מחקר זה ממומן על ידי משרד המחקר הביולוגיים הסביבה, משרד האנרגיה של ארה&quot;ב ועל ידי מענק למימון ממועצת העמים של וירג&#39;יניה לחקר בריאות. Oak Ridge National Laboratory מנוהלת על ידי UT-באטל, LLC, עבור משרד האנרגיה של ארה&quot;ב תחת חוזה מס &#39;DE-AC05-00OR22725.

1. מיכה הכנה: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חותכים את מיקה (מדעי אלקטרונים מיקרוסקופית) עם מספריים לגודל הדרוש כדי להתאים את מיקרוסקופ AFM (בערך 22 × 30 מ&quot;מ). </li><li style=";text-align:right;direction:rtl"> קליב מיקה בשני הצדדים, בדרך כלל באמצעות קלטת כדי להסיר את השכבה החיצונית, עד שרק שכבות רצוף וחלק נשארים. </li></ol> 2. הכנת פתרון ג&#39;לטין: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מוסיפים 100 מ&quot;ל מים מזוקקים כדי בקבוק מעבדה. </li><li style=";text-align:right;direction:rtl"> מחממים את הבקבוק במיקרוגל עד שהמים מתחילים לרתוח. (אם במיקרוגל אינו זמין, המים ניתן לחמם בכוס על פלטה חשמלית). </li><li style=";text-align:right;direction:rtl"> לשקול את 0.5 גרם של ג&#39;לטין (Sigma-G-6144, G-2625 או G-2500), ולהוסיף למים מזוקקים חמים. </li><li style=";text-align:right;direction:rtl"> מערבולת הבקבוק בעדינות עד הג&#39;לטין נמס לחלוטין. </li><li style=";text-align:right;direction:rtl"> מגניב הפתרון ג&#39;לטין על 60-70 ° C. </li><li style=";text-align:right;direction:rtl"> יוצקים כ 15 מ&quot;ל לתוך כוס קטנה (20 מ&quot;ל) שלתוכו מיקה לחתוך ביקע יכול להיות טבול. </li></ol> 3. מיכה ציפוי: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להטביע את הריבועים נציץ לתוך הפתרון ג&#39;לטין חימם ולסגת כזה מהר כי מיקה היא מצופה (איור 1 א). </li><li style=";text-align:right;direction:rtl"> לאחר ציפוי, לתמוך מיקה מיד, על קצה, על מגבת נייר לייבוש באוויר הסביבה (איור 1b). נציץ מצופה ג&#39;לטין יכול לשמש לפחות 2 שבועות. הפתרון ג&#39;לטין עודף יכולים להישמר בקירור בשימוש במשך חודש בערך, פשוט על ידי reheating הפתרון המניות בין 60-70 ° C. יש להקפיד על מנת להבטיח כי ג&#39;לטין אינו מבושל במהלך reheating. </li></ol> 4. הרכבה חיידקים על נציץ מצופה ג&#39;לטין: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> גלולה 1 מ&quot;ל של התרבות חיידקים (0.5-1.0 OD ב 600 ננומטר) בשעה RCF 800 עד 4500. בהתאם חיידקים, השתמש RCF המינימום גלולה יהיה התאים ~ 5 דקות. </li><li style=";text-align:right;direction:rtl"> שטפו את גלולה בהיקף זהה של מים מסוננים או deionized חיץ. </li><li style=";text-align:right;direction:rtl"> איסוף התאים שוב על ידי צנטריפוגה. </li><li style=";text-align:right;direction:rtl"> Resuspend את הכדור מיד ב 500 μL מים deionized חיץ nanopure או. (השעיה חיידקי צריך להיות עכור בעליל כדי שיהיה ריכוז מספקת של תאים עבור הדמיה AFM). </li><li style=";text-align:right;direction:rtl"> החל את ההשעיה תא (10-20 μL) כדי מיקה ג&#39;לטין מצופה ולהתפשט על פני השטח באמצעות קצה פיפטה (איור 1 ג&#39;). יש להקפיד לא לגעת פיזית את פני השטח ג&#39;לטין עם קצה פיפטה תוך יישום או להפיץ את פתרון חיידקי. </li><li style=";text-align:right;direction:rtl"> אפשר המשטח לנוח למשך 10 דקות ולשטוף עם זרם מים או חיץ (איור 1 cd). לאורך כל ההליך, דואגים להבטיח כי המדגם אינו רשאי יבש. </li></ol> 5. נציג תוצאות: תרשים 2 מציג תמונות דוגמה תאים חיידקיים כי כבר רכוב על מיקה ג&#39;לטין מצופה הדמיה על ידי AFM. במעבדות שלנו, דגימות הם צילמו בדרך כלל באמצעות מיקרוסקופ כוח אטומי PicoPlus (Agilent Technologies, בית המקדש, AZ) לבוש עם ראש מיקרומטר 100 סריקה. המכשיר מופעל על 128-512 פיקסלים לכל לסרוק קו במהירות סריקה של 0.5-1.0 קווי / s. Cantilevers המשמשים בדרך כלל הם Veeco בדיקות סיליקון ניטריד (MLCT-AUHW, Veeco, סנטה ברברה, קליפורניה) עם קבועי קפיץ הנומינלי החל 0.01-0.1 NN / ננומטר. <img src="/files/ftp_upload/2880/2880fig1.jpg" alt="איור 1"/> באיור 1. לטין בטמפרטורה של 60-70 מעלות צלזיוס ממוקם כוס 20 מ&quot;ל. בעזרת פינצטה טרי ביקע ריבועים מיקה שקועים לגמרי לתוך הג&#39;לטין (א) מכונס העמידו על מגבת נייר נשען על מדף צנטריפוגות מיקרו (ב). אגל של המדגם חיידקי מושם על מיקה ושל התפשטות עם טיפ pipet הן X ו Y כיוונים (ג). לאחר המאפשר המדגם כדי לדגור על פני השטח מיקה לפחות 10 דקות, לשטוף את המדגם בזרם של מים מזוקקים. אם immobilization עבדו תוכל לראות את המקום אטום על מיקה כפי שמוצג בלוח השמאלי של (ד). אם immobilization לא לעבוד תקבלו חתיכת ברורה של מיקה כמו בלוח השמאלי. הנקודה אטום על המאזניים מיקה ריכוז של חיידקים בתוך אגל לך מקום על מיקה התייחסו ג&#39;לטין. <img src="/files/ftp_upload/2880/2880fig2.jpg" alt="איור 2"/> 2. איור AFM תמונות של א coli. בלוח (א), התאים הם רכובים על מיקה ג&#39;לטין מצופה הדמיה ב 0.005 M-PBS. לשם השוואה, דימוי של תאים חיידקיים רכוב על מיקה ג&#39;לטין מצופה הדמיה באוויר מוצג בלוח (ב). למרות septa הם בבירור בתמונה שנאספו נוזלי, ברזולוציה גדולה יותר, כפי שצוין על ידי נוכחות של פילי (פאנל הבלעה (ב)), ניתן להשיג באוויר.

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גורמים שונים יכולים להשפיע על התא של חיידקים הרכבה הדמיה באמצעות AFM. ג&#39;לטין המשמש לציפוי מיקה חשוב. מסחרי ג&#39;לטין מבודד ממספר חוליות כולל דגים, פרות וחזירים. שניהם מקורם ואת שיטת עיבוד לקבוע התאמתו של ג&#39;לטין עבור immobilization חיידקי. מקורות רבים וסוגי ג&#39;לטין הוערכו ע...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם </td><td> חברה </td><td> מספר קטלוגי </td></tr><tr><td> לטין </td><td> סיגמא, סנט לואיס, מיזורי </td><td> G6144, G2625 או G2500 </td></tr><tr><td> PicoPlus מיקרוסקופ כוח אטומי </td><td> Agilent Technologies, בטמפה, AZ </td><td></td></tr><tr><td> AFM cantilevers </td><td> Veeco, סנטה ברברה, קליפורניה </td><td> MLCT-AUHW </td></tr></tbody></table>

bacterial immobilization for imaging by atomic force microscopy

AFM הינה ברזולוציה גבוהה (בסולם ננומטר) כלי הדמיה כי מכנית בדיקות משטח. יש לו את היכולת תאים התמונה ביומולקולות, בסביבה נוזלית, ללא צורך כימית לטפל המדגם. כדי להשיג מטרה זו, המדגם חייב דיו לדבוק פני השטח גובר על מנת למנוע הסרה של כוחות המופעלים על ידי קצה שלוחה סריקה AFM. במקרים רבים, הדמיה מוצלח תלוי immobilization של המדגם על פני השטח גובר. באופן אופטימלי, צריך להיות immobilization פולשנית כדי לדגום את כזה תהליכים מטבוליים ותכונות פונקציונליות אינם בסכנה. עד ביקע טרי ציפוי משטחים מיקה עם חזירי (חזיר) ג&#39;לטין, חיידקים בעלי מטען שלילי יכול להיות משותקת על פני השטח הדמיה בנוזל על ידי AFM. Immobilization של תאים חיידקיים על מיקה ג&#39;לטין מצופה קרוב לוודאי בשל אינטראקציה אלקטרוסטטית בין חיידקים בעלי מטען שלילי ואת הג&#39;לטין מטען חשמלי חיובי. מספר גורמים יכולים להפריע immobilization חיידקים, כולל מרכיבים כימיים של הנוזל שבו חיידקים מושעים, זמן הדגירה של החיידק על ג&#39;לטין מצופה, נציץ תכונות פני השטח של זן חיידקים המדיום שבו הם צילמו את החיידקים. באופן כללי, השימוש מיקה ג&#39;לטין מצופה יימצא החלים בדרך כלל תאים מיקרוביאליים הדמיה.

Watch this Scientific Journal Video about Bacterial Immobilization for Imaging by Atomic Force Microscopy at JoVE.com

Bacterial Immobilization for Imaging by Atomic Force Microscopy

חיים חיידקים גראם שליליים גראם חיוביים יכולים להיות משותקת על מיקה ג&#39;לטין מצופה הדמיה בנוזל באמצעות מיקרוסקופ כוח אטומי (AFM).

Immobilization חיידקית עבור הדמיה על ידי מיקרוסקופית כוח אטומי

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

bacterial-immobilization-for-imaging-by-atomic-force-microscopy

Research

JoVE Journal

Bioengineering

17.2K Views.  Oak Ridge National Laboratory. חיים חיידקים גראם שליליים גראם חיוביים יכולים להיות משותקת על מיקה ג'לטין מצופה הדמיה בנוזל באמצעות מיקרוסקופ כוח אטומי (AFM).

Video: Bacterial Immobilization for Imaging by Atomic Force Microscopy

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 ...

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. 
The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3,such as Ni2+ or Mg2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

ויזואליזציה של דנ&quot;א רקומביננטי ו קומפלקסים חלבונים באמצעות מיקרוסקופ כוח אטומי

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

כימות תכונות המכאניות של Glycocalyx האנדותל עם מיקרוסקופי כוח אטומי

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

מדידת התכונות המכאניות של תאי חיים באמצעות מיקרוסקופ כוח אטומי

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

רזולוציה גבוהה ניתוח Spatiotemporal של קולטן Dynamics ידי המולקולה בודדת מיקרוסקופ פלואורסצנטי

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson&#8217;s disease and beta-amyloid and tau in Alzheimer&#8217;s disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.

Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

רומן הכוח האטומי מיקרוסקופית מבוסס Biopanning לבידוד של חומרים כימיים מסוימים מורפולוגיה נגד גרסאות TDP-43 בטרשת לרוחב amyotrophic

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

כוח האטומי מיקרוסקופית הדמיה וחיל ספקטרוסקופיה של bilayers שומנים הנתמכים

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

אנגיוגנזה להמחיש באמצעות מיקרוסקופ Multiphoton Vivo ב מהונדסים גנטית לגרדום 3D-PLGA/nHAp Calvarial תיקון פגם קריטי של העצם

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

להמחיש אדהזיה היווצרות בתאים באמצעות מתקדם ספינינג מיקרוסקופ פלורסצנטיות השתקפות פנימית דיסק-סיכום

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

יישום של מיקרוסקופ כוח אטומי כדי לזהות אוסטיאוארתריטיס מוקדם

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...