system results by setting the injection volume and concentration of fluorophore conjugated antibodies in tissue and approximating the time interval where maximum specific binding occurs and the tumor can be imaged.

Animals↗
PubMed2001

Noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice.

The ability to monitor reporter gene expression noninvasively offers significant advantages over current techniques such as postmortem tissue staining or enzyme activity assays. Here we demonstrate a novel method of repetitively tracking in vivo gene expression of firefly luciferase (FL) in skeletal muscles of mice using a cooled charged coupled device (CCD) camera. We first show that the cooled CCD camera provides consistent and reproducible results within +/-8% standard deviation from mean values, and a detection sensitivity (range tested: 1 x 10(4) - 1 x 10(9) plaque form-ing units (pfu)) of 1 x 10(6) pfu of E1-deleted adenovirus expressing FL driven by a cytomegalovirus promoter (Ad-CMV-FL). The duration and magnitude of adenoviral mediated (1 x 10(9) pfu) FL gene expression were then followed over time. FL gene expression in immunocompetent Swiss Webster mice peaks within the first 48 hours, falls by 98% after 20 days, and persists for >150 days. In contrast, FL activity in nude mice remains elevated for >110 days. Finally, transduced Swiss Webster and nude mice were sacrificed to show that the in vivo CCD signals correlate well with in vitro luciferase enzyme assays (r(2)=0.91 and 0.96, respectively). Our findings demonstrate the ability of the cooled CCD camera to sensitively and noninvasively track the location, magnitude, and persistence of FL gene expression. Monitoring of gene therapy studies in small animals may be aided considerably with further extensions of this technique.

Adenoviridae↗