이 프로토콜을 사용하여 연구는 심장과 뇌졸중 재단 (G-18-0020952), 크레이그 H. 닐슨 재단 (542675), 캐나다 자연 과학 및 공학 연구 위원회 (RGPIN-2014-05498), 연구, 혁신 및 과학부, 온타리오 (ER16-12-013)에 의해 투자되었다.

연구 결과는 대학 건강 네트워크의 연구 윤리 위원회에 의해 승인되었습니다. 서명된 정보에 입각한 동의는 연구에 등록하기 전에 각 참가자로부터 얻어졌습니다. 비디오 녹화에 등장하는 간병인 이나 가정 구성원으로부터 서명 된 동의를 얻었습니다.
1. 개인에 대한 프로토콜 적용 가능성 확인
참고: 이 프로토콜은 손상되었지만 완전히 결석하지 않은 개인에게 적용하기 위한 것입니다(특정 기준은 인구 및/또는 관심 문제에 맞게 조정할 수 있음).
<ol><li>참가자들에게 영향을 받는 손이 AD를 수행하는 능력에 영향을 미치는지 물어보십시오. 참고: 참가자에게 영향을 받는 손으로 독립적으로 수행할 수 있고 수행할 수 없는 작업의 몇 가지 예를 제공하도록 요청하는 것이 좋습니다.</li>
	<li>몬트리올 인지 평가(MoCA)의 총 점수가 21을 초과하는지 확인하여 프로토콜 절차를 이해하고 따르는 잠재적 어려움을 방지합니다.</li>
</ol>2. 참가자의 일상의 결정
<ol><li>참가자들에게 지난 2주 동안 의 일상을 회상해 보라고 한다. 수행되는 일일 작업, 기간 및 대략 몇 시에 문서화합니다.</li>
	<li>참가자와 공동으로 동영상을 녹화하는 동안 각각 1.5h의 3회 를 선택합니다. 주 중 다른 날에 걸쳐 분산되는 타임슬롯을 선택하고 손을 포함하는 AD가 일반적으로 순서대로 수행될 때 수행됩니다. 참고: 선택한 AD는 각 참가자의 일반적인 활동을 대표해야 하며 의미 있는 것으로 인식되어야 합니다. 다양한 날에 기록 기간을 예약하는 것은 기록된 AL의 다양성을 높이고 유용하고 의미 있는 데이터의 캡처를 촉진하기 위한 것입니다. 참고: 기록 시간대는 기록 효율성을 위해 예약되어 있지만 참가자는 언제 시작하고 녹화를 중지해야 하는지 완전히 제어할 수 있음을 이해해야 합니다.</li>
</ol>3. 녹화 일정에 대한 합의 및 참가자와의 비디오 콘텐츠 타겟팅
<ol><li>기록 일정에 대한 각 참가자의 동의를 얻고, 그들이 가질 수있는 우려를 논의 한 후.</li>
	<li>2주 동안 3h의 동영상 목표를 설정합니다. 동영상이 부족하면 녹화 기간이 연장될 수 있음을 참가자에게 알립니다.</li>
</ol>4. AD를 자연스럽게 수행하는 것의 중요성을 강조합니다.
<ol><li>참가자에게 기록할 특정 활동을 지정하는 대신 사실적인 루틴을 캡처하는 데 집중하도록 지시합니다. 명령의 의도는 참가자가 일반적인 것보다 더 많은 양의 특정 활동을 인위적으로 기록하지 못하게하는 것입니다.</li>
</ol>5. 집에서 녹음 하는 동안 잠재적인 개인 정보 보호 문제에 대 한 통지
<ol><li>참가자는 모든 기록이 개인 정보 보호 문제를 피하기 위해 공공 장소가 아닌 집 안에서 수행되어야한다는 것을 이해해야합니다.</li>
	<li>목욕, 드레싱/드레싱, 기밀 정보 확인 등 개인 정보 보호 문제를 제기할 수 있는 몇 가지 예를 제시합니다. 참가자들에게 녹음에 얼굴을 표시할 수 있는 거울을 알고 있음을 상기시킨다.</li>
	<li>참가자는 비디오에서 가능한 한 가족 구성원이나 간병인과 같은 다른 사람들의 존재를 피할 것을 제안합니다. 참고: 연구 연구의 맥락에서, 다른 사람의 존재가 불가피한 경우, 그 개인에게서 통보된 동의를 얻어야 합니다.</li>
</ol>6. 카메라 및 태블릿 명령
참고: 참가자가 초기 접촉 중에 많은 일상적인 필요에 대한 간병인의 도움이 필요하다고 표시하는 경우, 간병인은 또한 연구 방문에 참석하고 장비 사용에 대한 교육을 받아 나중에 참가자를 도울 수 있도록 권장됩니다.
<ol><li>참가자에게 자기 중심카메라(재료 표)를사용하는 방법을 시연합니다.<ol>
<li>카메라를 켜고 끄는 방법을 보여 줍니다.</li>
		<li>카메라를 사용하여 녹화(시작, 일시 중지, 중지)를 제어하는 방법을 보여 줍니다.</li>
	</ol>
</li>
	<li>태블릿(재료 테이블)을사전 설치된 카메라 앱과 함께 사용하여 녹화를 제어하는 방법을 보여 준다. 참고: 데모에는 카메라 앱의 녹화 제어뿐만 아니라 녹화된 비디오를 재생 및 편집(예: 트림 또는 삭제)하는 것이 포함됩니다. 카메라 리모컨은 처음에는 고려되었지만(보충파일)을고려했지만, 실제로는 참가자가 카메라 나 태블릿을 사용하여 녹화를 시작하고 중지하는 것이 편안했기 때문에 사용되지 않았습니다.<ol>
<li>태블릿을 켜고 끄는 방법을 보여 줍니다.</li>
		<li>카메라 앱을 통해 태블릿을 카메라에 연결하는 방법을 보여 줍니다.</li>
		<li>카메라 앱에서 녹화를 제어하는 방법을 보여 줍니다.</li>
		<li>카메라 앱에서 녹화된 동영상을 검토하는 방법을 보여 줍니다.</li>
		<li>카메라 앱에서 비디오를 잘라내거나 삭제하는 방법을 보여 줍니다.</li>
	</ol>
</li>
	<li>참가자의 머리에 조절가능한 탄성 헤드밴드를 사용하여 카메라를 착용하고 벗는 방법을 시연합니다. 참고: 그림 1을참조하십시오. <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61898/61898fig01.jpg"> 그림 1. 웨어러블 카메라 설정.  (A) 자기 중심 카메라의 위치. (B) 카메라에서 각광을 받고 있습니다. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61898/61898fig01large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>
	<ol>
<li>참가자의 이마에 카메라를 놓습니다. 헤드밴드를 조정하여 카메라를 편안하고 꾸준히 착용합니다.</li>
		<li>이마에 대하여 카메라의 최적의 각도를 보장합니다.</li>
		<li>참가자에게 앞에서 손을 움직이고 객체(예: 태블릿)를 조작하는 동안 짧은 비디오 세그먼트를 기록하도록 요청합니다.</li>
		<li>녹화된 비디오를 검토하고 조작 작업을 수행하는 동안 장면의 중앙 영역에서 두 손이 명확하게 표시되는지 확인합니다.</li>
		<li>참가자 및 간병인과 함께 카메라와 태블릿을 사용하여 숙련도를 입증할 때까지 연습합니다.</li>
	</ol>
</li>
</ol>7. 장비 제공
<ol><li>참가자들에게 모든 장비가 있는 키트를 제공하여 집에서 AD를 기록하십시오. 카메라와 태블릿 외에도 추가 카메라 배터리, 카메라와 태블릿용 배터리 충전기, 충전 케이블, 카메라 헤드밴드 및 카메라 사용에 대한 인쇄된 지침 세트가 포함되어 있습니다(보충 자료참조).</li>
</ol>8. 실험적 문제 해결 및 후속 조치
<ol><li>집에서 실제 녹음 하는 동안 장애물을 해결 하는 데 도움이 연구원의 연락처 정보를 제공 합니다. 1주일 후, 연구원들은 참가자들에게 전화를 걸어 기록 진행 상황을 기록하고 잠재적인 기술적 문제를 해결합니다.</li>
</ol>9. 장비 및 비디오 검색
<ol><li>참가자의 모든 장비와 비디오를 직접 또는 선불 우편 소포를 통해 재구성합니다.</li>
	<li>참가자가 반환된 모든 동영상을 공유하는 데 동의해야 합니다. 참가자는 수집된 모든 동영상을 연구팀에 반환하기 전에 검토하고 공유하고 싶지 않은 부분을 삭제하는 것이 좋습니다.</li>
	<li>연구 연구의 경우 반환된 비디오를 검토하고 동의없이 비디오에 누군가가 나타나는지 확인하십시오. 이 경우 동의서를 보내거나 동영상에 등장하는 개인에게 전화하여 동영상 사용에 대한 동의를 얻습니다. 개인이 도달 할 수없는 경우, 그들이 나타나는 비디오의 부분은 연구원에 의해 삭제됩니다.</li>
</ol>

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F., Wang, R. H., Zariffa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalizability+of+Hand-Object+Interaction+Detection+in+Egocentric+Video+across+Populations+with+Hand+Impairment.">Generalizability of Hand-Object Interaction Detection in Egocentric Video across Populations with Hand Impairment.</a> 2020 42nd Annual International Conference of the IEEE Engineering in Medicine &amp; Biology Society (EMBC). , 3228-3231 (2020).</li><li>Bandini, A., Dousty, M., Zariffa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+wearable+vision-based+system+for+detecting+hand-object+interactions+in+individuals+with+cervical+spinal+cord+injury:+First+results+in+the+home+environment.">A wearable vision-based system for detecting hand-object interactions in individuals with cervical spinal cord injury: First results in the home environment.</a> 2020 42nd Annual International Conference of the IEEE Engineering in Medicine &amp; Biology Society (EMBC). , 2159-2162 (2020).</li><li>Dousty, M., Zariffa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+Clustering+Hand+Grasps+of+Individuals+with+Spinal+Cord+Injury+in+Egocentric+Video.">Towards Clustering Hand Grasps of Individuals with Spinal Cord Injury in Egocentric Video.</a> 2020 42nd Annual International Conference of the IEEE Engineering in Medicine &amp; Biology Society (EMBC). , 2151-2154 (2020).</li><li>Bandini, A., Zariffa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+hands+in+egocentric+vision:+A+survey.">Analysis of the hands in egocentric vision: A survey.</a> IEEE Transactions on Pattern Analysis and Machine Intelligence. , (2020).</li><li>Likitlersuang, J., Sumitro, E. R., Theventhiran, P., Kalsi-Ryan, S., Zariffa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Views+of+individuals+with+spinal+cord+injury+on+the+use+of+wearable+cameras+to+monitor+upper+limb+function+in+the+home+and+community.">Views of individuals with spinal cord injury on the use of wearable cameras to monitor upper limb function in the home and community.</a> Journal of Spinal Cord Medicine. 40 (6), 706-714 (2017).</li></ol>

저자는 공개 할 것이 없습니다.

우리는 cSCI 및 치기와 같은 상반신 장애가있는 개인의 웨어러블 카메라를 사용하여 집에서 ADL 비디오를 녹화하기위한 프로토콜을 발표했습니다. 이 프로토콜은 유연하며 특정 ADL에서 손 기능 성능을 캡처하거나 집에 거주하는 사람들의 원격으로 재활 진행 상황을 추적하는 것을 목표로 지시할 수 있습니다. 자기 중심의 비전 패러다임은 지역 사회에 살고있는 개인의 손 기능을 원격 모니터링하?...

손 기능은 상지 장애를 가진 임상 인구에 걸쳐 독립성과 삶의 질을 결정합니다1,2. 집에서 손 장애를 가진 개인의 손 기능을 캡처하는 것은 재활 중 및 후 일상 생활 (ADL)의 활동을 수행 할 수있는 능력의 진행 상황을 평가하는 데 필수적입니다. 대부분의 임상손 기능 평가는 가정3,4보다는임상 또는 실험실 환경에서 수행된다. 가정에서 ADL에 미치는 영향을 포착하고자 하는 기존 임상 손 기능 평가는 설문지이며 주관적인 자체 보고 등급5,6,7에의존한다. 재활이 가정에서 손 기능에 미치는 궁극적인 영향을 평가하는 객관적인 평가는 여전히 사용할 수 없습니다.
최근 몇 년 동안, 실제 환경에서 상지 기능을 캡처하기 위해 많은 웨어러블 기술이 개발및 구현되었습니다. 가속도계 및 관성 측정 장치(ImUS)와 같은 웨어러블 센서는 일상 생활에서 상지 의 움직임을 측정하는 데 일반적으로 사용되어 왔습니다. 그러나, 이들 장치는 전형적으로 검출된 시대가 기능상 상반두운동8,9에속하는지, 원하는 작업을 완료하기 위한 의도적인 움직임으로 정의되는지 여부를 구별하지 않는다. 예를 들어, 일부 웨어러블 센서는 보행 중 팔다리 의 존재에 민감하며, 이는 상지의 기능적 움직임이 아닙니다. 또한 손목착용 가속도계는 팔다리 의 상부 움직임을 포착하지만 실제 환경에서는 손 기능의 세부 사항을 캡처할 수 없습니다. 감각 장갑은 손 조작에 대한 자세한 정보를 캡처 할 수있습니다 10,하지만 손 기능과 감각이 이미 손상된 사람들에게 는 성가신 수 있습니다. 웨어러블 접근법은 또한 자기 측정또는 손가락 마모 가속도계11,12,13을통해 손가락 움직임을 포착하기 위해 제안되었지만, 이러한 움직임의 기능적 해석은 여전히 도전적이다14. 따라서 이전에 제안된 웨어러블 장치는 작고 사용하기 편리하지만 손 사용의 세부 사항 및 기능적 맥락을 설명하기에는 충분하지 않습니다.
웨어러블 카메라는 이러한 간격을 채우고 신경 재활 응용 프로그램15,16,17,18,19에대한 가정에서 ADLs 동안 손 기능의 세부 사항을 캡처하기 위해 제안되었습니다. 컴퓨터 비전을 사용하여 자기 중심 비디오를 자동화하여 손 자체와 실제 ADL20에서수행 된 작업에 대한 정보를 제공함으로써 컨텍스트에서 손 기능을 정량화 할 수있는 상당한 잠재력을 가지고 있습니다. 한편, 연속 기록의 지속 시간은 전형적으로 배터리, 저장 및 편안함 고려 사항에 의해 약 1~1.5h로 제한됩니다. 여기서 이러한 제약 조건 내에서 는 개인의 일상 생활을 대표하는 데이터와 손 기능 평가를 위한 유익한 데이터를 얻기 위한 자기 중심적인 비디오 수집 프로토콜을 제시합니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Egocentric camera</td>
			<td>GoPro Inc., CA, USA</td>
			<td>GoPro Hero 4 and 5</td>
			<td>A camera that records from a first-person angle.</td>
		</tr>
		<tr>
			<td>Battery chager and batteries</td>
			<td>GoPro Inc., CA, USA</td>
			<td>MAX Dual Battery Charger + Battery</td>
			<td>Extra batteries for the camera and battery charger</td>
		</tr>
		<tr>
			<td>Camera charger</td>
			<td>GoPro Inc., CA, USA</td>
			<td>Supercharger</td>
			<td>This charger is connected to the camera directly without disassembling the camera frame.</td>
		</tr>
		<tr>
			<td>Camera frame</td>
			<td>GoPro Inc., CA, USA</td>
			<td>The Frame</td>
			<td>The hinge of the camera frame can be used to adjust the angle of view of the camera.</td>
		</tr>
		<tr>
			<td>Headband for the camera</td>
			<td>GoPro Inc., CA, USA</td>
			<td>Head Strap + QuickClip</td>
			<td></td>
		</tr>
		<tr>
			<td>SD card</td>
			<td>SanDisk, CA, USA</td>
			<td>32GB microSD</td>
			<td></td>
		</tr>
		<tr>
			<td>Tablet</td>
			<td>ASUSTeK Computer Inc., Taiwan</td>
			<td>ZenPad 8.0 Z380M</td>
			<td>The tablet is installed with the GoPro App in order to connect with the camera.</td>
		</tr>
	</tbody>
</table>

capturing representative hand use at home using egocentric video in individuals with upper limb impairment

신경 손상 후 손상 된 손 기능 독립성과 삶의 질에 큰 영향을 미칠 수 있습니다. 대부분의 기존 상반신 평가는 직접 수행되며, 이는 항상 지역 사회에서 손 사용을 나타내는 것은 아닙니다. 재활 개입의 진정한 영향을 측정하기 위해서는 일상 생활에서 손 기능을 포착하기 위한 새로운 접근법이 필요합니다. 자동화된 분석을 위한 컴퓨터 비전과 결합된 자기 중심 비디오는 가정에서 손 사용을 평가하기 위해 제안되었습니다. 그러나 연속 녹음 기간에는 제한이 있습니다. 획득한 동영상이 참가자의 프라이버시를 존중하면서 일상을 대표하도록 설계된 프로토콜을 제시합니다. 
대표적인 기록 일정은 연구자와 참가자 간의 협업 프로세스를 통해 선택되어 비디오가 자연스러운 작업과 성능을 포착할 수 있도록 하는 동시에 손 평가에 유용합니다. 장비 및 절차의 사용은 참가자에게 입증됩니다. 총 3h의 비디오 녹화가 2주 동안 예정되어 있습니다. 개인 정보 보호 문제를 줄이기 위해 참가자는 녹화를 시작하고 중지할 수 있는 모든 권한을 가지며, 이를 연구팀으로 되돌리기 전에 동영상을 편집할 수 있습니다. 미리 알림이 제공되며 필요한 경우 전화 및 가정 방문을 도와줍니다. 
이 프로토콜은 9명의 뇌졸중 생존자와 자궁 경부 척수 손상을 입은 14명의 개인으로 테스트되었습니다. 입수한 동영상에는 식사 준비, 식기 세척, 뜨개질 등 다양한 활동이 포함되어 있습니다. 평균 3.11 ± 0.98 h의 비디오가 입수되었습니다. 기록 기간은 질병이나 예기치 않은 사건으로 인해 12-69 d에서 다양합니다. 23명 중 22명이 데이터를 성공적으로 입수했으며, 6명의 참가자는 가정 기록 기간 동안 조사관의 도움을 받아야 합니다. 이 프로토콜은 신경 학적 부상 후 집에서 손 기능에 대한 귀중한 정보가 포함 된 비디오를 수집하는 데 효과적이었다.

참가자 인구 통계 및 포함 기준 이 연구 결과를 위해 23명의 참가자의 견본은 모집되었습니다: 9 치기 생존자 (6 남자, 3 여자) 및 cSCI를 가진 14명의 개별 (12 남자, 2 여자). 모집된 표본에 대한 요약 인구 통계 학적 및 임상 정보는 표 1에보고된다.
<table border="1" fo:keep-together.within-page="always"><tbody>
<tr>
<td rowspan="2"></td>
			<td rowspan="2...

Watch this Scientific Journal Video about Capturing Representative Hand Use at Home Using Egocentric Video in Individuals with Upper Limb Impairment at JoVE.com

Capturing Representative Hand Use at Home Using Egocentric Video in Individuals with Upper Limb Impairment

프로토콜은 자기 중심의 카메라를 사용하여 일상 생활 중에 손 장애가있는 개인의 자연스러운 손 기능을 캡처하는 것이 좋습니다. 프로토콜의 목표는 집에서 일상 생활의 활동 중에 녹음이 개인의 전형적인 손 사용을 대표하는지 확인하는 것입니다.

팔다리 장애가 있는 개인의 자기 중심 비디오를 사용하여 집에서 대표손 사용 캡처

Impaired hand function after neurological injuries can have a major impact on independence and quality of life. Most existing upper limb assessments are ...

capturing-representative-hand-use-at-home-using-egocentric-video

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

2.5K 조회수.  University of Toronto. 프로토콜은 자기 중심의 카메라를 사용하여 일상 생활 중에 손 장애가있는 개인의 자연스러운 손 기능을 캡처하는 것이 좋습니다. 프로토콜의 목표는 집에서 일상 생활의 활동 중에 녹음이 개인의 전형적인 손 사용을 대표하는지 확인하는 것입니다.

비디오: Capturing Representative Hand Use at Home Using Egocentric Video in Individuals with Upper Limb Impairment

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

멀티 채널 Microfluidic 장치에서 분리 비즈와 세포 Dielectrophoresis와 층류를 사용하여

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

연구

생체공학

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

비디오 - 속도 검색 공촛점 현미경과 Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to investigate mechanisms governing cell adhesion under dynamic flow conditions. For instance, cancer cells labeled with multiple fluorophores can be perfused over a potentially reactive substrate to model mechanisms of cancer metastasis. However, multi-channel single camera systems and color cameras exhibit shortcomings in image acquisition for real-time live cell analysis. To overcome these limitations, we used a dual camera emission splitting system to simultaneously capture real-time image sequences of fluorescently labeled cells in the flow chamber. Dual camera emission splitting systems filter defined wavelength ranges into two monochrome CCD cameras, thereby simultaneously capturing two spatially identical but fluorophore-specific images. Subsequently, psuedocolored one-channel images are combined into a single real-time merged sequence that can reveal multiple target molecules on cells moving rapidly across a region of interest.

Dual camera emission splitting systems for two-color fluorescence microscopy generate real-time image sequences with exceptional optical and temporal resolution, a requirement of certain live cell assays including parallel plate flow chamber adhesion assays. When software is employed to merge images from simultaneously acquired emission channels, pseudocolored image sequences are produced.

동시에 듀얼 카메라 방출 분할 시스템을 사용하여 두 개의 배출 채널의 실시간 이미지를 캡처 : 응용 프로그램은 접착 셀에

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

전체 피부 결함 모델은 생체의 혈관 신생을 평가하기<em&gt; 생체</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

소형 동물 모델에서의 동정맥 (AV) 루프는 혈관 신생과 혈관을 조성한 조직 공학을 공부하기

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

물 정화를위한 막으로 방오 특성을 가진 히드로 겔의 합성

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

세포질 기계 화학 Epigenetic 한정자를 리디렉션하여 억압 유전자 전사

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

물 유지와 울트라입니다 미세 칩을 사용 하 여 라이브 꼬마 선 충 의 동원 정지

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Home-Based Monitor for Gait and Activity Analysis

Current outcomes in neuromuscular disorder clinical trials include motor function scales, timed tests, and strength measures performed by trained clinical evaluators. These measures are slightly subjective and are performed during a visit to a clinic or hospital and constitute therefore a point assessment. Point assessments can be influenced by daily patient condition or factors such as fatigue, motivation, and intercurrent illness. To enable home-based monitoring of gait and activity, a wearable magneto-inertial sensor (WMIS) has been developed. This device is a movement monitor composed of two very light watch-like sensors and a docking station. Each sensor contains a tri-axial accelerometer, gyroscope, magnetometer, and a barometer that record linear acceleration, angular velocity, the magnetic field of the movement in all directions, and barometric altitude, respectively. The sensors can be worn on the wrist, ankle, or wheelchair to record the subject&#8217;s movements during the day. The docking station enables data uploading and recharging of sensor batteries during the night. Data are analyzed using proprietary algorithms to compute parameters representative of the type and intensity of the performed movement. This WMIS can record a set of digital biomarkers, including cumulative variables, such as total number of meters walked, and descriptive gait variables, such as the percentage of the most rapid or longest stride that represents the top performance of patient over a predefined period of time.

This innovative device uses magneto-inertial sensors to permit gait and activity analysis in uncontrolled environments. Currently in the qualification process as an outcome measure in the European Medical Agency, one of the applications will be to serve as a clinical endpoint in clinical trials in neuromuscular diseases.

걸음걸이 및 활동 분석을 위한 홈 기반 모니터

Current outcomes in neuromuscular disorder clinical trials include motor function scales, timed tests, and strength measures performed by trained clinical ...

Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate structures&#160;while processing and histological observation provide limited information through cross-sectional imaging. Micro X-ray computed tomography (microCT) is an effective tool for obtaining three-dimensional information without damaging specimens. However, it shows limited efficiency in differentiating soft tissue parts. Use of contrast-enhancing agents, like phosphotungstic acid (PTA), can solve this problem by improving soft tissue contrast. We implemented microCT with PTA to investigate the human orbicularis retaining ligament (ORL), which is a delicate structure in the orbit area. In this method, harvested specimens are fixed in formalin, dehydrated in serial ethanol solutions, and stained with a PTA solution. After staining, microCT scanning, 3D reconstruction, and analysis are performed. Skin, ligaments, and muscles can be clearly visualized using this method. The specimen size and duration of staining are essential features of the method. The suitable specimen thickness was about 5&#8211;7 mm, above which the process was slowed, and the optimum duration was 5&#8211;7 days, below which an empty hole in the central area occasionally occurred. To maintain the location and direction of small pieces during cutting, sewing on the same region of each part is recommended. Furthermore, preliminary analyses of the anatomical structure are needed to correctly identify each piece. Parafilm can be used to prevent drying, but care should be taken to prevent specimen distortion. Our multidirectional observation showed that the ORL is composed of a multilayered meshwork of continuous plates, rather than thread-like fibers, as reported previously. These results suggest that microCT scanning with PTA is useful for examining specific compartments within complex structures of human tissue. It may be helpful in the analyses of cancer tissues, nerve tissues, and various organs, like the heart and liver.

Micro X-ray computed tomography is effective in obtaining three-dimensional information from undamaged human specimens but has limited success in observing soft tissues. The use of phosphotungstic acid contrast agent can resolve this issue. We implemented this contrast agent to examine human delicate fibromuscular tissues (the orbicularis retaining ligament).

인간 섬유근육 조직을 시각화하기 위해 인산화산 제제를 사용한 마이크로 X선 컴퓨터 단층 촬영 사용

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate ...