이 작품은 호주 연구위원회 링키지 인프라 장비 및 시설 그랜트 LEO668421 및 호주 연구위원회 링키지 프로젝트 부여 LP0669471에 의해 지원되었다. 저자는 기꺼이 KIT - 맥쿼리 브레인 연구소를 설립에 기술과 요코 전기 주식 회사의 가나자와 연구소의 협력을 인정합니다.

이 지침은 장비와 magnetoencephalography (MEG)를 사용하여 유아의 두뇌인지 기능을 측정하기위한 절차를 설명합니다. 첫째, 우리는 어린이인지 기능의 실험 테스트를 들고 때 고려해야 할 일반적인 지침과해야 문제를 토론합니다. 둘째, 우리는 4 년 된 아이들의 동기 부여와 행동 용량 MEG 데이터 수집의 요구 사항을 정렬하기 위해 개발된 프로토콜을 설명합니다. 1. MEG와 어린 자녀를 공부에 대한 일반적인 고려 사항 깨어 건강한 아이와 함께 작업할 때 메그 연구자들은 독특한 도전의 숫자를 얼굴. 먼저, 스탁 및 기능 실습 환경은 어린 아이들을 위협하거나 놀라게 할 가능성이 높습니다. 둘째, 실험 MEG 연구의 기본 요건은 운동의 제한을 포함하고 적극적으로 많은 성인들이 지루하고 불편 발견되는, 장기간 작업 조건을 참석. 자녀를 공부하면 그것은 자녀의 능력과 한계에 대한 실험 절차를 적응하기 위해 필수적됩니다. 이 섹션에서는, 우리는 데이터 수집 세션에 참여하기 위해 주제를 준비에 관련된 초기 단계를 설명합니다. 1.1 친하게 함 / 교육 세션 : 유치원 어린이들을위한 친하게 함 / 교육 세션 전에 실제 데이터 수집 세션 예정입니다. 이 세션은 아이가 자신의 속도로 환경 연구원에 익숙해질 수 있습니다. 우리는 연구자에 아이와 부모를 소개하고 실습 환경 및 루틴으로 그들을 숙지. 소개 세션이 끝날으로 부모와 자녀가 참여에 관련된 모든 단계를 이해하고 그들이 데이터를 기록하는 동안 기대해야하는지 알고 있습니다. 처음 방문하는 동안, 아이가 수줍음이나 불안 나타나는 경우들이 더 편안하게 때까지, 우리는 놀이의 기간이 있습니다. 그렇다면, 우리는 모든 단계의 도입과 함께 진행합니다. 환경 설명하기 1.2 : 자녀에게 MEG 환경을 설명하려면, 우리는 주변 환경에 대한 어린이 친화적인 정당화를 제공하는 테마를 사용합니다. 예를 들어, MEG 시스템은 일부 생소한 이벤트가 발생할 수 있습니다 우주 모험에 아이들을 데리고 기다리고 우주선이다. 실험실은 또한 벽 스티커, 장난감 (그림 1)으로 장식하고 있으며, 그것은 전용 놀이방이 있습니다. 자녀에게 이야기하면, 우리는 어린이들이 쉽게 이러한 대신 &#39;마커 코일 모자&#39;대신 &#39;최소화 운동&#39;의 &#39;동결&#39;과 &#39;우주 비행사 헬멧&#39;으로 이해할 수있는 그 언어를 사용합니다. 1.3 아이와 부모에게 절차를 설명 : 우리는 아이와 부모 자녀가 자기 (磁气) 차폐 실 (MSR) 내부되기 전에 촬영하는 단계를 보여줍니다. 첫 번째 단계 중 하나는 제목의 머리에 다섯 마커 - 코일 모자를 맞게되며 이러한 코일은 MEG 헬멧 안에 주제의 머리의 위치를​​ 나타냅니다. 아이의 도움으로, 우리는 디지털 인형의 머리 모양, 그리고 우리는 아동과 MSR / 우주선에 인형을 동반 부모를 초대합니다. 일단 MSR 안에서, 우리는 영화를 감상하기 위해 헬멧 내부에 인형의 머리를 놓으십시오. 그들은 인형과 함께 영화를보고 싶은 경우 다음 아이를 부탁드립니다. 아이가 편안하고 편안하고 부모가 우리의 프로토콜에 만족하는 경우, 우리는 그들이 우주선 안에서 게임을하고 그들이 우리 실험실을 방문 다음 시간을 초대합니다. 1.4 통합 문서 : 우리 아이가 테스트 세션 동안 걸립니다 단계를 보여주는 공간 모험 테마를 기반으로 통합 문서를했습니다. 아동은 가정을 가지고 다음 방문에 대비하여 자신의 부모와 그들을 통해 작동하도록 이러한 worksbooks 제공됩니다. 통합 문서의 목적은 테스트를 위해 세 가지 주요 요구 사항의 중요성을 강조하는 것입니다 :) MSR 안에 어떤 철재료의 소지를 없애기, B) 정확한 데이터를 공동 등록 및 C를 보장하기 위해 마커 코일 모자의 배치)과 디지털화 및 데이터 수집 중에 여전히 남아의 중요성. 2. 예비 학교 세 어린이 MEG 신호를 측정 프로토콜 데이터 수집 및 실험 작업을하는 동안 사용되는 2.1 장비 마커 - 코일 총액 : 제목의 구조 정보 (예 : MRI 또는 디지털 머리 모양) 다섯 코일의 집합 MEG 데이터를 정렬하기 위해서는 자녀가 MEG 헬멧 안에 착용하는 수영 모자에 배치됩니다. 이러한 코일은 기준점 역할과 위치는 제목의 머리가 인수하는 동안 이동이 얼마나 측정하는 작업 전후 비교됩니다. 디지타이저 : 데이터 수집하기 전에 다섯 마커 코일과 피사체의 머리 모양의 위치가 Polhemus의 F를 사용하여 디지털이다astrak ® 디지타이저 (콜체스터, VT). 디지타이저는 펜 디지타이저 세 수신기, 송신기 및 모션 추적 장치로 구성되어 있습니다. 이 프로세스는 코일의 위치를​​ 측정합니다. 나중에 해당 위치는 취득한 자기 데이터와 머리 모양을 정렬하는 MEG에 의해 얻은 위치 정보에 대해 일치하고 있습니다. 어린이 MEG 시스템 : 데이터 수집이 전체 헤드 아이 MEG 시스템 (KIT, 카나자와, 일본)를 사용하여 수행됩니다 [2]. 데이터는 0.03 Hz에서와 200Hz 사이의 필터 온라인 밴드 패스와 1000Hz의 샘플링 레이트에서 연속적으로 취득됩니다. 마커 코일 위치는 머리의 움직임을 측정하는 음반 이전과 이후를 얻을 수있다. 머리의 움직임은 5mm보다 큰 경우에는 데이터가 거부됩니다. MEG 시스템 주변 장치 : 실험 자극은 프레 젠 테이션을 사용하여 PC와 제어 ® (Neurobehavioral 시스템, 알바니, CA). 영상 입력은 MSR 밖에 앉아 및 MSR 화면에 반영하는 프로젝터로 전달됩니다. 청각 자극이 Etymotic 연구 모델로 전달됩니다 ER - 30 아기 크기의 거품 earpieces (Etymotic 연구 주식 회사, 엘크 그로브 빌리지, IL)가 장착되어 삽입 이어폰. 아이가 지속적으로 폐쇄 회로 카메라에 의해 감시와 실험자가 마이크를 통해 자녀와 통신합니다. 2.2 데이터 수집 : 데이터 레코딩 세션은 다음 단계를 포함 : I를 단계 : 전에 자녀의 도착 그것은 모든 컴퓨터, 소프트웨어 및 장비 마자 아이가 도착하는대로 실험을 시작할 준비를하는 것이 중요합니다. 아이와 부모가 실험실에있는 동안 일반적인 추천으로, 두 경험이 연구자들은 존재한다. 연구원, 부모와 자녀 사이에 명확한 커뮤니케이 션이 실험의 성공을 위해 매우 중요합니다. 단계 II : 아이가 도착한 후 <ol><li> 동의 양식에 서명하고 부모가 질문이 있으시면 경험이 있는지 확인합니다. </li><li> 아이가 첫 회의 동안이나 책자에 소개된 모든 단계를 기억하고 기억하지 않는 어떤 reintroduces 경우 실험자가 확인합니다. 디지털화 및 데이터 수집 동안 운동을 동결하거나 최소화의 중요성이 강조됩니다. 이것은 인형을 사용하여 시연 수 있습니다. </li><li> 아이가 (번쩍 이는 패치 의류, 보석, 주머니 안에서 잊혀진 장난감, 머리 핀 등) 어떤 철재료를 가지고있다면 그 다음, 실험자가 확인합니다. 그들이 철재료를 포함 의복의 부분을 가지고있다면, 우리는 MEG 안전 복장으로 그들을 제공합니다. 부모가 실험 중에 자녀를 동반하고자한다면, 우리는 그들이 또한 철재료를 제거되었는지 확인합니다. </li></ol> 단계 III : MSR 내부 작업을 수행할 수있는 아이가 준비하기 <ol><li> 실험자들은 5 마커 코일을 보유하고 뚜껑을 맞는 데요. </li><li> 실험자의 마커 코일의 위치와 아이의 머리 모양을 디지털화. 고정된 위치에 헤드를 유지하기 위해, 우리는 세 수신기가 장착되어되었습니다 목 괄호를 사용합니다. 정기적으로 좋은 머리 모양을 얻기 위해 운동 또는 &#39;동결&#39;을 최소화의 개념을 강화하는 것이 중요합니다. 그 아이는 &#39;동결&#39;에서 좋은 일을위한 스티커로 보상을받을 수 있습니다. </li><li> 그 아이는 MSR를 입력 초대합니다. 이어폰이 삽입되고 아이가 위로 향한 위치에 MEG 듀어 헬멧에 위치합니다. </li><li> 아이가 MEG 헬멧 내부의 머리를 가진 편안하게 누워되면, 마커 코일들은 전원 장치에 연결되고 MSR 도어가 닫힙니다. 한 실험자는 전체 세션 동안 방에있는 (그리고 원하는 경우에도 부모) 남아 있습니다. </li><li> 설치하는 동안 데이터 수집 블록 사이 엔터테인먼트, 영화나 만화는 MSR 다시 프로젝션 스크린에 투영 수 있습니다. </li></ol> 단계 IV : 실험 실행 <ol><li> MSR 도어가 닫혀되면, 오징어는 약혼 및 데이터 수집이 시작됩니다. </li><li> 어린이 영화 coregistration의 사전 시험 MEG 신호가 디지털 헤드 모양 coregistered 수 있는지 확인하기 위해 실시됩니다 시계로. 이 pretest은 MEG 시스템 표시된 위치와 디지털 동안 얻은 마커 코일의 위치를​​ 일치로 구성되어 있습니다. </li><li> 아이의 머리가 제대로 헬멧 내부에 위치되는 첫 번째 테스트 프로그램되면, 연구자는 실험을 시작할 수 있습니다 : (A) 영화가 음소거되어, 그들은 &quot;게임을&quot;준비하는 경우 (B) 연구원이 아이를 묻습니다; 게임이 시작되면 (C), 실험자 정기적으로 피드백과 어린이를 제공합니다. 예를 들어, 아이가 문장의 연속을 듣고, 그리고 바로 그들이 소리로 &#39;치즈&#39;같은 목표 단어를 반복하라는 요청을받을 수 있습니다. 이러한 대상 단어 잡기 트라이의 하위 집합에 포함된탐사선이 작업에 배려를 계속하지만 ALS가 최종 분석에 포함되지 않습니다. </li><li> 작업이 완료되면, 다른 마커 코일 측정은 머리 위치와 움직임을 모니터하는 수행됩니다. </li><li> 오징어가 해지되면, MSR 도어가 열립니다와 자녀들은 좋은 작품을위한 상품을 선택할 수있는 놀이방에 초대합니다. </li></ol> 3. 대표 결과 저희 연구실의 대표적인 연구는 아동 언어 논리의 개발 연구이 프로토콜을 사용합니다. 3-4년 세 스물 여섯 어린이 (4 세 세 5개월을 의미)은 적어도 하나의 인수 세션에 참여했습니다. 그 아이들의 17 두 번째 테스트 세션에 반환 다섯 세 번째 테스트 세션에 왔어요. 평균 머리의 움직임은 잘 5mm 임계값 내에서 미만 3.7 mm (SD 3.1 mm)였다. 5mm보다 큰 움직임 유사 5.5 mm에서 15mm (= 8.6mm, SD = 2.5 mm를 평균)까지로, 세션의 4.5 %에서 발생했습니다. <img src="/files/ftp_upload/1693/1693fig1.jpg" alt="그림 1" /> 그림은 1. MSR과 MEG 투구는 스티커와 부드러운 장난감을 장식하고 있습니다. <img src="/files/ftp_upload/1693/1693fig2.jpg" alt="그림 2" /> 그림 2. 성인 크기의 MEG (왼쪽)에서 네 살짜리 소녀. 움직임과 머리와 헬멧 사이의 거리를 최소화하기 위해 그녀의 머리의 상단에있는 패딩을 확인합니다. 오른쪽 그림은 아이 MEG에서 같은 소녀를 보여줍니다.

<ol>
	<li>H&#228;m&#228;l&#228;inen, M., Hari, R., Ilmoniemi, R. J., Knuutila, J., Lounasmaa, O. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetoencephalography-+theory,+instrumentation,+and+applications+to+noninvasive+studies+of+the+working+human+brain.">Magnetoencephalography- theory, instrumentation, and applications to noninvasive studies of the working human brain.</a> Rev. Mod. Physics. 65 (2), 413-497 (1993).</li><li>Johnson, B. W., Crain, S., Thornton, R., Tesan, G., Reid, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+brain+function+in+pre-school+children+using+a+custom+sized+whole-head+MEG+sensor+array.">Measurement of brain function in pre-school children using a custom sized whole-head MEG sensor array.</a> Clin. Neurophysiol. , (2009).</li><li>Kado, H., Higuchi, M., Shimogawara, M., Haruta, Y., Adachi, Y., Kawai, J., Ogata, H., Uehara, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetoencephalogram+system+developed+at+KIT.">Magnetoencephalogram system developed at KIT.</a> IEEE Trans. Appl. Supercond. 9, 4057-4062 (1999).</li><li>Uehara, G., Adachi, Y., Kawai, J., Shimogawara, M., Higuchi, M., Haruta, Y., Ogata, H., Kado, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-Channel+SQUID+Systems+for+Biomagnetic+Measurement.">Multi-Channel SQUID Systems for Biomagnetic Measurement.</a> IEICE Trans. Electr. E86-C, 43-54 (2003).</li></ol>

이 프로토콜은 건강한 정신 사년 된 아이인지 뇌 기능의 측정을 용이하게하기 위해 설계되었습니다. 우리는 사전에 학교 세 어린이의 머리에 맞게 설계된 사용자 정의 크기 MEG 시스템을 사용합니다. 이 시스템을 통해 어린이 친화적인 실험실 환경 및 능력과 아동의 동기에 적응 프로토콜, 우리는 아주 작은 데이터가 현재가되는 연령 범위에서인지 뇌 기능을 측정 할 수 있습니다. KIT - 맥쿼리 시스템은 사전에 학교 세 아이들과 함께 사용하기 위해 크기의 첫 번째 전체 헤드 시스템을 사용자 정의합니다. 자녀를 공부에 대한 사용자 정의 헬멧 듀어의 세 가지 주요 장점이 있습니다. 첫째, 센서 구성의 작은 반경 아이의 머리 neuromagnetic 신호의 범위에 오징어 센서를 제공합니다. 둘째, 작은 헬멧은 듀어에 아이의 머리의 전체 삽입하실 수 있습니다. 전체 삽입 때문에 (그림을 참조하십시오. 2) 어린이에 거리를 어깨에 작은 왕관의 성인 듀어 헬멧에 방지합니다. neuromagnetic 신호가 거리로 급속히 감쇠하기 때문에이 두 요소 MEG를 사용하여 두뇌 활동을 기록에 기초하고 있습니다. 셋째, 맞춤형 아동 헬멧 머리의 대칭 위치에 에이즈 및 듀어 내에서 아이의 머리 움직임의 자유를 제한합니다. 이러한 기능은 크게 설정, 포지셔닝 및 어린이 MEG 신호의 측정을 용이하게합니다. 사용자 정의 크기의 자녀 MEG 시스템의 출현은인지 신경 과학의 중요한 발전이다. 방사성 물질이나 강한 자기장을 고용 대체 기능 neuroimaging 기술은 어린 아이와 함께 일상에서 사용하기에 적합하지 않을 수 있습니다. MEG가 완전히 수동적 측정 기법이기 때문에 조직을 개발하기 위해 더 생각할 수있는 위험이 없습니다. 기존의 성인 MEG 시스템은 어린이와 함께 사용하기 위해 적합하지 않기 때문에 건강한 취학전 세 어린이의 아주 작은 MEG 데이터는 현재 있습니다. 어린이 MEG 시스템은 우리가인지 기능의 정상 및 병적인 개발에 새로운 통찰력을 제공 뇌 활동의 극적인 구조가 발생 연령에서에서 뇌의 기능을 연구하실 수 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>Whole head child MEG</td>
<td>&nbsp;</td>
<td>Kanazawa Institute of Technology (Kanazawa) and Yokogawa Electric Corporation (Tokyo)</td>
<td>Model PQ1064R-N2m</td>
<td>&nbsp;</td>
<tr>
<td>Magnetically shielded room</td>
<td>&nbsp;</td>
<td>Fujihara Co. Ltd., (Tokyo)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Digitiser</td>
<td>&nbsp;</td>
<td>Polhemus (Colchester, VT)</td>
<td>Fastrack</td>
<td>&nbsp;</td>
<tr>
<td>Experimental control software</td>
<td>&nbsp;</td>
<td>Neurobehavioral Systems (Albany, CA)</td>
<td>Presentation</td>
<td>&nbsp;</td>
<tr>
<td>Earphones</td>
<td>&nbsp;</td>
<td>Etymotic Research Inc. (Elk Grove Village, IL)</td>
<td>ER-30</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

measurement of neuromagnetic brain function in pre-school children with custom sized meg

Magnetoencephalography 대뇌 피질의 활동 [1]와 관련된 자기장을 감지하는 기술입니다. 두뇌의 electrophysiological 활동은 전기 필드를 생성 - 그들 수반하는 자기장 - - electroencephalography을 (EEG)를 사용하여 기록 수 MEG에 의해 감지. MEG 신호를 양자 간섭 장치 (오징어)을 초전도로 알려진 특수 센서에 의해 감지됩니다. 초전도 센서는 -270에서 액체 헬륨과 냉각 요구 ° C. 그들은 vacumm - 절연 헬멧 액체로 가득입니다 듀어라는 안에 포함되어 있습니다. 오징어는 헬륨 냉각수의 헬멧 듀어 내부 고정 위치에 배치하고 있으며, 주제의 머리는 MEG 측정을위한 헬멧 듀어 내부에 배치됩니다. 헬멧 듀어는 반대 제약을 모두 만족하는 크기 여야합니다. 확실히, 그것은 대부분 또는 모두 공부하게 될 인구의 머리에 맞게 충분히 크게해야합니다. 그러나 헬멧은 또한 그들이 측정되는 작은 대뇌 분야의 범위 내에서 오징어 센서의 대부분을 유지하기 위해 충분히 작은되어야합니다. 기존의 전체 헤드 MEG 시스템은 성인 머리의 90 % 이상을 수용할 수 있도록 설계되었습니다. 그러나 어른이 시스템은 잘 그의 머리가 성인보다 작은 반경 몇 cm를 미리 학교 chidren에서 뇌 기능을 측정하기에 적합하지 않습니다. 맥쿼리 대학교 KIT - 맥쿼리 브레인 연구소는 사전에 학교 아이들의 머리에 맞게 크기 MEG 시스템 사용자 정의를 사용합니다. 이 아이 시스템은 50mm의 기준 64 1 차 축 gradiometers있다 [2]와 일반 성인 사이즈 MEG 시스템과 함께 자기 (磁 气) - 차폐된 방 (MSR) 안에 포함되어있다 [3,4]. 자녀를 공부에 대한 사용자 정의 헬멧 듀어의 세 가지 주요 장점이 있습니다. 첫째, 센서 구성의 작은 반경 아이의 머리의 neuromagnetic 신호의 범위에 오징어 센서를 제공합니다. 둘째, 작은 헬멧은 듀어에 아이의 머리의 전체 삽입하실 수 있습니다. 전체 삽입 때문에 어린이 거리를 어깨에 작은 왕관의 성인 듀어 헬멧에 방지합니다. neuromagnetic 신호가 거리로 급속히 감쇠하기 때문에이 두 요소 MEG를 사용하여 두뇌 활동을 기록에 기초하고 있습니다. 셋째, 맞춤형 아동 헬멧 머리의 대칭 위치에 에이즈 및 듀어 내에서 아이의 머리 움직임의 자유를 제한합니다. 아이들의 동기 부여와 행동 용량 데이터 수집의 요구 사항을 일치 프로토콜과 함께 사용할 경우, 이러한 기능은 크게 설정, 포지셔닝 및 MEG 신호의 측정을 용이하게합니다.

Watch this Scientific Journal Video about Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG at JoVE.com

Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

어린이를위한 사이즈 MEG 시스템의 출현은 두뇌 개발을 연구하는 것이 중요합니다 새로운 기회를 엽니다. 함께 아이들의 용량 실험 요구 사항을 일치 프로토콜과 새로운 시스템은, 6-3 세 건강, 깨어있는 아이인지 및 언어 과정을 연구하는 데 사용할 수 있습니다.

사용자 정의 크기 MEG 사전 - 학교 어린이 Neuromagnetic 뇌 기능의 측정

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain ...

measurement-neuromagnetic-brain-function-pre-school-children-with

Research

JoVE Journal

Biology

16.8K 조회수.  Macquarie University. 어린이를위한 사이즈 MEG 시스템의 출현은 두뇌 개발을 연구하는 것이 중요합니다 새로운 기회를 엽니다. 함께 아이들의 용량 실험 요구 사항을 일치 프로토콜과 새로운 시스템은, 6-3 세 건강, 깨어있는 아이인지 및 언어 과정을 연구하는 데 사용할 수 있습니다.

비디오: Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

와이드 필드 현미경을 사용하여 장기 이미징 포유 동물 세포

연구

생물학

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 ...

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

가까운 적외선 분광학과 함께 아기, 어린이 및 성인의인지 기능을 탐색

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

CompoZr 맞춤 아연 손가락 Nucleases와 게놈 편집 (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

ECIS / 택시와 셀룰러 Chemotaxis 측정

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

Zebrafish의 유충의 근육 기능을 평가하기 위해 수축하는 동안 힘 측정

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

약리학 연구에 고립 된 조직 목욕-애플리케이션에서 부드러운 근육 기능의 측정

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

정제 된 야생형과 돌연변이 체 CFTR 단백질의 기능의 재구성 및 채널 활동 측정

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

샘플 준비 및 전송 전자 현미경 검사 법에 의해 Exosomes의 영상

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

비 트랜스제닉 무척추 동물의 단일 신경 해상도를 가진 네트워크 역학을 기록하기위한 포토다이오드 기반 광학 이미징

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...