Z 양 및 Y 왕의 작품은 NIAMS / NIH에서 교부금 K02 AR051181, 근육질 영양 장애 협회와 Y 왕에 대한 하버드 줄기 세포 연구소에서 부여에 의해 가능하게되었다. Q Zeng 및 X 쑤의 작품은 브리검 앤드 위민스 병원에서 광학 이미징 연구실에게 수여 프로그램 부여에 의해 지원됩니다. 저자는 셀 작업에 기술적인 도움을 마취학과, BWH에서 웬 리우 감사하고 싶습니다.

셀 준비 <ol><li> 위성 세포 분리 <ol style="list-style-type:lower-alpha;"><li> 마취에서 GFP 형질 전환 마우스 (C57BL / 카 - βactin - EGFP)의 사지 근육이 제거됩니다. 작은 조각으로 절단 후, 위성 세포 37 1 시간 동안 2 % dispase의 collagenase 솔루션 다진 조직을 잠복기 ° C.를하여 효소 dissociated 아르 </li><li> 햄 F - 10 20 % FBS를 포함하는 성장 매체와 효소 소화를 중화, 파스퇴르 피펫과 반복 triturating하여 근육을 떼어 놓다. 세포는 세포 스트레이너 (ø70 - 100μm)를 통해 필터링 후 37에서 1 시간 동안 비 코팅 접시 ° C.에 비쳐지게 만들려합니다 </li><li> 부드럽게 콜라겐 코팅 접시에 뜨는와 플레이트를 제거합니다. myoblasts 정화 나중에이 단계 1 시간 반복합니다. (미리 판) </li><li> 시간에 미디어 및 플레이트 세포를 변경합니다. 최종 정화 myoblasts는 매체 햄 F - 10, 20 % FBS, 100 U / ML 페니실린, 37 ° C 5 % CO2와 95 %의 공기를 100 μg / ML 스트렙토 마이신 (의 성장 매체)이있는에 양식 있습니다. </li></ol> </li><li> 문화 10 NG / ML의 기본 FGF의 추가와 함께 성장 매체의 기본 myoblasts을 확장합니다. </li><li> 생체내 실험의 모든을 위해, 우리는 TA의 근육 당 5x10 5 세포를 주입. </li><li> myoblasts을 수확하면, 우리는 0.25 % 트립신 / EDTA (에틸렌 다이아 민 테트라 초산)를 사용하여 세포를 분리하여 두 번 PBS로 그들을 씻으십시오. 휴대폰 번호는 hemocytometer로 집계되고 세포는 5x10 4 셀 / μl의 농도에 HBSS에 resuspended 있습니다. HBSS에 집중 세포는 얼음에 게재 및 수집 후 1 시간 이내에 주입하고 있습니다. </li></ol> VIVO 이미징에 <ol><li> 4~6주의 나이에 남자 MDX 마우스는 잭슨 실험실에서 구입 동물 주거 시설에서 유지 관리됩니다. </li><li> 환경에 숙박 시설 중 하나에 대한 주 후 생쥐는 교양 세포와 이식 준비하고 있습니다. </li><li> 세포 이식하기 전에 마우스 케타민을 (100 MG / kg)과 Xylazine (10 MG / kg)의 혼합물의 intraperitoneal (IP) 주입에 의해 anesthetized 있습니다. 다음 TA 근육 주변 머리가 다리에 나이르를 적용하고 증류수로 깨끗이 닦아 30 초 동안 대기에 의해 제거됩니다. </li><li> 마우스가 마취하에 여전히 있지만, 10μl HBSS에 5x10 5 전지 천천히 30 게이지 바늘을 사용하여 3 점 각 TA 근육에 주입하고 있습니다. </li><li> 4 단계 후, 마우스는 정기적으로 생체내 형광 이미징에 의해 몇 군데하실 수 있습니다. 높은 민감한 전하 결합 카메라가 장착된 형광 이미징 역 NightOwl LB981 (베르톨드 기술 주식 회사)는, 생쥐의 방해 다리의 이미지를 얻는 데 사용됩니다. </li><li> 생체내 이미징에의 날, 우리는 먼저 TA 근육에 머리 regrowth를 확인하십시오. 필요한 경우, 우리는 케타민을과 Xylazine의 혼합으로 마우스를 anesthetizing 후, 위에 설명된대로 머리를 제거하는 나이르를 다시 적용됩니다. </li><li> 마우스가 마취하는 동안, 우리는 검은 비 형광 종이 (스트라 스에서 검은 Artagain 용지)의 작품에 자주 발생하는 위치에 마우스를 놓으십시오. 충분히 더 나은 이미지를위한 TA 근육을 노출하기 위해, 우리는 종이로 뒷다리 발톱 테이프. </li><li> 우리는 이미징 챔버와 위치 카메라의 시야의 중심에 몇 군데로 뒷다리 다리에 마우스를 놓으십시오. 우리는 처음 5 초 노출 시간과 다리의 전통 회색 스케일 사진 사진을 찍을 수있는 방사 필터를 전환합니다. 필요한 경우 카메라의 마우스와 초점의 위치를​​ 재조정. 노출 시간 (1250 MS), 표본의 높이 (0.9 ㎝) 등 이미징 매개 변수는 실험 과정에서 지속적으로 보관됩니다. </li><li> 그러면 녹색 형광 단백질 파장에 해당하는 방출 필터에 스위치. 우리는 1250 MS와 1 비닝 번호 노출 시간 형광 이미지를 가져가라. 이미징 후, 마우스 회복을 기다릴 수있는 케이지에 이미징 챔버에서 제거됩니다. </li><li> 분석을 위해, 우리는 초당 광자의 관점에서 그들의 강도를 측정하는 형광 신호를 통해 관심 영역 (ROI)을 그립니다. 우리는 또한 다른 부량 값으로 픽셀의 측면에서 투자 수익 (ROI)의 크기를 측정합니다. 세로 데이터를 취득하기 위해 필요한 경우 이미징 실험은 몇 개월 동안 더 자주 매주 반복하거나 수 있습니다. </li></ol> 실험의 마지막에는, 마우스 euthanized되고 TA 근육은 조직학 분석에 수확합니다.

<ol>
	<li>Ballou, B., Ernst, L. A., Waggoner, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+imaging+of+tumors+in+vivo.">Fluorescence imaging of tumors in vivo.</a> Curr Med Chem. 12, 795-805 (2005).</li><li>Green, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fluorescent+protein+as+a+marker+for+gene+expression.">fluorescent protein as a marker for gene expression.</a> Science. 263, 802-805 (1994).</li><li>Godavarty, A., Sevick-Muraca, E. M., Eppstein, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+fluorescence+lifetime+tomography.">Three-dimensional fluorescence lifetime tomography.</a> Med Phys. 32, 992-1000 (2005).</li><li>Graves, E. E., Weissleder, R., Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+molecular+imaging+of+small+animal+tumor+models.">Fluorescence molecular imaging of small animal tumor models.</a> Curr Mol Med. 4, 419-430 (2004).</li><li>Graves, E. E., Yessayan, D., Turner, G., Weissleder, R., Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+in+vivo+fluorochrome+concentrations+measured+using+fluorescence+molecular+tomography.">Validation of in vivo fluorochrome concentrations measured using fluorescence molecular tomography.</a> J Biomed Opt. 10, 44019-44 (2005).</li><li>Gurfinkel, M., Ke, S., Wen, X., Li, C., Sevick-Muraca, E. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+fluorescence+enhanced+optical+tomography+using+referenced+frequency-domain+photon+migration+measurements+at+emission+and+excitation+wavelengths.">Three-dimensional fluorescence enhanced optical tomography using referenced frequency-domain photon migration measurements at emission and excitation wavelengths.</a> J Opt Soc Am A Opt Image Sci Vis. 19, 759-771 (2002).</li><li>Xu, X., Yang, Z., Liu, Q., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+fluorescence+imaging+of+muscle+regeneration+by+transplanted+eGFP-labeled+myoblasts.">In vivo fluorescence imaging of muscle regeneration by transplanted eGFP-labeled myoblasts.</a> , (1985).</li><li>Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cell+biology+of+cancer+cells+visualized+with+fluorescent+proteins.">In vivo cell biology of cancer cells visualized with fluorescent proteins.</a> Curr Top Dev Biol. 70, 121-144 (2005).</li><li>Coulton, G. R., Morgan, J. E., Partridge, T. A., Sloper, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mdx+mouse+skeletal+muscle+myopathy:+I.+A+histological,+morphometric+and+biochemical+investigation.">The mdx mouse skeletal muscle myopathy: I. A histological, morphometric and biochemical investigation.</a> Neuropathol Appl Neurobiol. 14, 53-70 (1988).</li><li>Morgan, J. E., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+satellite+cells.">Muscle satellite cells.</a> Int J Biochem Cell Biol. 35, 1151-116 (2003).</li><li>Mendell, J. R., Kissel, J. T., Amato, A. A., King, W., Signore, L., Prior, T. W., Sahenk, Z., Benson, S., McAndrew, P. E., Rice, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoblast+transfer+in+the+treatment+of+Duchenne's+muscular+dystrophy.">Myoblast transfer in the treatment of Duchenne's muscular dystrophy.</a> N Engl J Med. 333, 832-838 (1995).</li><li>Bogdanovich, S., Perkins, K. J., Krag, T. O., Khurana, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutics+for+Duchenne+muscular+dystrophy:+current+approaches+and+future+directions.">Therapeutics for Duchenne muscular dystrophy: current approaches and future directions.</a> J Mol Med. 82, 102-115 (2004).</li><li>Morgan, J. E., Coulton, G. R., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mdx+muscle+grafts+retain+the+mdx+phenotype+in+normal+hosts.">Mdx muscle grafts retain the mdx phenotype in normal hosts.</a> Muscle Nerve. 12, 401-409 (1989).</li><li>Sherwood, R. I., Christensen, J. L., Weissman, I. L., Wagers, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+skeletal+muscle+contributions+from+circulating+cells,+bone+marrow+cells,+and+hematopoietic+stem+cells.">Determinants of skeletal muscle contributions from circulating cells, bone marrow cells, and hematopoietic stem cells.</a> Stem Cells. 22, 1292-1304 (2004).</li><li>Cerletti, M., Jurga, S., Witczak, C. A., Hirshman, M. F., Shadrach, J. L., Goodyear, L. J., Wagers, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient,+functional+engraftment+of+skeletal+muscle+stem+cells+in+dystrophic+muscles.">Highly efficient, functional engraftment of skeletal muscle stem cells in dystrophic muscles.</a> Cell. 134, 37-47 (2008).</li></ol>

우리는이 연구에서 호스트 골격근에 형광 표시 이식 세포를 추적하기위한 안정적이고 안정적​​인 이미지 플랫폼을 설정할 수 있습니다. GFP 유전자 변형 마우스 GFP - 라벨 myoblasts은 향후 세포 치료를위한 후보 것입 성인 줄기 세포의 유형을 나타냅니다. 휴대폰 번호, 휴대 사출, 명확한 배경 이미지 위치 및 기계 매개 변수를 포함하여 지속적인 조작은 높은 품질의 이미지를 확보와 특히 정량 분석​​을 위해 중요합니다. 형광 이미징은 사용하는 비침습, 쉬운으로 생물 의학 연구에 많은 응용 프로그램을 가지고 있으며, 높은 처리량을하고 있습니다. 또한 형광 이미징의 산란, 감쇠 및 흡수에 의한하지만 광자 건의에 따라 양적 측정을 제공할 수 불빛이 기법의 단점이다 조직에 큰 거리를 침투할 수 없습니다. 노력은 빛의 침투 깊이를 높이기 위해 빨간색이나 가까운 적외선 기자를 사용하여 진행되었습니다. 형광 이미징의 또 다른 장점은 특정 형광 기자가 인간의 사용 승인을하는 경우가 클리닉에 번역되어있다. 그것이 고가의 하드웨어 및 이미징 에이전트를 필요로하지 않으므로 MRI, CT 및 PET과 비교, 형광 이미징 높은 유연성을했다. 한 예로 클리닉에서 사용되었습니다 형광 기반의 내시경 및 마이크로 내시경이다. 다른 modalities와 형광 이미징을 결합함으로써, 우리는 기본적인 생물 학적 과정에 대한 해부학 및 기능 정보를 모두 얻을 수있는 기능을 달성하기 위해 기대하고 있습니다.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NightOwl LB981</td>
<td>&nbsp;</td>
<td>Berthold Technologies</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

tracking dynamics of muscle engraftment in small animals by in vivo fluorescent imaging

근육 dystrophies는 수축성 근육 세포의 점진적 상실의 특징 퇴행성 근육 질환의 그룹입니다. 현재 사용 가능한 치료 치료가 없습니다. 줄기 세포 생물학의 최근 발전은 이러한 질병을 치료하는 효과적인 세포 기반 치료법​​의 개발을위한 새로운 희망을 생성합니다. dystrophic 동물 모델의 근육에 형광 단백질로 표시 줄기 세포의 다양한 종류의 이식이 분야에 광범위하게 사용되고 있습니다. 이식 세포의 운명을 추적하는 기능이있는 비침습 기술은 길이 방향보다 정확하고 효율적으로 이식 세포 근육 engraftment을 평가하는 우리의 능력이 더 있습니다. 현재 연구에서 우리는 생체내 형광 이미징에서 이식 GFP (녹색 형광 단백질) 표시 마우스를 골격 근육에있는 세포를 추적 민감하고 안정적인 방법입니다 보여줍니다. 형광 단백질 여기에 필요한 외부 조명의 사용으로 인해 배경에 대한 우려에도 불구하고, 우리는 어느 누드 ​​마우스를 사용하거나 머리 제거 시약과 머리를 제거하여 더이 문제가 제거되고있는 것으로 나타났습니다. CCD 카메라를 사용하여 형광 신호는 GFP 형질 전환 마우스 또는 eGFP가 myoblast 문화를 transduced 중 하나에서 5 × 10 5 세포의 주입 후에 정강뼈 앞쪽에 (TA) 근육에서 발견하실 수 있습니다. 같은 더 많은 외면적인 근육에 대한 신근의 digitorum longus의 (편집 판단리스트), 적은 수의 세포의 주입은 감지 신호를 생성합니다. 신호 강도가 측정 및이자 (ROI)의 지역에서 초당 방출되는 광자의 개수로 계량하실 수 있습니다. 획득 이미지가 engrafted 지역 demarcating 명확한 경계를 표시 때문에, 투자 수익 (ROI)의 크기뿐만 아니라 측정할 수 있습니다. 다리가 지속적으로 때마다 위치하는 경우, 근육 및 투자 수익 (ROI)의 크기 초당 당 광자의 총수의 변경 engrafted 세포의 숫자의 변화와 engrafted 영역의 크기를 반영합니다. 따라서 시간이 지남에 따라 같은 근육의 변화가 같지는 있습니다. 생체내에서 형광 이미징 기술이 tumorogenic 세포의 성장을 추적하기 위해 주로 사용되고 있습니다, 우리의 연구는 이식 줄기 세포의 운명을 추적하기 위해 우리를있게 해주는 강력한 도구입니다 보여줍니다.

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

우리는 설명을<em&gt; 생체내에</em&gt; 형광 이미징 프로토콜은 건강하고 dystrophic 두 생쥐의 골격 근육에 이식 후 GFP - 라벨 myoblasts 의한 근육 재생을 모니터링합니다. 이 프로토콜은 세포의 다른 유형의 이식에 의해 다른 근육의 조건뿐만 아니라 근육 재생 연구를 적용할 수 있습니다.

작은 동물의 근육 Engraftment의 역학에 의해 추적 VIVO에서 형광 이미징

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

tracking-dynamics-muscle-engraftment-small-animals-vivo-fluorescent

Research

JoVE Journal

Biology

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

9.7K 조회수.  Brigham and Woman's Hospital. 우리는 설명을 생체내에 형광 이미징 프로토콜은 건강하고 dystrophic 두 생쥐의 골격 근육에 이식 후 GFP - 라벨 myoblasts 의한 근육 재생을 모니터링합니다. 이 프로토콜은 세포의 다른 유형의 이식에 의해 다른 근육의 조건뿐만 아니라 근육 재생 연구를 적용할 수 있습니다.

비디오: 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.

내부 핵심 현미경에 의해 골격근의 생체내 미세 순환 측정

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

연구

생물학

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.

이런 산화물로 hESCs과 hMSCs을 라벨링하는 MR 영상과 생체내 추적 비침습에 대한 Nanoparticles

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).

형광등을 통해 F - 굴지 역학 라이브 셀 이미징은 현미경을 스페클 (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.

절연 골격근의 수축성, Fatigability 및 Alternans의 예 VIVO 평가

 
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.

공 초점 및 다중 광자 현미경을 사용하여 다섯 형광 단백질에 의해 표시됨 조혈 줄기 및 전구 세포의 생체 내 클론 추적에서

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.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

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.

고해상도 시간 경과 이미징 및 생활 인간 제대 정맥 내피 세포의 미세 소관 역학의 자동 분석

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

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

동적 세포 돌출부에서 소프트웨어를 이용한 단백질 농도 추적을위한 그래픽 사용자 인터페이스

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

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.

레이저 스캐닝 현미경 검사 법에 의해 눈의 앞쪽 챔버에 Thymocytes의 비보에 추적 실시간

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.

형광 라이브 세포 현미경 검사법에 의한 미토틱 및 Meiotic 핵분열 효모 핵 역학의 검사

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 ...