وأيد هذا البحث عن طريق منحة من المعاهد الوطنية للصحة: ​​R21AG042066. ونود أن نشكر فيليب شولتز لمساهماته في خلق أشرطة الفيديو القبض على الشاشة.

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The authors have nothing to disclose.

Protein variants have been shown to be involved in the progression of many neurodegenerative diseases such as Alzheimer&#8217;s, Parkinson&#8217;s, ALS and FTD1,2,3,4,5,6,7,8,9,10,11. Isolation of antibodies that can selectively recognize these different protein variant targets can be effective reagents to study, diagnose and potentially treat such ailments. To generate such variant specific antibodies we have developed a novel biopanning process that utilizes atomic force microscopy to monitor the progress ...

وجود بدائل البروتين وقد تورط كعامل في تطور العديد من الأمراض بما فيها الأمراض العصبية مثل الزهايمر والشلل الرعاش، ALS والجبهي الصدغي الخرف (FTD) 1،2،3،4،5،6،7،8،9 ، 10،11. ويعتقد أشكال بلازميدة قليلة القسيمات من البروتينات بيتا اميلويد وألفا synuclein أن تكون من الأنواع السامة المسؤولة عن مرض الزهايمر والشلل الرعاش، على التوالي 2،3،4،5. وقد تم ربط المجاميع من البروتين 43 (TDP-43) TAR DNA ملزم لALS وFTD 12،13،14. لذلك الكواشف مثل الأجسام المضادة التي يمكن أن تستهدف بشكل انتقائي يمكن للمتغيرات بروتين مختلفة أن تكون أدوات قوية لخدمة علامات تشخيصية وعلاجية محتملة. في هذه الدراسة، وركزنا على تطوير الكواشف التي تربط بشكل انتقائي أنواع من البروتين TDP-43 المتورطين في ALS، ولكن التقنية الواردة في هذه الورقة يجب أن تكون قابلة للتطبيق إلى عزل الكواشف ضد طائفة واسعة من البروتوكول الاضافيعين المتغيرات. وقد تم تحديد تجميع حشوية من TDP-43 كسمة المرضية في ALS 15،16،17،18،19. وعادة ما يتم العثور TDP-43 في نواة كل الخلايا من فرد العادي، على الرغم من أنه يميل للتنقل بين العصارة الخلوية ونواة 15،17. تم الكشف عن أشكال ومع ذلك، في ALS تجميعها من TDP-43 في سيتوبلازم الخلايا العصبية والدبقية مختارة مع تركيز أقل وجدت في النواة مما يدل على أن حركة TDP-43 من النواة إلى السيتوبلازم أثناء تطور المرض 16،20. في حين تم العثور على تجميع TDP-43 في معظم الحالات ALS، فإنه لا حساب لجميع الحالات منذ 1٪ -2٪ من إجمالي الحالات ALS (أو 15٪ -20٪ من الحالات الأسرية ALS) ترتبط طفرات في ديسموتاز الفائق 1 (SOD1) 15،17 الجين. نظرا للدور الهام للTDP-43 في الغالبية العظمى من الحالات ALS، وهنا علينا أن نركز على تطوير الكواشف الأجسام المضادة مقرها التي يمكن أن تربط بشكل انتقائي لTDP-43 المتغيرات التي هيموجودة في الإنسان ALS أنسجة المخ باستخدام لدينا رواية AFM تقنيات biopanning مقرها. في البداية نحن بحاجة إلى ذخيرة متنوعة من الأجسام المضادة ملزمة المجالات. نحن الجمع بين ثلاثة نماذج مختلفة من عرض فج سلسلة واحدة نطاق متغير الأضداد شظية (scFvs) مكتبات (توملينسون الأول وJ والمكتبات صفائح 21). وتنقسم عملية بالغسل إلى مراحل بالغسل السلبية والإيجابية. وتعرض فاجات من المكتبات أولا إلى عملية بالغسل السلبية خلالها فاجات رد الفعل ليتم استبعاد متعددة المستضدات بعيدا عن الهدف. بعد الانتهاء من كل جولة من بالغسل السلبي ضد كل مستضد بعيدا عن الهدف، ويتم رصد هذه العملية عن طريق AFM التصوير لضمان أن كل فج ملزمة المستضدات بعيدا عن الهدف قد أزيلت. إلا بعد التحقق من AFM التصوير التي تتم إزالة كافة فاجات رد الفعل لم نشرع في الهدف التالي. لعزل الكواشف ضد TDP-43 المتغيرات المتورطين في ALS نحن الاستفادة من المستضدات بالغسل السلبية التالية:<html> 1) BSA لإزالة فج التي تربط ضعيفة أو غير خصيصا لبروتينات. 2) مجمعة ألفا synuclein لإزالة الفيروس التي ربط العناصر الهيكلية العامة للبروتينات المجمعة. 3) الخليط أنسجة المخ البشري لإزالة الفيروس التي ربط أي البروتينات أو المكونات الأخرى الموجودة في عينات التشريح من صحة أنسجة المخ البشري؛ 4) immunoprecipitated TDP-43 من الدماغ البشري صحي لإزالة فج التي تربط لجميع TDP-43 الأشكال المرتبطة الدماغ البشري صحي. و5) immunoprecipitated TDP-43 معزولة عن الخليط الدماغ FTD لإزالة فج التي تربط TDP-43 المتغيرات المرتبطة غير ALS-علم الأمراض. بعد إزالة كل فج رد الفعل لجميع المستضدات بعيدا عن الهدف، ثم انتقلنا بعد ذلك إلى المرحلة بالغسل إيجابية خلالها شظايا الأجسام المضادة التي تربط مستضد من الفائدة معزولة، في هذه الحالة TDP-43 immunoprecipitated من أنسجة المخ ALS الإنسان. قد تكون هذه الأجسام المضادة معزولة رد الفعل إلى أشكال تجميعها أو معدلة من TDP-43. &quot;&gt; التقليدي فج biopanning يركز أساسا على المرحلة بالغسل إيجابية 22،23. وأضافت مكتبة فج وعادة ما يتم يجمد الهدف من الفائدة ومزال فاجات منضم. وتتضخم وفاجات ثم وإضافتها إلى الهدف مرة أخرى. هذه عملية التضخيم وحضانة وعادة ما تتكرر عدة مرات لزيادة نسبة فج ملزم إيجابي. وفي حين أن الاختلافات من هذه العملية وقد استخدمت على نطاق واسع لعزل الكواشف الأجسام المضادة ضد طائفة واسعة من المستضدات الهدف، فإنها تتطلب عادة كميات كبيرة من النقى الهدف مستضد 24،25،26، 27، في حين أن عمليتنا يتطلب تتبع سوى كميات من المستضد الهدف. بروتوكول الموصوفة هنا يمكن أن تستخدم لعزل الكواشف أن المستضدات الهدف على ربط بشكل انتقائي التي تكون موجودة بتركيزات منخفضة جدا، دون الحاجة لتنقية وبالغسل يمكن القيام بها مباشرة ضد مستضد موجودة في عينات الأنسجة المعقدة. إن استخدام بروتوكولات بالغسل السلبية شاملة كما تم التحقق منهابواسطة AFM يضمن أن الحيوانات المستنسخة معزولة ضد مستضد إيجابي يجب ربط بشكل انتقائي الهدف حتى عندما لا تنقيته أو المخصب. ونفذت كاستوريرانجان وزملاؤه (2003) من السلبية والإيجابية لعملية biopanning مماثلة لعزل الأجسام المضادة تفاعلية للبلازميدة قليلة القسيمات بيتا اميلويد باستخدام تركيز نانوغرام من الهدف 5. نحن هنا التوسع في هذه العملية لتمكين جيل من الكواشف التي تربط بشكل انتقائي البروتين بأمراض محددة المتغيرات مباشرة من عينات الأنسجة البشرية. في الدراسات المستقبلية نعتزم مواصلة التحقيق ليس فقط القيمة التشخيصية الكواشف معزولة هنا ولكن أيضا تقييم أهميتها العلاجية لعلاج ALS. وعموما، يجب أن يكون لدينا رواية AFM التكنولوجيا biopanning مقرها ينطبق على عزل أي مرض معين البروتين البديل في أي مادة بيولوجية دون الحاجة لتنقية البروتين أو التعديل، حتى عندما يكون الهدف مستضد concentratioنانوثانية منخفضة للغاية.

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novel atomic force microscopy based biopanning for isolation of morphology specific reagents against tdp-43 variants in amyotrophic lateral sclerosis

بسبب المتغيرات البروتين تلعب أدوارا حاسمة في كثير من الأمراض بما في ذلك TDP-43 في الضموري العضلي الجانبي التصلب (ALS)، ألفا synuclein في باركنسون مرض وبيتا اميلويد وتاو في مرض الزهايمر، من المهم للغاية لتطوير الكواشف التشكل المحددة التي يمكن أن تستهدف بشكل انتقائي هذه البروتينات بأمراض محددة المتغيرات لدراسة دور هذه المتغيرات في علم الأمراض والتطبيقات التشخيصية والعلاجية المحتملة. لقد قمنا بتطوير مجهر القوة الذرية (AFM) تقنيات biopanning رواية تقوم العزلة التي تمكن من الكواشف التي تعترف بشكل انتقائي المتغيرات البروتين بأمراض محددة. هناك مرحلتين الرئيسية المشاركة في هذه العملية، ومراحل بالغسل السلبية والإيجابية. خلال المرحلة بالغسل السلبية، يتم استبعاد فاجات التي هي رد الفعل لمستضدات خارج الهدف من خلال جولات متعددة من بالغسل مطروح باستخدام سلسلة من تم اختيارهم بعناية بعيدا عن الهدف المستضدات. ومن السمات الرئيسية في السلبيةالمرحلة بالغسل هو الاستفادة التصوير AFM لمراقبة العملية والتأكد من أن تتم إزالة جميع الجزيئات فج غير مرغوب فيها. وبالنسبة للمرحلة بالغسل إيجابية، يتم إصلاح المستضد الهدف من الفائدة على سطح الميكا ومزال فاجات ملزمة وفحص لتحديد فاجات التي تربط بشكل انتقائي المستضد الهدف. لا يحتاج البديل البروتين الهدف المراد تنقيتها توفير استخدمت الضوابط بالغسل السلبية المناسبة. حتى استهداف المتغيرات البروتين التي تكون موجودة فقط في تركيزات منخفضة جدا في المواد البيولوجية المعقدة يمكن استخدامها في الخطوة بالغسل إيجابية. من خلال تطبيق هذه التكنولوجيا، وحصلنا على أجسام مضادة لبروتين من المتغيرات TDP-43 التي تم العثور عليها بشكل انتقائي في أنسجة المخ ALS الإنسان. نحن نتوقع أن هذا البروتوكول ينبغي أن تكون قابلة للتطبيق على الكواشف توليد التي تربط بشكل انتقائي المتغيرات البروتين الموجودة في مجموعة واسعة من العمليات والأمراض البيولوجية المختلفة.

في الشكل 1، يوضح التخطيطي عملية بالغسل السلبية التي أزلنا فج ملزم بعيدا عن الهدف مستضدات من مكتبتنا باستخدام immunotubes. بدأنا في البداية مع BSA لأن هذا هو عامل مشترك وحجب أي فج التي سترد nonspecifically مع هذا الهدف سيكون إشكالية في المناعية في المستقبل. المقبل، ونحن إزا?...

Watch this Scientific Journal Video about Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis at JoVE.

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

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 ...

novel-atomic-force-microscopy-based-biopanning-for-isolation

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JoVE Journal

Bioengineering

8.7K Views.  Arizona State University. 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.

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

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 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.

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 ...

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, ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

طريقة مبتكرة لإضفاء الطابع المحلي على مراسل نيون الخرز بالقرب من سطح الثقافة الخليوي لقوة الجر المجهري

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

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 ...

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved oxygen concentration). Nevertheless, the morphology of cells can be a suitable indicator for optimal conditions, since it changes with dependence on the growth state, product accumulation and cell stress. Furthermore, the single-cell size distribution provides not only information about the cultivation conditions, but also about the population heterogeneity. To gain such information, a photo-optical in situ microscopy device1 was developed to enable the monitoring of the single-cell size distribution directly in the cell suspension in bioreactors. An automated image analysis is coupled to the microscopy based on a neural network model, which is trained with user-annotated images. Several parameters, which are gained from the captures of the microscope, are correlated to process relevant features of the cells, like their metabolic activity. Until now, the presented in situ microscopy probe series was applied to measure the pellet size in filamentous fungi suspensions. It was used to distinguish the single-cell size in microalgae cultivation and relate it to lipid accumulation. The shape of cellular particles was related to budding in yeast cultures. The microscopy analysis can be generally split into three steps: (i) image acquisition, (ii) particle identification, and (iii) data analysis, respectively. All steps have to be adapted to the organism, and therefore specific annotated information is required in order to achieve reliable results. The ability to monitor changes in cell morphology directly in line or on line (in a by-pass) enables real-time values for monitoring and control, in process development as well as in production scale. If the off line data correlates with the real-time data, the current tedious off line measurements with unknown influences on the cell size become needless.

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

A photo-optical in situ microscopy device was developed to monitor the size of single cells directly in the cell suspension. The real-time measurement is conducted by coupling the photo-optical sterilizable probe to an automated image analysis. Morphological changes appear with dependence on the growth state and cultivation conditions.

في الموقع المجهر لتحديد في الوقت الحقيقي من المورفولوجية خليه واحده في العمليات الحيوية

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved ...

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 ...

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

This protocol aims to automate AFM measurements on hundreds of microbial cells. First, microbes are immobilized into PDMS stamp microstructures and then force spectroscopy measurements are performed automatically on hundreds of immobilized cells.

أتمتة قياسات المجهر القوة الحيوية الذرية على مئات من خلايا ألبيكانس C.

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record ...

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 ...