drosophila-preparation-longitudinal-imaging-heart-function-vivo-using

<em>Drosophila</em> Preparation and Longitudinal Imaging of Heart Function <em>In Vivo</em> Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

Lehigh University

We report noninvasive imaging of the brain of adult zebrafish (Danio rerio) using real time optical coherence tomography (OCT) capable of acquiring cross sectional 2D OCT images @ 8 frames/sec. Anatomic features such as telencephalon, tectum opticum, eminentia Granularis and cerebellum were clearly resolved in the OCT images. A 3D model of zebrafish brain was reconstructed, for the first time to our knowledge, using these 2D OCT images.

Real-time in vivo imaging of adult zebrafish brain using optical coherence tomography.

The capability of optical coherence tomography (OCT) to perform "optical biopsy" of tissues within a depth range of 1 to 2 mm with micron-scale resolution in real time makes it a promising biomedical imaging modality for dermatologic applications. Three high-speed, spectrometer-based frequency-domain OCT systems operating at 800 nm (20,000 A-scans/s), 1060 nm, and 1300 nm (both 47,000 A-scans/s) at comparable signal-to-noise ratio (SNR), SNR roll-off with scanning depth, and transverse resolution (<15 microm) were used to acquire 3-D tomograms of glabrous and hairy human skin in vivo. Images obtained using these three systems were compared in terms of penetration depth, resolution, and contrast. Normal as well as abnormal sites like moles and scar tissue were examined. In this preliminary study, skin pigmentation had little effect on penetration accomplished at three different wavelengths. The epidermis and dermal-epidermal junction could be properly delineated using OCT at 800 nm, and this wavelength offered better contrast over the other two wavelength regions. OCT at 1300 nm permits imaging of deeper dermal layers, critical for detecting deeper tumor boundaries and other deeper skin pathologies. The performance at 1060 nm compromises between the other wavelengths in terms of penetration depth and image contrast.

Multispectral in vivo three-dimensional optical coherence tomography of human skin.

To gather sub-surface in situ images of microneedle-treated human skin, in vivo, using optical coherence tomography (OCT). This is the first study to utilise OCT to investigate the architectural changes that are induced in skin following microneedle application.

In vivo, in situ imaging of microneedle insertion into the skin of human volunteers using optical coherence tomography.

This study evaluated in vivo the potential of optical coherence tomography (OCT) to determine changes in thickness of the epidermis in response to the topically applied anti-psoriatics betamethasone dipropionate (BD), salicylic acid (SA) and also fish oil (FO). GsdmA3Dfl/+ mice have an inflammatory hair loss phenotype that includes hyperproliferation and epidermal thickening, hence a potential psoriasis model. Changes in epidermal thickness were evaluated over a period of 10 days, with the mice treated with combined BD + SA, FO + SA and BD + FO + SA. The data were validated with conventional measurement using H&E staining coupled with microscopy. Initial baseline measurement revealed an average epidermal thickness of 26.92 ± 1.17 μm. After 10 days of treatment with BD, the average epidermal thickness was reduced by 38.8% (P = 0.0001), and inversely, treatment with FO resulted in an unexpected 105% increase (P = 0.0001) in epidermal thickness. Combined BD + FO treatment did not cause any significant change (P = 0.3755) and may further indicate opposing effects on keratinocyte proliferation. The data obtained using OCT were statistically the same as those obtained by H&E/microscopy (P = 0.4325), supporting a greater role for OCT in dermatological studies, while also allowing a reduction in the number of animals used in such studies as sacrifice at individual timepoints is not necessary.

In vivo response of GsdmA3Dfl/+ mice to topically applied anti-psoriatic agents: effects on epidermal thickness, as determined by optical coherence tomography and H&E staining.

A noninvasive, multimodal photoacoustic and optical coherence tomography (PAT/OCT) scanner for three-dimensional in vivo (3D) skin imaging is described. The system employs an integrated, all optical detection scheme for both modalities in backward mode utilizing a shared 2D optical scanner with a field-of-view of ~13 × 13 mm(2). The photoacoustic waves were detected using a Fabry Perot polymer film ultrasound sensor placed on the surface of the skin. The sensor is transparent in the spectral range 590-1200 nm. This permits the photoacoustic excitation beam (670-680 nm) and the OCT probe beam (1050 nm) to be transmitted through the sensor head and into the underlying tissue thus providing a backward mode imaging configuration. The respective OCT and PAT axial resolutions were 8 and 20 µm and the lateral resolutions were 18 and 50-100 µm. The system provides greater penetration depth than previous combined PA/OCT devices due to the longer wavelength of the OCT beam (1050 nm rather than 829-870 nm) and by operating in the tomographic rather than the optical resolution mode of photoacoustic imaging. Three-dimensional in vivo images of the vasculature and the surrounding tissue micro-morphology in murine and human skin were acquired. These studies demonstrated the complementary contrast and tissue information provided by each modality for high-resolution 3D imaging of vascular structures to depths of up to 5 mm. Potential applications include characterizing skin conditions such as tumors, vascular lesions, soft tissue damage such as burns and wounds, inflammatory conditions such as dermatitis and other superficial tissue abnormalities.

Multimodal photoacoustic and optical coherence tomography scanner using an all optical detection scheme for 3D morphological skin imaging.

A preliminary clinical trial using state-of-the-art multiphoton tomography (MPT) and optical coherence tomography (OCT) for three-dimensional (3D) multimodal in vivo imaging of normal skin, nevi, scars and pathologic skin lesions has been conducted. MPT enabled visualization of sub-cellular details with axial and transverse resolutions of <2 μm and <0.5 μm, respectively, from a volume of 0.35 × 0.35 × 0.2 mm(3) at a frame rate of 0.14 Hz (512 × 512 pixels). State-of-the-art OCT, operating at a center wavelength of 1300 nm, was capable of acquiring 3D images depicting the layered architecture of skin with axial and transverse resolutions ~8 μm and ~20 μm, respectively, from a volume of 7 × 3.5 × 1.5 mm(3) at a frame rate of 46 Hz (1024 × 1024 pixels). This study demonstrates the clinical diagnostic potential of MPT/OCT for pre-screening relatively large areas of skin using 3D OCT to identify suspicious regions at microscopic level and subsequently using high resolution MPT to obtain zoomed in, sub-cellular level information of the respective regions.

Three-dimensional multiphoton/optical coherence tomography for diagnostic applications in dermatology.

We demonstrate noninvasive structural and microvascular contrast imaging of different human skin diseases in vivo using an intensity difference analysis of OCT tomograms. The high-speed swept source OCT system operates at 1310 nm with 220 kHz A-scan rate. It provides an extended focus by employing a Bessel beam. The studied lesions were two cases of dermatitis and two cases of basal cell carcinoma. The lesions show characteristic vascular patterns that are significantly different from healthy skin. In case of inflammation, vessels are dilated and perfusion is increased. In case of basal cell carcinoma, the angiogram shows a denser network of unorganized vessels with large vessels close to the skin surface. Those results indicate that assessing vascular changes yields complementary information with important insight into the metabolic demand.

In situ structural and microangiographic assessment of human skin lesions with high-speed OCT.

To visualize the precorneal tear film with ultrahigh-resolution spectral domain optical coherence tomography, enabling quantification of tear film thickness in healthy subjects.

Measurement of tear film thickness using ultrahigh-resolution optical coherence tomography.

Eosinophilic esophagitis (EoE) is a food allergy-associated inflammatory disease characterized by esophageal eosinophilia. Current management strategies for EoE are nonspecific, and thus there is a need to identify specific immunological pathways that could be targeted to treat this disease. EoE is associated with polymorphisms in the gene that encodes thymic stromal lymphopoietin (TSLP), a cytokine that promotes allergic inflammation, but how TSLP might contribute to EoE disease pathogenesis has been unclear. Here, we describe a new mouse model of EoE-like disease that developed independently of IgE, but was dependent on TSLP and basophils, as targeting TSLP or basophils during the sensitization phase limited disease. Notably, therapeutic TSLP neutralization or basophil depletion also ameliorated established EoE-like disease. In human subjects with EoE, we observed elevated TSLP expression and exaggerated basophil responses in esophageal biopsies, and a gain-of-function TSLP polymorphism was associated with increased basophil responses in patients with EoE. Together, these data suggest that the TSLP-basophil axis contributes to the pathogenesis of EoE and could be therapeutically targeted to treat this disease.

Thymic stromal lymphopoietin-elicited basophil responses promote eosinophilic esophagitis.

High speed, high resolution and high sensitivity are desirable for optical coherence tomography (OCT). Here, we demonstrate a space-division multiplexing (SDM) technology that translates long coherence length of a commercially available wavelength tunable laser into high OCT imaging speed. We achieved an effective 800,000 A-scans/s imaging speed using a 100,000 Hz tunable vertical cavity surface-emitting laser (VCSEL). A sensitivity of 94.6 dB and a roll-off of < 2 dB over ~30 mm imaging depth were measured from a single channel in the prototype SDM-OCT system. An axial resolution of ~11 μm in air (or ~8.3 μm in tissue) was achieved throughout the entire depth range. An in vivo, 3D SDM-OCT volume of an entire Drosophila larva consisting of 400 x 605 A-scans was acquired in 0.37 seconds. Synchronized cross-sectional OCT imaging of three different segments of a beating Drosophila larva heart is demonstrated. The SDM technology provides a new orthogonal dimension for further speed improvement for OCT with favorable cost scaling. SDM-OCT also preserves image resolution and allows synchronized cross-sectional and three-dimensional (3D) imaging of biological samples, enabling new biomedical applications.

Space-division multiplexing optical coherence tomography.

Pre-clinical studies using murine models are critical for understanding the pathophysiological mechanisms underlying immune-mediated disorders such as Eosinophilic esophagitis (EoE). In this study, an optical coherence tomography (OCT) system capable of providing three-dimensional images with axial and transverse resolutions of 5 µm and 10 µm, respectively, was utilized to obtain esophageal images from a murine model of EoE-like disease ex vivo. Structural changes in the esophagus of wild-type (Tslpr(+/+) ) and mutant (Tslpr(-/-) ) mice with EoE-like disease were quantitatively evaluated and food impaction sites in the esophagus of diseased mice were monitored using OCT. Here, the capability of OCT as a label-free imaging tool devoid of tissue-processing artifacts to effectively characterize murine EoE-like disease models has been demonstrated.

Characterization of eosinophilic esophagitis murine models using optical coherence tomography.

Optical coherence tomography (OCT) is a noninvasive, three-dimensional imaging modality with several medical and industrial applications. Integrated photonics has the potential to enable mass production of OCT devices to significantly reduce size and cost, which can increase its use in established fields as well as enable new applications. Using silicon nitride (Si3N4) and silicon dioxide (SiO2) waveguides, we fabricated an integrated interferometer for spectrometer-based OCT. The integrated photonic circuit consists of four splitters and a 190 mm long reference arm with a foot-print of only 10 × 33 mm(2). It is used as the core of a spectral domain OCT system consisting of a superluminescent diode centered at 1320 nm with 100 nm bandwidth, a spectrometer with 1024 channels, and an x-y scanner. The sensitivity of the system was measured at 0.25 mm depth to be 65 dB with 0.1 mW on the sample. Using the system, we imaged human skin in vivo. With further optimization in design and fabrication technology, Si3N4/SiO2 waveguides have a potential to serve as a platform for passive photonic integrated circuits for OCT.

Photonic integrated Mach-Zehnder interferometer with an on-chip reference arm for optical coherence tomography.

Circadian rhythms are endogenous, entrainable oscillations of physical, mental and behavioural processes in response to local environmental cues such as daylight, which are present in the living beings, including humans. Circadian rhythms have been related to cardiovascular function and pathology. However, the role that circadian clock genes play in heart development and function in a whole animal in vivo are poorly understood. The Drosophila cryptochrome (dCry) is a circadian clock gene that encodes a major component of the circadian clock negative feedback loop. Compared to the embryonic stage, the relative expression levels of dCry showed a significant increase (>100-fold) in Drosophila during the pupa and adult stages. In this study, we utilized an ultrahigh resolution optical coherence microscopy (OCM) system to perform non-invasive and longitudinal analysis of functional and morphological changes in the Drosophila heart throughout its post-embryonic lifecycle for the first time. The Drosophila heart exhibited major morphological and functional alterations during its development. Notably, heart rate (HR) and cardiac activity period (CAP) of Drosophila showed significant variations during the pupa stage, when heart remodeling took place. From the M-mode (2D + time) OCM images, cardiac structural and functional parameters of Drosophila at different developmental stages were quantitatively determined. In order to study the functional role of dCry on Drosophila heart development, we silenced dCry by RNAi in the Drosophila heart and mesoderm, and quantitatively measured heart morphology and function in those flies throughout its development. Silencing of dCry resulted in slower HR, reduced CAP, smaller heart chamber size, pupal lethality and disrupted posterior segmentation that was related to increased expression of a posterior compartment protein, wingless. Collectively, our studies provided novel evidence that the circadian clock gene, dCry, plays an essential role in heart morphogenesis and function.

A Circadian Clock Gene, Cry, Affects Heart Morphogenesis and Function in Drosophila as Revealed by Optical Coherence Microscopy.

Electrical stimulation is currently the gold standard for cardiac pacing. However, it is invasive and nonspecific for cardiac tissues. We recently developed a noninvasive cardiac pacing technique using optogenetic tools, which are widely used in neuroscience. Optogenetic pacing of the heart provides high spatial and temporal precisions, is specific for cardiac tissues, avoids artifacts associated with electrical stimulation, and therefore promises to be a powerful tool in basic cardiac research. We demonstrated optogenetic control of heart rhythm in a well-established model organism, Drosophila melanogaster. We developed transgenic flies expressing a light-gated cation channel, channelrhodopsin-2 (ChR2), specifically in their hearts and demonstrated successful optogenetic pacing of ChR2-expressing Drosophila at different developmental stages, including the larva, pupa, and adult stages. A high-speed and ultrahigh-resolution optical coherence microscopy imaging system that is capable of providing images at a rate of 130 frames/s with axial and transverse resolutions of 1.5 and 3.9 μm, respectively, was used to noninvasively monitor Drosophila cardiac function and its response to pacing stimulation. The development of a noninvasive integrated optical pacing and imaging system provides a novel platform for performing research studies in developmental cardiology.

Optogenetic pacing in Drosophila melanogaster.

Eosinophilic esophagitis (EoE) is an immune-mediated disorder characterized by esophageal inflammation and related structural changes causing symptoms such as feeding difficulties and food impaction. The pathophysiological mechanisms underlying EoE remain poorly understood. Preclinical studies using mouse models have been critical in comprehending human disease mechanisms and associated pathways. In this chapter, we describe an experimental method using a noninvasive label-free optical imaging technique, optical coherence tomography, to characterize the pathophysiological changes in the esophagus of mice with EoE-like disease ex vivo.

Label-Free Imaging of Eosinophilic Esophagitis Mouse Models Using Optical Coherence Tomography.

Optical coherence tomography (OCT) is a promising research tool for brain imaging and developmental biology. Serving as a three-dimensional optical biopsy technique, OCT provides volumetric reconstruction of brain tissues and embryonic structures with micrometer resolution and video rate imaging speed. Functional OCT enables label-free monitoring of hemodynamic and metabolic changes in the brain in vitro and in vivo in animal models. Due to its non-invasiveness nature, OCT enables longitudinal imaging of developing specimens in vivo without potential damage from surgical operation, tissue fixation and processing, and staining with exogenous contrast agents. In this paper, various OCT applications in brain imaging and developmental biology are reviewed, with a particular focus on imaging heart development. In addition, we report findings on the effects of a circadian gene (Clock) and high-fat-diet on heart development in Drosophila melanogaster. These findings contribute to our understanding of the fundamental mechanisms connecting circadian genes and obesity to heart development and cardiac diseases.

Article	Year
Real-time in vivo imaging of adult zebrafish brain using optical coherence tomography. Journal of biophotonics\| PubMed ID: 19434615	2009
Multispectral in vivo three-dimensional optical coherence tomography of human skin. Journal of biomedical optics\| PubMed ID: 20459270	2010
In vivo, in situ imaging of microneedle insertion into the skin of human volunteers using optical coherence tomography. Pharmaceutical research\| PubMed ID: 20464461	2011
In vivo response of GsdmA3Dfl/+ mice to topically applied anti-psoriatic agents: effects on epidermal thickness, as determined by optical coherence tomography and H&E staining. Experimental dermatology\| PubMed ID: 21323746	2011
Multimodal photoacoustic and optical coherence tomography scanner using an all optical detection scheme for 3D morphological skin imaging. Biomedical optics express\| PubMed ID: 21833358	2011
Three-dimensional multiphoton/optical coherence tomography for diagnostic applications in dermatology. Journal of biophotonics\| PubMed ID: 22711418	2013
In situ structural and microangiographic assessment of human skin lesions with high-speed OCT. Biomedical optics express\| PubMed ID: 23082302	2012
Measurement of tear film thickness using ultrahigh-resolution optical coherence tomography. Investigative ophthalmology & visual science\| PubMed ID: 23847319	2013
Thymic stromal lymphopoietin-elicited basophil responses promote eosinophilic esophagitis. Nature medicine\| PubMed ID: 23872715	2013
Space-division multiplexing optical coherence tomography. Optics express\| PubMed ID: 23938839	2013
Characterization of eosinophilic esophagitis murine models using optical coherence tomography. Biomedical optics express\| PubMed ID: 24575353	2014
Photonic integrated Mach-Zehnder interferometer with an on-chip reference arm for optical coherence tomography. Biomedical optics express\| PubMed ID: 24761288	2014
A Circadian Clock Gene, Cry, Affects Heart Morphogenesis and Function in Drosophila as Revealed by Optical Coherence Microscopy. PloS one\| PubMed ID: 26348211	2015
Optogenetic pacing in Drosophila melanogaster. Science advances\| PubMed ID: 26601299	2015
Label-Free Imaging of Eosinophilic Esophagitis Mouse Models Using Optical Coherence Tomography. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 27246028	2016
Optical Coherence Tomography for Brain Imaging and Developmental Biology. IEEE journal of selected topics in quantum electronics : a publication of the IEEE Lasers and Electro-optics Society\| PubMed ID: 27721647	2016

Aneesh Alex

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