Name: coordinate mapping of hyolaryngeal mechanics in swallowing
Uploaded: 2014-05-06T15:30:00
Description: Watch this Scientific Journal Video about Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing at JoVE.com

저자는 Kendrea Focht, CSCD, CCC-SLP 및 음성에 대한 에블린 트라멜 연구소를 인정하고 MBS에게이 방법을 설명하는 데 사용되는 이미지 파일을 공유, 사우스 캐롤라이나 의과 대학에서 삼키는. (PI : 마틴 해리스) 번역 상 과학 선진화를위한 그랜트 번호 1K24DC12801에 의해 국립 센터에서 이러한 MBS 데이터는 부여 번호 TL1TR000061 (Focht PI)에 의해 투자 성 밖의 지원을 통해 수집 된 청각 장애에 국립 연구소 및 기타 의사 소통 장애에서, 그리고 마크와 에블린 트라멜 신탁 교내 지원. 난청 및 기타 통신 장애에 국립 연구소에서 부여 번호 F31DC011705에서 지원하는 동안 이러한 방법은 원래 수사 반장에 의해 개발되었다. 내용은 전적으로 저자의 책임이며 반드시 청각 장애에 국립 연구소 및 기타 의사 소통 장애 또는 국립 연구소의 공식 견해를 대변하지 않습니다건강.

1. 컴퓨터 구성 <ol><li> ImageJ에, MacX 비디오 컨버터 무료 판 (맥), 퀵타임 (소재 / 장비의 표 참조) : Macintosh의 경우, 다음과 같은 오픈 소스 또는 프리웨어 소프트웨어를 다운로드합니다. </li><li> ImageJ에, MP​​EG 및 Streamclip (PC), 퀵타임 (소재 / 장비의 표 참조) : PC를 들어, 다음과 같은 오픈 소스 또는 프리웨어 소프트웨어를 다운로드합니다. </li></ol> 2. 비디오 클립을 준비 <ol><li> ?...

<ol>
	<li>Leonard, R. J., Kendall, K. A., McKenzie, S., Gon&#231;alves, M. I., Walker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Displacements+in+Normal+Swallowing:+A+Videofluoroscopic+Study.">Structural Displacements in Normal Swallowing: A Videofluoroscopic Study.</a> Dysphagia. 15, 146-152 (2000).</li><li>Molfenter, S. M., Steele, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+Variability+in+the+Deglutition+Literature:+Hyoid+and+Laryngeal+Kinematics.">Physiological Variability in the Deglutition Literature: Hyoid and Laryngeal Kinematics.</a> Dysphagia. 26, 67-74 (2010).</li><li>Pearson, W. G., Hindson, D. F., Langmore, S. E., Zumwalt, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+Swallowing+Muscles+Essential+for+Hyolaryngeal+Elevation+by+Using+Muscle+Functional+Magnetic+Resonance+Imaging.">Evaluating Swallowing Muscles Essential for Hyolaryngeal Elevation by Using Muscle Functional Magnetic Resonance Imaging.</a> International Journal of Radiation Oncology* Biology* Physics. 85, 735-740 (2013).</li><li>Pearson, W. G., Langmore, S. E., Yu, L. B., Zumwalt, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Analysis+of+Muscles+Elevating+the+Hyolaryngeal+Complex.">Structural Analysis of Muscles Elevating the Hyolaryngeal Complex.</a> Dysphagia. 27, 445-451 (2012).</li><li>Kim, Y., McCullough, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximum+hyoid+displacement+in+normal+swallowing.">Maximum hyoid displacement in normal swallowing.</a> Dysphagia. 23, 274-279 (2008).</li><li>Steele, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+hyoid+and+laryngeal+displacement+and+swallowing+impairment.">The relationship between hyoid and laryngeal displacement and swallowing impairment.</a> Clin. Otolaryngol. 36, 30-36 (2011).</li><li>Logemann, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+Biomechanical+Characteristics+of+Oropharyngeal+Swallow+in+Younger+and+Older+Men.">Temporal and Biomechanical Characteristics of Oropharyngeal Swallow in Younger and Older Men.</a> Journal of Speech, Language and Hearing Research. 43, 1264-1274 (2000).</li><li>Pearson, W., Langmore, S., Zumwalt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+Structural+Properties+of+Suprahyoid+Muscles+and+their+Potential+for+Moving+the+Hyoid.">Evaluating the Structural Properties of Suprahyoid Muscles and their Potential for Moving the Hyoid.</a> Dysphagia. 26, 345-351 (2011).</li><li>Hopkins, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measures+of+reliability+in+sports+medicine+and+science.">Measures of reliability in sports medicine and science.</a> Sports Med. 30, 1-15 (2000).</li><li>Bingjie, L., Zhang, T., Sun, X., Xu, J., Jiang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+videofluoroscopic+analysis+of+penetration-aspiration+in+post-stroke+patients.">Quantitative videofluoroscopic analysis of penetration-aspiration in post-stroke patients.</a> Neurol. India. 58, 42-47 (2010).</li><li>Webster, M., Sheets, H. D., Alroy, J., Hunt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+introduction+to+landmark-based+geometric+morphometrics.">A practical introduction to landmark-based geometric morphometrics.</a> Quantitative Methods in Paleobiology. Paleontological Society Papers. 16, 163-188 (2010).</li><li>Inamoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+swallowing+using+320-detector-row+multislice+CT.+Part+II:+Kinematic+analysis+of+laryngeal+closure+during+normal+swallowing.">Evaluation of swallowing using 320-detector-row multislice CT. Part II: Kinematic analysis of laryngeal closure during normal swallowing.</a> Dysphagia. 26, 209-217 (2011).</li></ol>

이 연구는 삼키는 hyolaryngeal 운동의 여러 운동 측정을 계산하는 해부학 적 랜드 마크의 좌표 데이터를 사용하는 방법의 유용성을 보여줍니다. 좌표와 계산 된 변수에 대한 두 초보자 평가자, 등 6 평가자의 평가 자간 신뢰도 (ICC&gt; 0.90) 강했습니다. 건강한 비 dysphagic 성인의 무작위 표본의 대표 결과는 두 개의 알약 유형에 대한 응답으로 여러 가지 운동 학적 변수의 차이를 보여 주었다. 우리는 또?...

연하의 인두 단계기도를 보호하면서 식도로 구강으로부터 볼 루스를 전송하기 위해 20여 근육 및 골격 체류 요소를 포함하는 복잡한 과정이다. 인두 수축, hyolaryngeal 복잡한 요소 (설골, 후두, 상부 식도 괄약근을 포함하여 관련 구조) 이전은 소화 기관에 호흡 관을 변환하는 변위. 후두는 전방 멀리 다가오는 알약의 궤도에서 다시 위치하고 있으며, 상부 식도 괄약근이 열려 뻗어있다. (또한 MBS로 알려진 또는 바륨 삼키기 연구 수정) 비디오 투시 연하 연구에서 가져온 운동 학적 측정은 hyolaryngeal 1 단지의 여러 움직임을 정량화하기위한 기본 연구 방법론이다. 양적 비디오 투시 측정 삼키는 기능, 참조의 다른 축 측정을 위해 유용하지만및 운동 측정 (2)의 다양한 방법 중 호환되지 않는 연구 결과에서 측정 결과의 스칼라. 서, 임상의 제어하에 환자와 형광 투시경의 움직임의이 복잡한 생리 학적 과정을 측정의 정확도를 혼동. 더 중요한 것은, 운동 학적 측정은 반드시 무질서 삼키는을 평가하는 중요한 구조에 함수 관계를 반영하지 않습니다. 특히 설골의 운동학은 척추 정렬 해부학적인면을 참고로 전방 또는 우수한 방향으로 움직임을 추적하도록 설계되었습니다. 그러나이 구성 설골 중단 근육의 작용선을 대표하지 않는다. 삼키는의 인두 단계에서 hyolaryngeal 상승의 2 슬링 메커니즘 (그림 1) 3,4 확인되었습니다. suprahyoid 근육은 앞쪽에 근육 슬링을 구성하고, 긴 인두 근육 P를 포함근육 슬링 osterior. 이 근육은 설골, 후두, 상부 식도 괄약근을 형성하는 구조를 포함 hyolaryngeal 단지의 다양한 요소를 상승. hyolaryngeal 역학의 좌표 매핑은 전방 및 후방 근육 슬링 (그림 2)의 hyolaryngeal 복잡한 나타내는 연결 지점의 세 가지 골격 레버와 기능을 매핑 할 아홉 쉽게 식별 해부학 적 랜드 마크를 사용합니다. 삼키는 동안, hyolaryngeal 복잡한 각각의 골격 레버와 기능은 움직임입니다. 좌표를 수집하여, 시스템은 임의의 시간 프레임에서 캡처 할 수있다. 코디네이터의 삼각 변환 삼키는 동안 hyolaryngeal 움직임의 여러 운동 학적 측정을 생성하기 위해 사용될 수있다. 변수는 문헌에보고 된 결과와 비교하기 위해 계산 된, 또는 그 구조에 대한 함수 관계를 나타내는 새로운 측정을 생성하기 위해 사용될 수있다. 이 논문의 주요 목적은 수정 된 바륨 삼키기 (MBS)의 연구에서 수집 된 해부학 적 랜드 마크 좌표의 단일 세트에서 계산 된 여러 운동 측정을 생성하는 방법을 설명하는 것입니다. 우리는 하나의 전문적인 경험을 가진 세 평가자, 두 초보자를 포함하여 6 개의 다른 연구자의 상호 평가자 신뢰성을 결정하는 급내 상관 계수를 사용하여이 방법의 신뢰성을 문서화합니다. 운동 학적 결과에서, 알약의 일관성에 의해 역학을 삼키는의 차이가 평가됩니다. 마지막으로, 설골 움직임을 측정에 사용되는 기준 축의 중요성에 관한 Molefenter 및 스틸 제안한 문제는 해결된다. 우리가 척추를 참고로 두 러스 유형에 대한 좌표의 동일한 세트에서 계산 된 하악을 참조에 설골 소풍의 측정을 비교이 문제를 접근하는. 설골 움직임을 측정하는이 방법은 두 관계를 작동하는 동일한 구조를 나타내면엉덩이, 그 결과는 강력하게 상관 관계가되어야한다. 본 연구의 경우, 40 측면보기 MBS 연구는 무작위로 조지아 리전트 대학 기관 평가위원회 및 음성에 대한 에블린 트라멜 연구소와 연구 협력에 의해 승인의 의과 대학에서 삼키는 연구 프로토콜에 따라 139 일반적인 연구의 컬렉션에서 선택되었다 사우스 캐롤라이나. 이 방법의 유용성을 입증하기 위해, hyolaryngeal 역학의 특성을 열 변수는 좌표 데이터의 동일한 세트 (표 1)에서 계산 하였다. 이러한 계산 된 측정 중 7 이전 등의 문헌에 사용되었습니다 : 전방 및 척추 5를 참조 우수 설골의 거리 측정; 전방 또한 척추 6을 참고로 C2-4 길이의 비율, 우량한 설골 변위; 척추 7을 참조에있는 후두의 뛰어난 운동; hyolaryngeal approximat이온 1; 및 척추 1을 참고로 최대 설골 소풍. 또한, 세 가지 새로운 측정을 계산 하였다 : 인두 단축은 suprahyoid 근육 4,8의 첨부 파일을 근사 palatopharyngeus 근육의 첨부 파일, stylopharyngeus을 나타내는 행동의 라인을 다음과 후두 상승 및 설골 여행을 근사. 전문가의 머리와 목의 해부학자 (WP), 제한된 경험을 측정하기 (CJ, SR, TT), 두 초보자 연구자 (RS, JT) 세 조사는 아래에 설명 된 프로토콜을 사용하여 매핑 좌표 데이터를 얻을. 전문가 (WP)는 경험이있는 세 평가자 훈련, 이들은 차례로 두 초보자 평가자 훈련. 주제별로 좌표 계산 좌표 데이터와 결과의 평가 자간 신뢰도는 급내 상관 계수 (9)에 의해 결정되었다. 두 개의 꼬리 T-테스트는 statisticall를 결정하는 각 변수에 대해 수행되었다알약 타입의 Y 유의 한 차이. 피어슨 상관 계수와 결정 계수는 5 ㎖ 얇은 액체의 제비와 5 ㎖에 대한 참조의 축으로 하악골과 설골 소풍 대 기준 축으로 척추 계산 설골 소풍의 결과 사이의 계약을 평가하는 데 사용되었다 푸딩 제비.

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coordinate mapping of hyolaryngeal mechanics in swallowing

hyolaryngeal 움직임을 특성화 연하의 연구에 중요하다. 이전 방법은 관심의 여러 변수를 계산하는 좌표 중 하나 세트를 사용하여 수정 된 바륨 삼키기 (MBS)를 사용하여 hyolaryngeal 역학의 매핑 좌표 반면, 하나의 운동 측정을 얻기 위해 다수의 측정이 필요합니다. 데모 용으로 열 학적 측정은 두 가지 다른 알약 유형을 삼키는의 차이를 결정하는 좌표의 한 세트에서 생성되었다. 척추와 하악에 대한 설골 소풍의 계산은 기준 축의 중요성을 결정하기 위해 상관 관계. 매핑 방법을 좌표 설명하기 위해 40 MBS 연구는 무작위로 알려진 바 없음 연하 장애와 건강한 정상인의 데이터 집합에서 선정되었다. 5 ㎖ 얇은 액체 알약 및 5 ㎖ 푸딩 제비는 각 과목에서 측정 하였다. 두개골베이스, 하악골, 척추와 효의 요소에 매핑 구 좌표,후두 복합체는, 최소 및 최대 hyolaryngeal 소풍의 프레임으로 기록되었다. 좌표는 수학적으로 hyolaryngeal 역학 10 변수로 변환 하였다. 평가 자간 신뢰도는 급내 상관 계수 (ICC)에 의해 평가 하였다. 양측 t-테스트는 볼 루스 점도 운동학의 차이를 평가하기 위해 사용되었다. 참고 다른 축에 대한 설골 소풍 측정 한 상관 관계가 있었다. 0.97 - 18 좌표 여섯 평가자 간의 평가 자간 신뢰도는 ICC에서 = 0.90이었다. 열 운동 측정의 슬레이트는 여섯 평가자 사이의 대상으로 비교 하​​였다. 특이 치 거부, 나머지 신뢰성 점수의 평균치는 ICC = 0.91, 0.84이었다 - 0.96, 95 % CI. 열 학적 변수 (5 ㎖ 얇은 액체 대 5 ㎖ 푸딩 제비)를 비교 Bonferroni 보정과 양측 t-테스트는 설골 소풍, 우수한 후두 운동 및 인두 shortenin에서 통계적으로 유의 한 차이를 보였다G (P &lt;0.005). R = 0.62, R2 = 0.38, (얇은 액체) : 기준의 두 가지 축에서 설골 소풍 측정 피어슨 상관 관계가 있었다 R = 0.52, R2 = 0.27, (푸딩). 랜드 마크의 좌표를 얻기 연하 곤란 연구에 유용한 비디오 투시 영상에서 여러 운동 학적 변수를 생성하는 신뢰할 수있는 방법입니다.

급내 상관 계수 독립적 (80) 비디오 투시 파일 (40 과목에서 두 개의 알약 시험)을 분석 여섯 조사에 의해 수집 된 좌표 (ICC)는 원거리 ICC에서 = 0.90-0.97. 다음과 같이 그룹에 의해 ICC의 좌표의 고장입니다 : # 1 좌표 - 5 (최소 hyolaryngeal 소풍 골격 요소) 평균 = 0.93, 0.91-0.95, 95 % 신뢰 구간; ; - 9 (최소 hyolaryngeal 소풍에 hyolaryngeal 복잡한) 평균 = 0.94, 092 - 0.96, 95 % CI # 6 좌표 # 10 좌표 - 0.95, 95 % CI - 14 (최대 hyolary...

Watch this Scientific Journal Video about Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing at JoVE.com

Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing

좌표 매핑 연하의 인두 단계에서 hyolaryngeal 역학의 두드러진 특징을 문서화하는 방법이다. 이 방법론은 해부학 마크의 좌표를 기록하는 이미지 분석 소프트웨어를 사용한다. 이 좌표는 엑셀 매크로로 가져 및 연하 곤란의 연구에 유용 관심 학적 변수로 변환됩니다.

삼키는 Hyolaryngeal 기계의 매핑 좌표

Characterizing hyolaryngeal movement is important to dysphagia research. Prior methods require multiple measurements to obtain one kinematic measurement ...

coordinate-mapping-of-hyolaryngeal-mechanics-in-swallowing

Research

JoVE Journal

Medicine

Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC twin pregnancies develop complications arising from the shared placenta and associated vascular connections1. Severe twin-to-twin syndrome (TTTS) is reported to account for approximately 20% of these complications2,3. Inter-twin vascular connections occur in almost all MC placentas and are related to the prognosis and outcome of these high-risk twin pregnancies. The number, size and type of connections have been implicated in the development of TTTS and other MC twin conditions. Three types of inter-twin vascular connections occur: 1) artery to vein connections (AVs) in which a branch artery carrying deoxygenated blood from one twin courses along the fetal surface of the placenta and dives into a placental cotyledon. Blood flows via a deep intraparenchymal capillary network into a draining vein that emerges at the fetal surface of the placenta and brings oxygenated blood toward the other twin. There is unidirectional flow from the twin supplying the afferent artery toward the twin receiving the efferent vein; 2) artery to artery connections (AAs) in which a branch artery from each twin meets directly on the superficial placental surface resulting in a vessel with pulsatile bidirectional flow, and 3) vein to vein connections (VVs) in which a branch vein from each twin meets directly on the superficial placental surface allowing low pressure bidirectional flow. In utero obstetric sonography with targeted Doppler interrogation has been used to identify the presence of AV and AA connections4. Prenatally detected AAs that have been confirmed by postnatal placental injection studies have been shown to be associated with an improved prognosis for both twins5. Furthermore, fetoscopic laser ablation of inter-twin vascular connections on the fetal surface of the shared placenta is now the preferred treatment for early, severe TTTS. 
Postnatal placental injection studies provide a valuable method to confirm the accuracy of prenatal Doppler ultrasound findings and the efficacy of fetal laser therapy6. Using colored dyes separately hand-injected into the arterial and venous circulations of each twin, the technique highlights and delineates AVs, AAs, and VVs. This definitive demonstration of MC placental vascular anatomy may then be correlated with Doppler ultrasound findings and neonatal outcome to enhance our understanding of the pathophysiology of MC twinning and its sequelae. Here we demonstrate our placental injection technique.

Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins&#x2019; circulations. 
Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.

Monochorionic 트윈 Placentas의 혈관 매핑을위한 가이드 와이어 원호 Catheterization과 컬러 염료 사출

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC ...

연구

의학

High-Resolution Endocardial and Epicardial Optical Mapping in a Sheep Model of Stretch-Induced Atrial Fibrillation

Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance seen in clinical practice and its prevalence is expected to increase in the coming years.3 Increased intra-atrial pressure and dilatation have been long recognized to lead to AF,1,4 which highlights the relevance of using animal models and stretch to study AF dynamics. Understanding the mechanisms underlying AF requires visualization of the cardiac electrical waves with high spatial and temporal resolution. While high-temporal resolution can be achieved by conventional electrical mapping traditionally used in human electrophysiological studies, the small number of intra-atrial electrodes that can be used simultaneously limits the spatial resolution and precludes any detailed tracking of the electrical waves during the arrhythmia. The introduction of optical mapping in the early 90's enabled wide-field characterization of fibrillatory activity together with sub-millimeter spatial resolution in animal models5,6 and led to the identification of rapidly spinning electrical wave patterns (rotors) as the sources of the fibrillatory activity that may occur in the ventricles or the atria.7-9 Using combined time- and frequency-domain analyses of optical mapping it is possible to demonstrate discrete sites of high frequency periodic activity during AF, along with frequency gradients between left and right atrium. The region with fastest rotors activates at the highest frequency and drives the overall arrhythmia.10,11 The waves emanating from such rotor interact with either functional or anatomic obstacles in their path, resulting in the phenomenon of fibrillatory conduction.12 Mapping the endocardial surface of the posterior left atrium (PLA) allows the tracking of AF wave dynamics in the region with the highest rotor frequency. Importantly, the PLA is the region where intracavitary catheter-based ablative procedures are most successful terminating AF in patients,13 which underscores the relevance of studying AF dynamics from the interior of the left atrium. Here we describe a sheep model of acute stretch-induced AF, which resembles some of the characteristics of human paroxysmal AF. Epicardial mapping on the left atrium is complemented with endocardial mapping of the PLA using a dual-channel rigid borescope c-mounted to a CCD camera, which represents the most direct approach to visualize the patterns of activation in the most relevant region for AF maintenance.

This report provides a detailed description of the methodology and results of simultaneous endocardial and epicardial optical mapping of electrical excitation in the intact left atrium of a Langendorff-perfused sheep heart during stretch-induced atrial fibrillation.

스트레치 유도 심방세동 세동의 쉽 모델에서 고해상도 Endocardial 및 Epicardial 광학 매핑

 Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance ...

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

양적 Immunofluorescence를 사용하여 종양의 단백질 표현의 이질 매핑

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process.
cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles.
Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing crossover frequency of a particle or cell, the frequency at which the dielectrophoretic force is zero. Finally, we demonstrate the use of this technique for sorting a mixture of ovarian cancer cells and fluorescing microspheres (beads).

Contactless dielectrophoresis (cDEP) achieves sorting and enrichment of particles via their intrinsic dielectric properties. Fluidic electrode channels replace metallic electrodes traditional to DEP, suiting cDEP to non-damaging sterile characterization and sorting of biological particles. We demonstrate how to prepare a cDEP microdevice and conduct cell characterization and sorting experiments.

비접촉식 유전을 통해 라벨이없는 분리 및 세포의 농축

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to ...

MRI Mapping of Cerebrovascular Reactivity via Gas Inhalation Challenges

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. Therefore, the ability of the blood vessels to dilate or constrict, known as Cerebral-Vascular-Reactivity (CVR), represents an important domain of vascular function. An imaging marker representing this dynamic property will provide new information of cerebral vessels under normal and diseased conditions such as stroke, dementia, atherosclerosis, small vessel diseases, brain tumor, traumatic brain injury, and multiple sclerosis. In order to perform this type of measurement in humans, it is necessary to deliver a vasoactive stimulus such as CO2 and/or O2 gas mixture while quantitative brain magnetic resonance images (MRI) are being collected. In this work, we presented a MR compatible gas-delivery system and the associated protocol that allow the delivery of special gas mixtures (e.g., O2, CO2, N2, and their combinations) while the subject is lying inside the MRI scanner. This system is relatively simple, economical, and easy to use, and the experimental protocol allows accurate mapping of CVR in both healthy volunteers and patients with neurological disorders. This approach has the potential to be used in broad clinical applications and in better understanding of brain vascular pathophysiology. In the video, we demonstrate how to set up the system inside an MRI suite and how to perform a complete experiment on a human participant.

Non-invasive imaging of the brain vasculature&#8217;s ability to dilate or constrict may allow a better understanding of cerebrovascular pathophysiology in various neurological diseases. The present report describes a reproducible and patient-comfortable protocol to perform vascular reactivity imaging in humans using magnetic resonance imaging (MRI).

가스 흡입 도전을 통해 뇌 혈관 반응성의 MRI 매핑

The brain is a spatially heterogeneous and temporally dynamic organ, with different regions requiring different amount of blood supply at different time. ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

마우스와 위식도 역류의 외과 모델

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

고해상도 광학 매핑 Murine Atria electrophysiological 평가

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

양성자 자기 공명 영상 및 조 영제로 산소를 사용 하 여 인간의 폐에 특정 통풍의 정량 매핑

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

ElectroMap을 사용한 광학 매핑 데이터의 높은 처리량 분석

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells provide promising sources for cardiac disease modeling. For cardiomyopathies, sarcomere shortening is one of the standard physiological assessments that are used with adult cardiomyocytes to examine their disease phenotypes. However, the available methods are not appropriate to assess the contractility of PSC-CMs, as these cells have underdeveloped sarcomeres that are invisible under phase-contrast microscopy. To address this issue and to perform sarcomere shortening with PSC-CMs, fluorescent-tagged sarcomere proteins and fluorescent live-imaging were used. Thin Z-lines and an M-line reside at both ends and the center of a sarcomere, respectively. Z-line proteins &#8212;&#160;&#945;-Actinin (ACTN2), Telethonin (TCAP), and actin-associated LIM protein (PDLIM3) &#8212; and one M-line protein &#8212; Myomesin-2 (Myom2) &#8212;&#160;were tagged with fluorescent proteins. These tagged proteins can be expressed from endogenous alleles as knock-ins or from adeno-associated viruses (AAVs). Here, we introduce the methods to differentiate mouse and human pluripotent stem cells to cardiomyocytes, to produce AAVs, and to perform and analyze live-imaging. We also describe the methods for producing polydimethylsiloxane (PDMS) stamps for a patterned culture of PSC-CMs, which facilitates the analysis of sarcomere shortening with fluorescent-tagged proteins. To assess sarcomere shortening, time-lapse images of the beating cells were recorded at a high framerate (50-100 frames per second) under electrical stimulation (0.5-1 Hz). To analyze sarcomere length over the course of cell contraction, the recorded time-lapse images were subjected to SarcOptiM, a plug-in for ImageJ/Fiji. Our strategy provides a simple platform for investigating cardiac disease phenotypes in PSC-CMs.

This method can be used to examine sarcomere shortening using pluripotent stem cell-derived cardiomyocytes with fluorescent-tagged sarcomere proteins.

형광 태그 사코레 단백질을 사용하여 다능줄기 세포 유래 심근 세포의 Sarcomere 단축.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells ...