وقد تم تمويل هذا العمل من قبل المعاهد الوطنية للصحة GM086822 المنحة. فإن الكتاب أود أن أشكر الدكاترة. أليك Pakhomov والاس بول (جامعة تقنية النانو واشنطن مرفق المستخدم (NTUF)) واندريا سليد (بروكر AXS) لما قدموه من مساعدة تقنية من الخبراء. وقد أجريت DNA فؤاد التصوير في جامعة. واشنطن NTUF ، وهو عضو في شبكة تقنية النانو البنية التحتية الوطنية. ويعود الفضل في البلازميد 2xGal4 - 2xGRE - luciferease من قبل المختبر الدكتور كيث ياماموتو (جامعة كاليفورنيا ، سان فرانسيسكو).

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly,+tuning+and+use+of+an+apertureless+near+field+infrared+microscope+for+protein+imaging.">Assembly, tuning and use of an apertureless near field infrared microscope for protein imaging.</a> J Vis Exp. , (2009).</li><li>Cretu, A., Castagnino, P., Assoian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+the+effects+of+matrix+stiffness+on+cellular+function+using+acrylamide-based+hydrogels.">Studying the effects of matrix stiffness on cellular function using acrylamide-based hydrogels.</a> J Vis Exp. , (2010).</li><li>Brunger, A. 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الإعلان عن أي تضارب في المصالح.

فؤاد يوفر تقنية فريدة من نوعها مجهرية قادرة على الجزيئات الحيوية الفردية غير المصقول في التصوير المائي وقرب حلول فيزيولوجي مخزنة في الوقت الحقيقي. وهذا يسمح للتصور من البروتينات الفردية وجزيئات الحمض النووي ، فضلا عن المجمعات والمجمعات multiprotein بالبروتين الحمض النووي. يمكن أن جزيئات الجزيئات التصوير مع فؤاد مفيدا لتقييم تجانس العينة وتحديد الترتيب الفعلي للعناصر المكونة للجسيمات ملاحظتها. ويمكن ملاحظة هذا النهج من جزيئات الجزيئات الفردية أن تكون إضافة مفيدة لتقنيات الكيمياء الحيوية التقليدية ، مثل فحوصات مناعي ، بولي أكريلاميد الكهربائي للهلام ، واللوني حجم العمود الاستبعاد ، والتي توفر بيانات تلخيصي كبيرة تمثل 10 3 11 -10 مجمعات الجزيئية البيولوجية لمصلحة فرد موجود في العينة. يمكن لجميع البحوث في رياضيات الكيمياء متعدد البروتينات ، والتفاعلات الجزيئية البيولوجية ، ومتطلبات العامل المساعد يتم التحقيق باستخدام فؤاد. ويمكن تكييف الطريقة المعروضة هنا لاستيعاب الأسئلة البيوفيزيائية محددة من الفائدة. على سبيل المثال ، وذلك باستخدام مسبار خلية السائل حامل قادرة على تبادل العازلة ، فمن الممكن لمتطلبات الفحص العامل المساعد لتكوين البروتين المعقدة. يمكن تشكيل الحمض النووي والبروتينات وفصلها في التجمعات القريبة من الوقت الحقيقي ، وحتى في الوقت الذي يجري تصويره. يمكن توليد الحمض النووي مع تسلسل محدد من الفائدة (على سبيل المثال عناصر الاستجابة الظني ، أو رواية تسلسل البروتين ملزمة) ، مختلطة مع بروتين الربط الافتراض ، وتصوير لتقديم أدلة مباشرة على التفاعلات بين الجزيئات. التنصت على وضع التصوير فؤاد هو الى حد كبير أقل تعقيدا إذا تستخدم العينات الجافة بدلا من العينات في المحاليل المائية ، وتقف على الحمض النووي DNA الخطية والبلازميدات دائرة مغلقة على حد سواء تم تصويره في هذا السبيل 3،9. الأسلوب المقدمة هنا تستخدم عينات مخزنة في الحلول من أجل توفير بيئة التصوير أكثر الفسيولوجية. وينبغي أيضا أساليب أخرى جديرة بالذكر من البروتينات التصوير النظر فيها ، بما في ذلك مجال الأشعة تحت الحمراء القريبة من المجهري 10. الاستخدام المتكامل للامتزاز مناعي في القرين مع فؤاد يوفر فرصة لإعداد مجموعة كبيرة من المجمعات البروتين ، وذلك باستخدام تقنيات راسخة البيوكيميائية ، وتصور لهم ثم بعد الإفراج عن بيليه الأجسام المضادة حبة immunoadsorbing. على سبيل المثال ، كان يعاير GR immunoadsorbed لارتباطه مع البروتين hsp70 كوصي الجزيئية (1) ، وكذلك البروتين السيارات داينين ​​2 باستخدام هذا النهج من أجل تقدير حجم التعقيد وstoichiometries. حجم الجسيمات وخصائص الفيزيائية الحيوية (مثل الصلابة) كانت مفيدة في التحقق من هوية الجزيئات الحيوية التي لوحظت في عينات من تصوير فؤاد 11،12. ومن الممكن أيضا لتأكيد الهوية جزيء حيوي عن طريق إضافة لجزيء حيوي التفاعل (مثل ليجند أو ريتوكسيماب) ، التي من شأنها ربط هدفها وتسبب زيادة حجم الجسيمات إذا كان الهدف هو جزيء حيوي الحالي.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> اسم كاشف </th><th> شركة </th><th> فهرس العدد </th></tr><tr><td> البعد 3100 </td><td> بروكر </td><td> البعد 3100 </td></tr><tr><td> البعد السوائل الخليوي </td><td> بروكر </td><td> DTFML - DD - سعادة </td></tr><tr><td> رافعة حادة نيتريد (SNL) السيليكون نيتريد فؤاد التحقيق </td><td> بروكر </td><td> SNL - 10 </td></tr><tr><td> Microlever حادة رافعة نيتريد (MSNL) السيليكون نيتريد فؤاد التحقيق </td><td> بروكر </td><td> MSNL - 10 </td></tr><tr><td> NanoScope البرمجيات فؤاد الصك </td><td> بروكر </td><td> 004-132-000 </td></tr><tr><td> أقراص معدنية فؤاد العينة </td><td> تيد بيلا </td><td> 16208 </td></tr><tr><td> الصف V1 أقراص ميكا ، 12 مم </td><td> تيد بيلا </td><td> 50-12 </td></tr></tbody></table>

visualization of recombinant dna and protein complexes using atomic force microscopy

قوة ذرية المجهر (فؤاد) يسمح للتصور الفردي للبروتينات ، جزيئات الحمض النووي ، ومجمعات البروتين البروتين ، والمجمعات الحمض النووي والبروتينات. في نهاية ناتئ المجهر هو تحقيق نانو النطاق الذي يخترق المناطق صورة تتراوح بين نانومتر وميكرومتر ، وقياس الارتفاع من الجزيئات يستريح على سطح الركيزة في أي لحظة معينة. القوات الكهروستاتيكية تسبب البروتينات والدهون والأحماض النووية لإرفاق فضفاضة إلى الركيزة في التوجهات العشوائية والسماح التصوير. البيانات التي تم إنشاؤها تشبه الخريطة الطوبوغرافية ، حيث حل والجزيئات ثلاثية الأبعاد من أحجام الجسيمات منفصلة (الشكل 1) 1،2. التنصت وضع فؤاد ينطوي على التذبذب المتكرر للتعزية ، والذي يسمح التصوير من المواد الحيوية لينة نسبيا مثل الحمض النووي والبروتينات. واحدة من فوائد ملحوظة من فؤاد على غيرها من تقنيات النانو المجهري هو قدرتها على التكيف النسبي لتصور البروتينات الفردية والجزيئات في مجمعات المخازن المائية ، بما في ذلك شبه فيزيولوجي ظروف تخزينها مؤقتا ، في الوقت الحقيقي ، ودون تلوين أو طلاء العينة ليتم طباعتها. الطريقة المعروضة هنا يصف التصوير من الحمض النووي وعامل النسخ immunoadsorbed (أي مستقبلات جلايكورتيكود ، GR) مخزنة في محلول (الشكل 2). ويمكن فصل البروتينات والمجمعات Immunoadsorbed البروتين من بيليه الأجسام المضادة immunoadsorbing حبة من روح التنافس مع الأجسام المضادة وحاتمة ثم التقط (الشكل 2A). وهذا يسمح للتلاعب البيوكيميائية من الجزيئات الحيوية التي تهم قبل التصوير. المنقى مرة واحدة ، يمكن أن تكون مختلطة الحمض النووي والبروتينات ويمكن تصوير مجمع التفاعل الناتجة كذلك. الربط من الحمض النووي لالميكا يتطلب الموجبة ثنائي التكافؤ 3 ، مثل Ni 2 + 2 + أو Mg ، والتي يمكن أن تضاف إلى مخازن العينة بعد الحفاظ على نشاط البروتين. باستخدام نهج مماثل ، وقد استخدمت لتصوير فؤاد الانزيمات الفردية ، بما في ذلك بوليميريز الحمض النووي الريبي (4) وانزيم تصليح 5 ، منضما إلى جدائل الحمض النووي الفردية. هذه التجارب تعطي تفسيرا واضحا في البروتين البروتين والحمض النووي والبروتينات البيوفيزيائية التفاعلات التي تحدث على المستوى الجزيئي. يمكن للجسيمات التصوير الجزيئات الفردية مع فؤاد مفيدا لتحديد الجسيمات التجانس ولتحديد الترتيب الفعلي للعناصر المكونة للجسيمات تصوير. بينما تم تطوير الطريقة الحالية لرؤية خيوط GR - البروتين والحمض النووي كوصي 1،2 المجمعات التي يمكن ربط الموارد الوراثية ، ويمكن تطبيقه على نطاق واسع لعينات الحمض النووي والتصوير والبروتين من مصادر متنوعة.

Watch this Scientific Journal Video about Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy at JoVE.com

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

ووصف التنصت وضع مجهر القوة الذرية (AFM) طريقة لتصور DNA البلازميد ، بروتينات حشوية ، والمجمعات الحمض النووي والبروتينات. الأسلوب يشمل النهج البديل لإعداد العينات للتصوير فؤاد التالية التلاعب البيوكيميائية. ويلاحظ والتي تحتوي على الحمض النووي محددة التفاعل البروتين المناطق العازلة في ظروف شبه فيزيولوجي.

التصور من الحمض النووي المؤتلف والمجمعات البروتين باستخدام الميكروسكوب القوة الذرية

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

visualization-recombinant-dna-protein-complexes-using-atomic-force

Research

JoVE Journal

Bioengineering

22.3K Views.  Seattle University. ووصف التنصت وضع مجهر القوة الذرية (AFM) طريقة لتصور DNA البلازميد ، بروتينات حشوية ، والمجمعات الحمض النووي والبروتينات. الأسلوب يشمل النهج البديل لإعداد العينات للتصوير فؤاد التالية التلاعب البيوكيميائية. ويلاحظ والتي تحتوي على الحمض النووي محددة التفاعل البروت...

Video: Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Bacterial Immobilization for Imaging by Atomic Force Microscopy

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 environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

الشلل البكتيرية للتصوير بواسطة الفحص المجهري القوة الذرية

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 ...

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

تصور منظمة اللحاء وحيوية في الكائنات الدقيقة، وذلك باستخدام المجموع الإسفار التأمل الداخلي المجهر

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

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.

قياس الخواص الميكانيكية للالكنان السكري غشائي مع مجهر القوة الذرية

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).

قياس الخواص الميكانيكية للخلايا الحية باستخدام المجهر القوة الذرية

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

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.

Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces.

سطح القياس المحتمل للبكتيريا عن طريق كلفن التحقيق قوة المجهر

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. ...

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.

رواية القوة الذرية المجهر Biopanning استنادا لعزل مورفولوجيا الكواشف محددة ضد المتغيرات TDP-43 في الضموري العضلي الجانبي التصلب

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.

قوة الذرية المجهر التصوير وقوة التحليل الطيفي لدعم الدهن طبقات ثنائية

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

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

التصور جيرمينوسوميس و "الغشاء الداخلي" في الجراثيم العصوية نجحت

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

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).

تطبيق المجهر القوة الذرية للكشف عن هشاشة العظام في وقت مبكر

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

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

قرب متزامنة ليزر المسح Confocal والقوة الذرية المجهرية (Conpokal) على الخلايا الحية

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...