The authors acknowledge Howard Berg for the gift of the bead-tracking microscope/photomultipliers and the Texas A&amp;M Engineering Experiment Station for funds.

1. 셀 준비 <ol><li> 10 mL의 신선한 TB 100 희석 : 1에 접종 한 다음 트립 톤 국물 (TB, 1 % 펩톤, 0.5 % 염화나트륨)에 대립 유전자 15, 22 끈적 끈적한 플릭를 들고 원하는 균주의 야간 문화를 성장. OD 600 = 0.5까지 진탕 배양기에서 33 ° C에서 문화를 성장. </li><li> 1,500 XG에서 세포 펠렛 5-7 분, 재 분산 펠렛 적극적으로 필터 살균 운동 버퍼의 10 ㎖의 (MB를 10 mM의 인산 완충액 : 0.05-0.06 M 염화나트륨, 10-4 M EDTA, 1 μm의 메티오닌, pH가 7.0). </li><li> 단계를 반복 1.2 두 번 더 1 ML의 MB의 마지막 펠릿을 재 분산. </li><li> (- 12cm 길이, 0.58 mm 내경 7) 폴리에틸렌 튜브로 연결된 21 ~ 23 게이지 어댑터와 함께 두 주사기 사이에 앞뒤로 ~ 75 번 전달하여 정지 전단. 45 초 - (30) 전단의 총 시간을 제한합니다. </li><li> 5-7 분 동안 1,500 XG에서 전단 된 세포를 원심 분리하여 다시 분산MB의 500 μL - 100 펠렛. </li></ol> 2. 슬라이드 준비 <ol><li> 커버 슬립 현미경 슬라이드 사이에 두 개의 양면 접착 테이프를 개재하여 촬상 챔버를 준비한다. 화성 분석을 위해, MB, 화학적 자극의 교환을 가능하게하는 미세 유체 챔버를 사용합니다. </li><li> 챔버에서 0.01 % 폴리 -L- 라이신 솔루션을 추가하고 5 분 후 부드럽게 MB와 표면 세척 (80-100 μL)를. </li><li> 챔버 내로 세포 현탁액 40 μL의 추가, 유리 표면에 부착을위한 충분한 시간이 허용 (7-8 분). 다른 측면에서 여과지로 용액을 심지하면서 상기 챔버의 일측에 100 μL를 첨가하여 MB 떨어진 세포를 흐른다. </li><li> 챔버로 라텍스 구슬 15 μL와 비즈를 정착 세포에 부착 할 충분한 시간 수 - 10 추가 (7-8 분). 구슬 떨어진 제거하는 단계 2.3에 설명 된대로 조심스럽게, MB의 100 μL 씻어. 비드 실리콘의 범위를 사용하여좋은 대비를 사용할 정도로 오랫동안 실험 ZES. </li></ol> 3. 비드 추적 <ol><li> 현미경 스테이지에 샘플을 놓고 모터에 연결된 구슬 위해 표면을 검사합니다. 위상 현미경이 필요하지 않지만 관측을 만들기 위해 40X 위상 목적을 사용합니다. 또한, 너무 오래 충분한 대비가 명확하게 어두운 배경에 밝은 구슬을 구분하기 위해 유지되는 밝은 - 필드 이미징을 사용한다. </li><li> 비드가 선택되면,도 1b에 도시 된 바와 같이 소정의 모서리에 비드를 위치 시키도록 측 방향으로 상기 스테이지를 이동. 같은 코너에 위치 구슬은 구슬의 회전 방향이 제대로 알려져 있는지 확인합니다. 이상적인 비드 궤도는 대략 원형이지만, 타원형 궤도가 허용됩니다. </li><li> 에일리어싱과 연관된 에러를 방지하기 위해 모터의 회전 주파수의 두 배 이상의 샘플링 주파수를 높게 유지. 본 연구에서는으로 회전 된 모터를 사용10 시간 이상 (500 Hz에서)했다 주파수 50 Hz의 시료는 평활 한 신호를 획득한다. </li></ol> 4. 데이터 분석 <ol><li> 23 필요한 경우 센터는 아핀 변환과 궤적의 PMT 출력 전압과 정확한 타원의 크기를 조절합니다. 회전 속도 (17)를 결정하기 위해 전력 스펙트럼 분석을 사용한다. </li><li> 극 각도 θ (t) = ATAN (Y (t) / X (t))를 결정합니다. 모터 속도의 변화를 확인하고 ω를 계산하여 시간이 지남에 따라 전환 <img alt="식 (1)" src="/files/ftp_upload/55240/55240eq1.jpg" /> 14. </li><li> 모터 속도 데이터를 부드럽게 중앙값 필터를 사용한다. 이 전체 회전을 통해 필터 창 (23, 24)를 권장합니다. </li></ol>

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The authors have nothing to disclose.

테더 비드 추적 모터 토크의 정확한 추정을 용이하게하기 위해, 다음과 같은 정보가 검토되어야한다. flagellated 세포 이러한 측정을 수행 할 때, 전단은 중요한 단계입니다. 전단시켜 상기 모터의 점성 부하가 비드에 주로 기인하고 10 %의 오차 (16)에서 추정 될 수 있도록, 단순한 스터브로 편모 필라멘트를 감소시킨다. 양털 깎기도 밀접하게 분산 기행 (&lt;비드 직경 14)와 원형 궤적을 찾는 기회를 향상시킨다. 추적 및 점성 드래그를 계산할뿐만 아니라 열악한 신호 대 잡음비의 결과에 오류를 화합물 제멋대로 궤적 부적절한 전단 결과. 오실로스코프의 사용은 이러한 데이터를 신속하게 제거 할 수 있습니다. 편모 필라멘트의 생체 역학적 특성은 종에 따라 다양 할 것으로 예상되기 때문에, 전단 방법 가능성 t에 충분한 전단력을 보장하도록 구성 될 필요그는 관심의 박테리아. 셔링과 연관된 에러를 감소시키는 효과적인 방법은 필라멘트 단백질을 인코딩하는 유전자를 결여 세포에서 작동한다. 프로브 비드 다음 방지 훅 항체 통해 고리에 직접 부착 될 수있다. 적절 어려울 수 있습니다 닿는되는 구슬을 찾기. 시야 가장 비드 어느 균체 또는 유리 표면에 부착되기 때문이다. 이러한 구슬은 쉽게 날카로운 초점을하게 될 수있다. 다른 구슬 진동 나타나거나 큰 진폭 또는 대형 기발로 눈에 띄게 회전합니다 (&gt; 1.5 - 2 배 비드 직경). 이들은 일반적으로 완전히 전단 또는 초점면에 기울어 진 평면에서 회전되지 않은 필라멘트를 편모에 닿는입니다. 이러한 구슬의 샘플링은 일반적으로 높은 잡음이 발생할 것이며, 점성 부하의 시간 변화는 주어진 비드 크기의 모터 토크의 과소 평가 될 수 있습니다. 곁에 구슬의 작은 부분은 임의 받게 될 것이다운동; 이 단지 브라운 회전을 겪고있다. 구슬의 비율은 흐리게 표시되며 쉽게 초점 상태로 만들 수 없습니다. 이 적절하게 닿는 관심있는 모터입니다 된 모터가 될 가능성이 높습니다. 여기에 설명 된 바와 같은 추적 단일 모터의 한계 중 높은 처리량 실험을 수행 할 수 없다는 것이다. 그 이미지 고속 카메라는 관심의 큰 영역이 점에서 유리할 수있다. 다른 제한은 여러 신호가의 PMT의 시야에 밀접하게 이격 된 회전 구슬 인해 발생과 관련된 오류를 포함한다. 마지막으로, 두 개의 광전자 슬릿에 대하여 기록 된 비드의 정확한 위치 결정에 오차가 스위칭 역학의 부정확 한 추정을 초래한다. 여기에 설명 된 설정의 장점은 오랜 기간 동안 실시간으로 비드의 회전을 추적 할 수있는 능력을 포함한다. 이오류가 발생하기 쉬운 궤도의 신속한 제거, 아마 어려운 초고속 카메라를 달성하기 위해 뭔가를 할 수 있습니다. 또한 약간의 수정이 설정은 다양한 자극에 세포를 환자에게 설계된 분석법으로 통합 될 수있다. 열전기 냉각 (26)이 결합 된 기술은 열 자극에 대한 개별 모터의 응답을 측정하기 위해 사용될 수있다. 최근 6 완료 한 바와 같이 광학 핀셋과의 통합, 기계적 자극에 반응하여 개별 모터의 리모델링의 측정을 활성화 할 수 있습니다. 최종적으로, 화학적 자극에 모터의 적응 적절한 관류 챔버를 이용하여 측정하고, 11 펌프 될 수있다. 알려진 박테리아 종의 대부분은 운동성이 있고 편모 매개 운동은 자연 지배적이다. 여기서 보여준 방법의 구조적 리모델링 통찰력 개발 및 적응성 O 돕기 계속 예상편모 모터 (F).

편모 모터는 나선형 외 필라멘트를 회전하여 수영을 셀 수 있습니다. 모터가 편모 (즉, 점성 부하)의 소정 기간 동안 생성 할 수있는 토크의 양이 수영 속도를 결정한다. 한편, 회전 방향을 전환 할 수있는 능력은 화학 약품, 화학 주성으로 공지 된 과정에 응답하여 세포 이동을 제어한다. 화성과 운동성되는 독성은 1-3, 편모 모터는 년 4로 잘 특성화 된 요인. 증거 확보 해주기 모터가 mechanosensor 역할을 제안 - 그것은 기계적 고체 기판 -5,6-의 존재를 검출한다. 이 기능은 가능성이 표면 식민지 및 감염 5,7 트리거링에 도움이됩니다. 결과적으로, 모터의 표면과 동수 신호를 감지하여 그에 메커니즘의 중요성 8,9이다. 편모 모터는 쉽게 flagell를 테 더링으로 공부하실 수 있습니다UM 기판에 세포 회전을 관찰. 이러한 테 더링이 먼저 안티 - 후크 항체 (10)와 유리 기판에 polyhook의 대장균에서 돌연변이 성공적으로 연결된 후크와 협력 실버 사이먼에 의해 달성되었다. 속박 세포 분석은 화학 자극의 다양한 모터 스위치의 반응을 연구 연구 할 수 있었다. 예를 들어, Segall와 동료들은 화학적으로 이온 영동 피펫의 도움으로 닿는 세포를 자극. CW 바이어스에 해당 변경 화성 네트워크 (11, 12)에서 적응의 반응 속도를 측정하기 위해 그들을 사용 (시간 모터의 비율은 CW, 시계 방향으로 회전). 닿는 세포 분석은 스위치 응답을 공부에 효과적이지만, 단지 점성로드 (13)의 제한된 범위에서 모터 역학에 대한 통찰력을 제공 할 수 있었다. 이 문제를 극복하기 위해 류와 동료들은 표면에 붙어있는 세포 스텁 필라멘트 구면 라텍스 비즈 닿는. 비드이었다다음 약한 광 트랩 (14) 백 초점 간섭을 사용하여 추적. 서로 다른 크기의 구슬과 협력함으로써, 연구진은 부하의 훨씬 넓은 범위에 걸쳐 모터를 연구 할 수있다. 이 분석법 후에 레이저 암시 야 조명과 결합 된 광전자 계 비드 추적 기술을 개발 위안 베르그 향상했다. 외부 점성 저항이 회전 (15, 16)에 내부 점성 저항에 비해 낮았다 너무 작았 닿는 골드 나노 비드 (~ 60 ㎚)의 그들의 방법을 사용 추적. 이 대장균에서 달성 가능한 최대 속도 (~ 300 Hz에서)의 측정을했다. V.의 alginolyticus에서 유사한 비드 분석은 중간 점성 부하 (~ 700 Hz에서) (17)의 회전 속도를 측정 할 수 있었다. (제로로드에서 가까운 매점에) 점성 부하의 전체 가능한 범위에서 모터 반응의 측정을 가능하게함으로써, 비드 분석은 t을 이해하는 중요한 생물 리 학적 도구를 제공orque 생성 프로세스 (18, 19). 최근에, 우리는 각각의 모터 6 정밀 기계적인 자극을 적용 할 수있게 광 핀셋을 포함하도록 위안 - 버그 분석을 수정했습니다. 그들은 점성 부하의 변화에 ​​응답하여 개조 -이 기술을 이용하여, 모터 회전 힘 발전기 동적 mechanosensors이다 것으로 나타났다. 이 메커니즘 불분명 남아 있지만 이러한 하중 감지가 진을 치고 박테리아로 세포 분화를 유발하는 것이 가능하다. 직접적인 증거는 부족하지만 다른 종의 편모 모터는,도 20 mechanosensitive 것으로도 보인다. 여기, 우리는 편모 필라멘트 (15)에 닿는 라텍스 구슬의 회전을 추적하기위한 광전자 기반 (PMT) 접근 방식에 대해 설명합니다. 이 실시간으로 긴 경질 위에 단일 비드를 추적 비교적 간단하기 때문에 초고속 카메라 추적 비교에서, 광전자 - 설치 유리한TIONS. 때문에 환경 자극 (21)에 편모 모터 단지에 리모델링 긴 시간을 공부 할 때 특히 유용합니다. 구체적으로는 E. coli에 대한 우리 상세히 프로토콜이지만, 그들은 용이하게 다른 종 편모 모터 공부하도록 할 수있다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Poly-L-lysine Solution (0.1%)</td>
			<td>Sigma-Aldrich</td>
			<td>P8920</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/p8920?lang=en&#38;region=US</td>
		</tr>
		<tr>
			<td>Polybead Microspheres</td>
			<td>Polysciences, Inc.</td>
			<td>7307</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/p8920?lang=en&#38;region=US</td>
		</tr>
		<tr>
			<td>1 ml Luer Slip Tip Syringe</td>
			<td>Exel Int.</td>
			<td>26048</td>
			<td>http://www.exelint.com/tuberculin_syringes.php</td>
		</tr>
		<tr>
			<td>Clay Adams Intramedic Luer-Stub Adapter 23-gauge</td>
			<td>Becton, Dickinson and Company</td>
			<td>427565</td>
			<td>http://www.bd.com/ds/productCenter/ES-LuerStubAdaptors.asp</td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>Harvard Apparatus</td>
			<td>59-8325</td>
			<td>http://www.harvardapparatus.com/laboratory-polye-polyethylene-non-sterile-tubing.html</td>
		</tr>
		<tr>
			<td>Photomultiplier Tubes</td>
			<td>Hamamatsu</td>
			<td>R7400U-20</td>
			<td>Spectral response range of 300 to 920 nm, Peak wavelength 630 nm,&#160; 0.78 ns response time&#160; 
			http://pdf1.alldatasheet.com/datasheet-pdf/view/212308/HAMAMATSU/R7400U-20.html</td>
		</tr>
		<tr>
			<td>3x1 mm precision slits</td>
			<td>Edmund Optics</td>
			<td>NT39-908</td>
			<td>2 slits mounted at right angles to one another on photomultiplier tubes</td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>Tektronix</td>
			<td>TBS 1032B</td>
			<td>Alternative brands are acceptable. Digital Oscilloscope, TBS 1000B Series, 2 Analogue, 30 MHz, 500 MSPS, 2.5 kpts&#160; 
			http://www.tek.com/oscilloscope/tbs1000b-digital-storage-oscilloscope</td>
		</tr>
		<tr>
			<td>8 Pole LP/HP Filter</td>
			<td>Krohn-Hite</td>
			<td>3384</td>
			<td>Alternative brands are acceptable. A frequency range from 0.1 Hz to 200 kHz is recommended.&#160;&#160;&#160; 
			http://www.krohn-hite.com/htm/filters/PDF/3384Data.pdf</td>
		</tr>
		<tr>
			<td>Optiphot microscope</td>
			<td>Nikon</td>
			<td>NA</td>
			<td>Any upright or inverted phase microscope can be used. 
			https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=754</td>
		</tr>
		<tr>
			<td>50:50 (R:T) Cube Beamsplitter</td>
			<td>ThorLabs</td>
			<td>BS013</td>
			<td></td>
		</tr>
	</tbody>
</table>

biophysical characterization of flagellar motor functions

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel in response to a variety of environmental stimuli and are among the triggers for surface colonization and infections. The precise mechanisms by which motors remodel and promote cellular adaptation likely depend on key motor attributes. The photomultiplier-based bead-tracking technique presented here enables accurate biophysical characterization of motor functions, including adaptations in motor speeds and switch-dynamics. This approach offers the advantage of real-time tracking and the ability to probe motor behavior over extended durations. The protocols discussed can be readily extended to study flagellar motors in a variety of bacterial species.

광전자 증 배관 설치는 그림 1A에 표시됩니다. 이는 관심의 PMT가 비드에 의해 산란의 파장 범위에서 높은 감도를 갖는 것이 중요하다. 여기에서 사용되는 PMT가 가시 근적외선 범위에서 동작하고, 할로겐 광원에 의해 조명 비즈 산란광을 검출 할 수 있었다. 최적의 조명 조건 및 공급 전압을 하나의 설정마다 다를 것이다. 이 작업에 사용되는 설정하는 PMT 이득 ~ 들어 4월 10일에서 5월 10일까지 충분히 입증했다. 각각의 광전자는 광전자 앞에 위치하는 3 × 1mm 슬릿 제외 덮었다. 슬릿 광은 광전자 증 배관을 입력 할 수있는 세포 샘플의 영역을 제한하고,이 슬릿은 서로 직교한다. 회전 비드 올바른 위치 (도 1b)에 위치하는 경우, 광전자 증 증가 들어오는 빛의 양이 비드 온다그것의 원형 경로로보기 및 감소에 떨어져보기에서 그것을합니다. 정현파 PMT 전압 출력의 주파수는 회전 속도를 나타내며, 두 신호 사이의 위상 차이는 회전 방향을 나타낸다. PMT를 출력 표시 오실로스코프를 사용하면 실시간으로 비드 궤적의 시각화를 가능하게한다. 대표적인 모터로부터 시변 PMT 신호 Y (t)이고, x (t)는,도 2a에 도시된다. 두 슬릿의 직교성 두 신호 사이의 위상 지연을 도입한다. 신호 진폭은 신호 - 대 - 잡음비는 물론 회전 편심에 의존한다. 비드의 대응 궤적은 그림 2B에 표시됩니다. 최 회장의 대표 모터에서 측정 한 속도의 히스토그램 - 삭제 된 균주도 3a에 도시된다 </strong&gt;. 삭제는 편모 모터 스위치를 독점적으로 반 시계 방향 (CCW) 회전 할 수 있도록합니다. 특정 전동기의 평균 속도는 60 Hz에서이고 측정 속도는 1 ㎛ 비드 (25)가 닿는 모터 이전보고와 일치한다. 그림 1B의 회로도와 같이 비드 먼저, 오른쪽 하단에 위치했다. 해당 각속도는 그림 3B (상단 패널)에 표시됩니다. 인접한 왼쪽 하단에 구슬의 위치는 모터 속도에 기호 (하단 패널)의 반전 결과. 따라서, 인접한 코너로 구슬을 이동하는 모터 회전의 관측 방향을 변경합니다. 이와 관련하여, 대각선으로 대향하는 코너는 동일하다. 올바르게 스위칭 역학을 결정하기 위해 측정시 비드의 위치를 ​​알기 때문에 중요하다. 도 3c는 회전의 두 방향 사이의 야생형 모터 반복 천이를 나타낸다. <html> 데이터 수집 소프트웨어에 대한 사용자 지정 코드가 컴퓨터 (15) 상에 데이터를 기록하기 전에 작업에서 적응되었다. PMT를 출력 AC 결합하고, 저역 100 Hz의 컷오프 주파수로 여과 하였다. 실시간 추적은 오실로스코프에 필터링 된 출력을 연결하여 사용 하였다. <img alt="그림 1" src="/files/ftp_upload/55240/55240fig1.jpg" /> 그림 1 : 구슬-추적 설정. PMT를 기반 추적 설정의 A) 도식. B) 두 개의 수직 슬릿에 비드 (검정 구) 상대의 이상적인 위치. 궤적은 점선으로 표시된다. 편심 E는 점선 원의 반경이다. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55240/55240fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> ove_content &quot;FO : 유지-together.within 페이지 =&quot;1 &quot;&gt; <img alt="그림 2" src="/files/ftp_upload/55240/55240fig2.jpg" /> 그림 2 : PMT를 출력합니다. A) 로우 패스를 중심으로 / 스케일링 후, 두 광전자 증 배관에서 출력을 필터링. B) (3) (S)를 통해 샘플링 된 PMT 데이터로부터 얻어진 비드 궤적. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55240/55240fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/55240/55240fig3.jpg" /> 그림 3 : 구슬 궤적. A) 대표 모터의 CCW 전용 속도의 히스토그램. B) 오른쪽 하단 (상단 패널)에 군데 CCW 전용 모터의 회전 속도. 같은 모터의 회전 속도는 왼쪽 하단 (하단 패널)에 위치 할 때. 기음) </강한&gt; 대표적인 야생 형 모터로 전환. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55240/55240fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Biophysical Characterization of Flagellar Motor Functions at JoVE.com

Biophysical Characterization of Flagellar Motor Functions

Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.

편모 모터 기능의 생물 물리학 적 특성

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel ...

biophysical-characterization-of-flagellar-motor-functions

Nanyang Technological University

Research

JoVE Journal

Biology

8.2K 조회수.  Texas A&M University. Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.

비디오: Biophysical Characterization of Flagellar Motor Functions

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.

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

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 ...

연구

생물학

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

의 준비 Aplysia 감각 - 모터의 연결을 셀 문화

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

의 파라핀 - 임베디드 및 냉동 섹션 Drosophila 성인 근육

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.1 A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer 'epicuticular' layer as well as the blend consisting of the cutin biopolymer and 'intracuticular' waxes.2 Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Sch&auml;ffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) 13C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS 13C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH2O) chemical moieties.
Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state 13C NMR reveals differences in the relative numbers of CHO and CH2O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-&mu;m length scale.

Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.

분리 및 과일 손톱의 Biophysical 연구

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

게놈 DNA에서 도보로 5 hydroxymethylcytosine의 선택적 캡처

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

유전자 규정의 단일 분자 이미징<em&gt; 생체에</em&gt; 절단하여 Cotranslational 인증 (CoTrAC)를 사용

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

세균 본부 기계의 슈퍼 해상도 이미징

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.

전기 생리학을 바탕으로 솔리드 지원 막 소개

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

의 효율적인 생산 및 정제 재조합 쥐과 Kindlin-3 생물 물리학 연구 곤충 세포에서

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.