Z Y Yang ve Wang çalışmaları NIAMS / NIH hibe K02 AR051181 Müsküler Distrofi Derneği ve Harvard Kök Hücre Enstitüsü Y Wang için hibe ile mümkün olmuştur. , Q Zeng ve X Xu iş Brigham ve Kadın Hastanesi Optik Görüntüleme Laboratuvarı verilen bir program hibe ile desteklenmektedir. Yazarlar hücre çalışmalarına teknik yardım için Anestezi Anabilim Dalı, BWH Wen Liu teşekkür etmek istiyorum.

Hücre hazırlık <ol><li> Uydu hücre izolasyonu <ol style="list-style-type:lower-alpha;"><li> Anestezi altında, GFP transgenik fareler (C57BL / Ka-βactin-EGFP) bacak kasları kaldırılır. Küçük parçalar halinde kestikten sonra, uydu hücreler 37 1 saat süreyle% 2 dispase kollajenaz çözümü ile kıyılmış doku kuluçka ° C enzimatik ayrışmış </li><li> Ham F-10 ve% 20 FBS içeren büyüme medya ile enzim sindirimi nötralize Pasteur pipeti ile tekrar triturating kas ayrıştırmaları. Hücreleri hücre süzgecinden geçirilerek süzülür (Ø70-100μm) ve ardından 37 az 1 saat kaplı olmayan bir yemek ° C kaplama </li><li> Kollajen kaplı bir çanak süpernatant ve plaka onları hafifçe kaldırın. Matür arındırmak için bu adımı daha sonra 1 saat tekrarlayın. (Ön plaka) </li><li> Zaman içinde medya ve plaka hücrelerin değiştirin. Son saflaştırılmış matür orta Ham F-10,% 20 FBS, 100 U / ml penisilin ve 37 ° C,% 5 CO2,% 95 hava az 100 mcg / ml streptomisin (besi) içeren kültür. </li></ol> </li><li> Kültür ve 10 ng / ml temel FGF ilavesi ile büyüme ortamında birincil matür genişletmek. </li><li> In vivo deneyler için, TA kas başına 5x10 5 hücre enjekte edilir. </li><li> Matür hasat,% 0.25 &#39;lik tripsin / EDTA kullanarak hücreleri ayırmak ve iki kez PBS ile yıkayın. Hücre sayıları bir hemasitometre sayılır ve hücreleri 5x10 4 hücre / ml &#39;lik bir konsantrasyon HBSS yeniden bekletildi. HBSS içinde buz üzerinde konsantre hücreleri yerleştirilir ve toplandıktan sonra 1 saat içinde enjekte edilir. </li></ol> Vivo Görüntüleme <ol><li> 4-6 haftalık yaşta Erkek mdx farelerin Jackson laboratuvarı satın alınan ve bir hayvan barınağı tesiste muhafaza edilir. </li><li> Çevre konaklama yaklaşık bir hafta sonra, farelerin kültür hücreleri ile implante olmaya hazır. </li><li> Hücre nakli öncesinde, fare karışımı Ketamin (100 mg / kg) ve Xylazine (10 mg / kg) intraperitoneal (ip) enjeksiyonu ile anestezi. Sonra TA kas alanı çevresinde saç bacak için Nair ve distile su ile silerek temizleyin uygulayarak 30 saniye bekliyor kaldırılır. </li><li> Fare anestezi altında iken, 5x10 5 hücre 10μl HBSS 30 gauge iğne kullanılarak 3 puan yavaş yavaş her TA kas içine enjekte edilir . </li><li> 4. adımı sonra, fare, in vivo floresan görüntüleme tarafından düzenli olarak görüntülü olabilir . Yüksek duyarlı şarj çiftli kamera ile donatılmış floresan görüntüleme istasyonu Nightowl LB981 (Berthold Technologies, Inc.), Farelerin engel bacak görüntüler elde etmek için kullanılır. </li><li> In vivo görüntüleme gününde, ilk olarak saç regrowth TA kas kontrol edin. Gerekirse, biz fareyi Ketamin ve Xylazine karışımı ile anestezi sonrası, yukarıda açıklandığı gibi saç kaldırmak için Nair yeniden uygulayın. </li><li> Fare anestezi altında iken, fare siyah floresan olmayan kağıt (Strathmore siyah Artagain kağıt) bir parçası üzerinde yüzükoyun pozisyonda yerleştirin. TA kas daha iyi görüntüleme için tam göstermek için, kağıt arka pençeleri bantlayın. </li><li> Biz görüntüleme odası ve konumu, kameranın görüş alanının merkezinde görüntülü arka bacak fare yer. Biz ilk 5 saniyelik bir pozlama süresi ile bacak geleneksel bir gri-ölçekli fotoğraf görüntü almak için emisyon filtresi anahtarı. Fare ve gerekirse kameranın odak konumunu yeniden ayarlayınız. Pozlama süresi (1250 ms), örnek yüksekliği (0.9 cm) vb de dahil olmak üzere görüntüleme parametreleri deneysel süreç içinde sabit tutulur. </li><li> Daha sonra yeşil flüoresan protein dalga boyu için uygun emisyon filtresi anahtarı. Biz 1.250 ms ve 1 binning maruz kalma süresi ile floresan görüntü alır. Görüntüleme sonra, fare toparlanma beklemek için bir kafes görüntüleme odasından çıkarılır. </li><li> Analizi için, saniyede fotonların açısından yoğunluğunu ölçmek için floresan sinyalleri üzerinde bir ilgi bölgesi (ROI) çizin. Biz de başka bir ölçüm değeri olarak piksel sayısı açısından yatırım getirisini ölçmek. Görüntüleme deneyler, boylamsal veri elde etmek için, gerekirse bir kaç ay içinde her hafta daha sık tekrarlanan ya da olabilir. </li></ol> Deney sonunda, fare ötenazi ve TA kas histolojisi analizi için hasat edilir.

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+fluorescence+optical+imaging+and+tomography.+Dis+Markers.">Near-infrared fluorescence optical imaging and tomography. Dis Markers.</a> , 107-121 .</li><li>Lee, J., Sevick-Muraca, E. 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Biz bu çalışmada ana iskelet kası floresan etiketli nakledilen hücrelerin izlemek için güvenilir ve istikrarlı bir görüntüleme platformu kurdu. GFP transgenik fare GFP etiketli matür yetişkin kök hücreler gelecekte hücre tedavisi için aday olacak bir tür için duruyor. Hücre sayısı, hücre enjeksiyonu, açık arka plan, görüntüleme konumunu ve makine parametresi de dahil olmak üzere sürekli bir manipülasyon, yüksek kalitede görüntü elde ve özellikle kantitatif analiz için önemlidir. Floresans görüntüleme non-invaziv, kolay olduğu gibi biyomedikal araştırmalarda birçok uygulamalar vardır ve yüksek kapasiteli. Buna ek olarak floresan görüntüleme, saçılma, zayıflama ve emilimi sayesinde de olsa, foton sayımı, dayanan kantitatif bir ölçüm sağlayabilir, ışık, doku büyük bir mesafe, bir teknik eksiklik nüfuz edemez. Kırmızı ya da yakın-kızılötesi ışık penetrasyon derinliğini artırmak için muhabir kullanmak için çalışmalar devam etmekte. Floresan görüntüleme diğer bir avantajı, insan kullanımı için özel floresan muhabiri onaylanmıştır eğer klinikler çevrilebilir. Pahalı donanım ve görüntüleme ajanları gerektirmez, MR, CT, ve PET ile karşılaştırıldığında, floresan görüntüleme yüksek esnekliğe sahiptir. Bir örnek kliniklerde kullanılan floresan tabanlı endoskop ve mikro-endoskop. Floresan görüntüleme yöntemleri ile birleştirerek, biz altta yatan biyolojik süreçler ile ilgili anatomik ve fonksiyonel bilgi elde etme yeteneği elde etmek için bekliyoruz.

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<td>NightOwl LB981</td>
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tracking dynamics of muscle engraftment in small animals by in vivo fluorescent imaging

Musküler distrofiler kontraktil kas hücrelerinin progresif kaybı ile karakterize dejeneratif kas hastalıklarının bir grup. Şu anda herhangi bir iyileştirici bir tedavi yoktur. Kök hücre biyolojisi son gelişmeler bu hastalıkları tedavi etmek için etkili bir hücre bazlı tedavilerin geliştirilmesi için yeni umutlar yarattı. Distrofik hayvan modelleri kas içine floresan proteinleri ile etiketlenmiş kök hücrelerin çeşitli türleri Nakli alanında geniş anlamda kullanılmıştır. Nakledilen hücre kaderi takip etmek yeteneğine sahip bir non-invaziv bir tekniktir boyuna, daha doğru ve verimli bir şekilde nakledilen hücrelerin kas engraftman değerlendirmek için yetenek daha. Bu çalışmada, in vivo floresan görüntüleme nakledilen GFP (Yeşil Floresan Protein) etiketli fare iskelet kaslarında hücre izlemek için duyarlı ve güvenilir bir yöntem olduğunu göstermektedir. Floresan protein uyarma için gerekli harici bir ışık kullanımı nedeniyle geçmişi hakkında endişe rağmen, çıplak ya da fareyi kullanarak ya da epilasyon reaktifler saç ortadan kaldırarak çok bu sorunun ortadan olduğu bulundu. CCD kamera kullanarak, floresan sinyal GFP transgenik fareler veya eGFP transduced myoblast kültür ya da 5 x 10 5 hücre enjeksiyonu sonra tibialis anterior kas (TA) tespit edilebilir. Ekstansör digitorum longus (EDL), daha az hücre enjeksiyonu gibi daha yüzeysel kasları için saptanabilir bir sinyal üretir. Sinyal yoğunluğu ölçülür ve ilgi (ROI) bir bölgede saniyede fotonların sayısı olarak sayılabilir olabilir. Edinilen görüntüleri engrafted alan net sınırlarının çizilmesi, ROI boyutu olarak ölçülebilir. Bacaklarda sürekli her zaman konumda iseniz, kas ve ROI boyutu başına saniyede fotonların toplam sayısı değişiklikler engrafted hücrelerinin sayısında değişiklikler ve engrafted alanının boyutunu yansıtmaktadır. Bu nedenle zaman içinde aynı kas değişiklikleri ölçülebilir. In vivo floresan görüntüleme tekniği tumorogenic hücrelerinin büyümesini izlemek için öncelikle kullanılan olmuştur, bizim çalışmamızda, nakledilen kök hücrelerin kaderi takip etmek için bize sağlayan güçlü bir araç olduğunu göstermektedir.

Watch this Scientific Journal Video about Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging at JoVE.com

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Biz bir tarif<em&gt; In vivo</em&gt; Floresan görüntüleme protokolü, hem sağlıklı ve distrofik farelerin iskelet kas içine transplantasyonu sonrası GFP-etiketli matür kas yenilenme izlemek. Bu protokol, diğer tip hücre nakli ve diğer kaslı koşulların yanı sıra kas yenilenme çalışma adapte edilebilir.

Küçük Hayvan Kas Aşı dinamiği ile Takip In Vivo Floresan Görüntüleme

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

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Research

JoVE Journal

Biology

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

9.7K Görüntüleme.  Brigham and Woman's Hospital. Biz bir tarif In vivo Floresan görüntüleme protokolü, hem sağlıklı ve distrofik farelerin iskelet kas içine transplantasyonu sonrası GFP-etiketli matür kas yenilenme izlemek. Bu protokol, diğer tip hücre nakli ve diğer kaslı koşulların yanı sıra kas yenilenme çalışma adapte edilebilir.

Video: Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

İskelet kas-içi hayati Mikroskopi in vivo Mikro sirkülasyon Ölçümü

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Biyoloji

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Demir Oksit hESC ve hMSCs Etiketleme MR Görüntüleme in vivo Takip Non-Invasive Nanopartiküller

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

Floresan üzerinden F-aktin Dynamics Canlı Hücre Görüntüleme speckle Mikroskopi (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

İzole İskelet Kaslar Kasılma, çabuk yorulma ve Alternans Ex Vivo Değerlendirilmesi

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

Eşodaklı ve multiphoton Mikroskopi kullanarak beş Floresan Proteinleri ile işaretlenmiş Hematopoetik kök hücrelerin in vivo klonal Takip

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Yüksek çözünürlüklü Time-lapse Görüntüleme ve Yaşayan İnsan Umbilikal Ven Endotelyal Hücrelerinde Mikrotubul Dinamiği Otomatik Analizi

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult mice for in vivo longitudinal real-time monitoring of thymocytes&#180; dynamics within a vascularized thymus segment. Following the transplantation, laser scanning microscopy (LSM) through the cornea allows in vivo noninvasive repeated imaging at cellular resolution level. Importantly, the approach adds to previous intravital T-cell maturation imaging models the possibility for continuous progenitor cell recruitment and mature T-cell egress recordings in the same animal. Additional advantages of the system are the transparency of the grafted area, permitting macroscopic rapid monitoring of the implanted tissue, and the accessibility to the implant allowing for localized in addition to systemic treatments. The main limitation being the volume of the tissue that fits in the reduced space of the eye chamber which demands for lobe trimming. Organ integrity is maximized by dissecting thymus lobes in patterns previously shown to be functional for mature T-cell production. The technique is potentially suited to interrogate a milieu of medically relevant questions related to thymus function that include autoimmunity, immunodeficiency and central tolerance; processes which remain mechanistically poorly defined. The fine dissection of mechanisms guiding thymocyte migration, differentiation and selection should lead to novel therapeutic strategies targeting developing T cells.

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress.

Gerçek zamanlı timositleri mikroskobu tarama lazer göz ön odasında Vivo içinde Takibi

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Floresans canlı hücre mikroskobu ile mitotik ve meiotic Fission Maya nükleer dinamiklerinin incelenmesi

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

Toplam İç Yansıma Floresan Mikroskobu Ile Canlı Hücrelerdeki Hücre Yüzey Reseptörlerinin Oligomerizasyon Dinamiği Sayı ve Parlaklık Analizi ile Birlikte

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...