The authors acknowledge funding from the NIDCR (Grant No. DE019902) and from the Israeli Science Foundation (Grant No. 382/13).

<ol>
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The authors have nothing to disclose.

המטרה של גישות ההדמיה multimodal המתוארות כאן היא לאפשר חקירה מדוקדקת של ציר אנגיוגנזה-Osteogenesis בהקשר של השתלת עצם גולגולת. Neovascularization היה צילם באמצעות פרוטוקול μCT, שאיפשר הפגנת 3D ברזולוציה גבוהה מדויקת של עץ כלי הדם המזין את פגם גולגולת כולה. נתונים μCT ניתן לנתח בקלות באמצע...

איבוד עצם Craniofacial עקב טראומה, כריתת גידול, craniotomy decompressive, ופגם מולד נדיר מרפא בעצמו ומציג צורך קליני מסופק ברור. שתלי עצם אוטולוגית ושתלי עצם אלוגנאית נמצאים בשימוש נרחב לטיפול במצבים אלה 1. זה מקובל שOsteogenesis מצמיד באופן הדוק עם אנגיוגנזה 2,3. כך, המחקר של טיפול מוצע להתחדשות עצם המלא צריך לכלול חקירה מקיפה של עץ כלי הדם ויוצר בכל אתר המום כולו. ישנן מספר שיטות זמינות כדי לאפיין כלי דם במודלי מחקר. עץ כלי הדם יכול להיחקר על ידי ניתוח היסטולוגית. מאז היסטולוגיה מסתמכת על חתך רקמה, קיים סבירות גבוהה שהתמונה המתקבלת תהיה מעוותת. כדי לטפל בבעיה זו, מיקרוסקופיה intravital יכולה להתבצע לכלי דם בשלמות תמונה 4; עם זאת, שיטה זו היאמוגבל להדמיה אחד-מטוס. סריקת μCT של דגימות שהתקבלו מבעלי חי perfused עם חומר ניגוד מאפשרת הדמיה 3D של רשת כלי דם המזינה את אתר ההתחדשות 5. גישה זו מאפשרת להפגנה מפורטת מאוד של כלי הדם של איבר בכללותו, כמו גם ניתוח קפדני של הפצת כלי דם. יתר על כן, μCT מאפשר בידול בין קטרים ​​המגוונים של כלי דם, המאפיינים את תת-הסוגים השונים של כלי דם. חוקרים שערנו כי השתלה של autograft calvarial יגרום neovascularization גדול יותר מהשתלה של שתל, וזה עלה neovascularization יוביל, בתורו, לעצם משופר formation.To להמשיך השערה זו אנו מועסקים במגוון טכניקות. חקרנו דפוסים של עץ כלי דם החדש שנוצר על ידי ביצוע ניתוח מבוסס μCT. מדדנו זלוף דם באמצעות בדיקה דם ניאון-בריכה. , אנו ישבנים הבאמינרליזציה רקמת עצם SED ידי FLI של בדיקה מכוונת hydroxyapatite וניתוח μCT. לבסוף, אנו פיקוח גיוס תאי גזע ובידול, ביצוע BLI בעכברים מהונדסים שבי לוציפראז מתבטא בתאי osteocalcin-חיובי.

<table border="0" cellpadding="0" cellspacing="0" width="614">
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TCA6%</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">T6399</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Osteosense 680</td>
			<td style="width:112px;">PerkinElmar</td>
			<td style="width:119px;">NEV10020EX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Angiosense750</td>
			<td style="width:112px;">PerkinElmar</td>
			<td style="width:119px;">NEV10011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oxigen 100% medical grade</td>
			<td style="width:112px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">isoflurane (furane)</td>
			<td style="width:112px;">Baxter</td>
			<td align="right" style="width:119px;">1001936040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IVIS kinetics</td>
			<td style="width:112px;">Xenogen</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Beetle luciferin</td>
			<td style="width:112px;">Promega</td>
			<td style="width:119px;">E160A</td>
			<td></td>
		</tr>
	</tbody>
</table>

computed tomography and optical imaging of osteogenesis-angiogenesis coupling to assess integration of cranial bone autografts and allografts

פרמטר עיקרי קביעת ההצלחה של הליך השתלת עצם הוא כלי דם באזור המקיף את השתל. חוקרים שערנו כי השתלה של autograft עצם יגרום התחדשות עצם גדולה יותר על ידי היווצרות כלי דם בשפע. כדי לחקור את ההשפעה של השתל על neovascularization באתר הפגם, שפיתחנו טומוגרפיה גישה מחושב מיקרו (μCT) לאפיין חדש יוצרים כלי דם, הכולל זלוף המערכתי של בעלי החיים עם חומר ניגוד polymerizing. שיטה זו מאפשרת ניתוח מפורט של כלי דם איברים בשלמותו. בנוסף, זלוף הדם הוערך באמצעות הדמיה הקרינה (FLI) של סוכן ניאון שמקורן בדם. היווצרות עצם הייתה לכמת ידי FLI באמצעות בדיקה-ממוקד hydroxyapatite וניתוח μCT. גיוס תאי גזע היה פיקוח על ידי הדמיה פליטת אור (BLI) של עכברים הטרנסגניים המבטאות לוציפראז תחת השליטה של ​​אמרגן osteocalcin.כאן אנו מתארים ומדגימים הכנת השתל, ניתוח פגם calvarial, פרוטוקולי סריקת μCT למחקר neovascularization וניתוח היווצרות עצם (כולל זלוף vivo של חומר ניגוד), והפרוטוקול לניתוח נתונים. הניתוח ברזולוציה גבוהה 3D של כלי דם הפגין אנגיוגנזה הגדולה יותר באופן משמעותי בבעלי חיים עם autografts המושתל, במיוחד ביחס להיווצרות עורקיק. בהתאם לכך, זלוף הדם היה גבוה יותר משמעותי בקבוצת autograft מהיום ליום 7 בלאחר ניתוח. אנו נצפו מינרליזציה עצם מעולה ויצירת עצם גדולה יותר נמדדה בבעלי חיים שקיבלו autografts. השתלת autograft גיוס תאי גזע תושב מושרה לתפר עצם שתל-מארח, שבו התאים מובחנים לתאי יוצרי עצם בין ה -7 ל -10 היום לאחר ניתוח ה. ממצא זה אומר שיצירת עצם משופרת ניתן לייחס להאכלת כלי הדם המוגברת המאפיינת את השתלת autograft. השיטות מתוארות יכולות לשמש ככלי אופטימלי ללמוד התחדשות עצם במונחים של יצירת עצם מתוחמת בחוזקה וניאו-וסקולריזציה.

Neovascularization הוערך על ידי ניתוח נפח μCT ועל ידי FLI באמצעות סוכן שמקורן בדם ניאון לכמת זלוף דם. שבעה ימים לאחר הניתוח, סריקת μCT הפגינה נפח גבוה יותר באופן משמעותי של כלי דם קטנים ובינוני בקוטר בעכברים שקיבלו autografts מאשר בעכברים שקיבלו allografts נקטף מן C57BL / 6 (איור 3 א). מ?...

Watch this Scientific Journal Video about Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts at JoVE.com

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

השרשה של שתלי עצם אוטולוגית ואלוגנאית מהווה קיבלה גישות לטיפול באובדן עצם craniofacial גדול. עם זאת, ההשפעה של הרכב שתל על יחסי הגומלין בין neovascularization, התמיינות תאים והיווצרות עצם לא ברורה. אנו מציגים פרוטוקול הדמיה multimodal נועד על מנת להבהיר את התלות ההדדית אנגיוגנזה-Osteogenesis בקרבת השתל.

טומוגרפיה ממוחשבת והדמיה אופטית של צימוד Osteogenesis-אנגיוגנזה להערכת שילוב של Autografts עצם הגולגולת וAllografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

computed-tomography-optical-imaging-osteogenesis-angiogenesis

Research

JoVE Journal

Bioengineering

11.3K Views.  The Hebrew University–Hadassah Faculty of Dental Medicine.  השרשה של שתלי עצם אוטולוגית ואלוגנאית מהווה קיבלה גישות לטיפול באובדן עצם craniofacial גדול. עם זאת, ההשפעה של הרכב שתל על יחסי הגומלין בין neovascularization, התמיינות תאים והיווצרות עצם לא ברורה. אנו מציגים פרוטוקול הדמיה multimodal נועד על מנת להבהיר את התל...

Video: Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

תלת ממדית ברזולוציה אופטית photoacoustic מיקרוסקופית

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.1-5 These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.2,6-7 While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.8-9
WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.6,10-12 The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.13 Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.2,14-15
To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.2 Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.8,16 Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

הוספת בדיקות ביולוגיות Biosensors סיליקה אופטי באמצעות סוכני צימוד silane

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography1, confocal scanning laser ophthalmoscopy (cSLO)2, and optical coherence tomography (OCT)3, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.
Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts 6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases 9 such as diabetic retinopathy and neovascular age-related macular degeneration.
Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

Ophthalmoscopy photoacoustic משולב וטומוגרפיה אופטית קוהרנטיות Spectral-תחום

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

Wideband גלאי אופטיים של אולטראסאונד ליישומי הדמיה רפואית

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces -&#160;namely numerical determinants of surface roughness -&#160;are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces -&#160;but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

מכונות בדיקת שיטה משופרת להערכת Anchorage עצם השתל

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

In vivo כמותי הערכת מבנה שריר הלב, תפקוד, זלוף ואת הכדאיות שימוש טומוגרפיה לב מיקרו שחושב

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high resolutions in live animal models. This allows for repeated imaging of the same rodent over time. This feature not only reduces the total number of rodents required in an experimental design and thereby reduces the inter-subject variation that can arise, but also allows researchers to assess longitudinal or life-long responses to an intervention. To acquire high quality images that can be processed and analyzed to more accurately quantify outcomes of bone micro-architecture, users of in vivo &#181;CT scanners must properly anesthetize the rat, and position and restrain the hind limb. To do this, it is imperative that the rat be anesthetized to a level of complete relaxation, and that pedal reflexes are lost. These guidelines may be modified for each individual rat, as the rate of isoflurane metabolism can vary depending on strain and body size. Proper technique for in vivo &#181;CT image acquisition enables accurate and consistent measurement of bone micro-architecture within and across studies.

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

This paper instructs users of in vivo micro-computed tomography (&#181;CT) scanners how to anesthetize, correctly position and restrain the hind limb of a rat for minimal movement during high-resolution imaging of the tibia. The result is high quality images that can be processed to accurately quantify bone micro-architecture.

מיקום מתאים ואיפוק של איבר הינד עכברוש עבור הדמיה ברזולוציה גבוהה ממוקד של עצם באמצעות מיקרו-ארכיטקטורה In Vivo שחושב מיקרו טומוגרפיה

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

עם מודאלים מרובים נפח הרשתית הדמיה על ידי עקיפה סריקת לייזר Ophthalmoscopy (אוסלו) ו טומוגרפיה אופטית קוהרנטית (אוקטובר)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate structures&#160;while processing and histological observation provide limited information through cross-sectional imaging. Micro X-ray computed tomography (microCT) is an effective tool for obtaining three-dimensional information without damaging specimens. However, it shows limited efficiency in differentiating soft tissue parts. Use of contrast-enhancing agents, like phosphotungstic acid (PTA), can solve this problem by improving soft tissue contrast. We implemented microCT with PTA to investigate the human orbicularis retaining ligament (ORL), which is a delicate structure in the orbit area. In this method, harvested specimens are fixed in formalin, dehydrated in serial ethanol solutions, and stained with a PTA solution. After staining, microCT scanning, 3D reconstruction, and analysis are performed. Skin, ligaments, and muscles can be clearly visualized using this method. The specimen size and duration of staining are essential features of the method. The suitable specimen thickness was about 5&#8211;7 mm, above which the process was slowed, and the optimum duration was 5&#8211;7 days, below which an empty hole in the central area occasionally occurred. To maintain the location and direction of small pieces during cutting, sewing on the same region of each part is recommended. Furthermore, preliminary analyses of the anatomical structure are needed to correctly identify each piece. Parafilm can be used to prevent drying, but care should be taken to prevent specimen distortion. Our multidirectional observation showed that the ORL is composed of a multilayered meshwork of continuous plates, rather than thread-like fibers, as reported previously. These results suggest that microCT scanning with PTA is useful for examining specific compartments within complex structures of human tissue. It may be helpful in the analyses of cancer tissues, nerve tissues, and various organs, like the heart and liver.

Micro X-ray computed tomography is effective in obtaining three-dimensional information from undamaged human specimens but has limited success in observing soft tissues. The use of phosphotungstic acid contrast agent can resolve this issue. We implemented this contrast agent to examine human delicate fibromuscular tissues (the orbicularis retaining ligament).

שימוש מיקרו רנטגן טומוגרפיה ממוחשבת עם חומצה Phosphotungstic הכנה לדמיין את רקמת האדם האנושי

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

הדמיה אופטית של מודציטים מבודדים המוח החדרית

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...