The authors have no acknowledgements.

주 : 동물의 사용과 관련된 모든 절차는 로컬 입법에 따라 실시된다. 우리의 작업은 시드니 지역 보건 지구 동물 복지위원회에 의해 승인됩니다. 허가 절차는 프로젝트 라이센스 문서에 설명 된 모든 시간에 동물의 복지를 보장 지침을 준수하고 있습니다. 작업이 수행되는 나라의 동물 실험에 법령 준수를 확인합니다. 1. 프로토콜 EM 정착액을 준비하기위한 <ol><li> 정착액 100 ㎖를 들어, 65 ºC로 삼각 플라스크에 증류수 25 ml의 열에 파라 포름 알데히드 분말 2g을 추가 자석 교반기로 일정하게 교반 파라 포름 알데히드를 준비합니다. 참고 : 글루 타르 알데히드, 파라 포름 알데히드 나트륨 cacodylate 버퍼가 모든 유해 물질이며 항상 fumehood 처리해야합니다. </li><li> 다음 냉각 할 수 있도록 65 ºC 분 2에서 열. 필요한 경우 수산화 나트륨 용액을 추가 dropwISE는 완전히 솔루션을 취소 교반하면서. 참고 : 수산화 나트륨이 위험합니다, 취급에주의. </li><li> EM 등급 스톡 글루 타르 알데하이드 10 ㎖, 50 0.2 M 나트륨 cacodylate 버퍼 ml의 자당의 2g을 추가하고 자석 교반기로 저어. 1.0 M CaCl2를 2 ㎖를 추가합니다. </li><li> 증류수 100 ml의 용액을 만들기 이전에 pH를 7.4로 조정한다. 최종 정착은 2.5 % 글루 타르 알데히드, 2 % 파라 포름 알데히드, 2 밀리몰 염화칼슘 2, 0.1 M cacodylate 버퍼에 2 % 자당이 포함되어 있습니다. 총 삼투압이 440 mOsmol / L 있는지 확인하십시오. 준비 24 시간 내에 사용하십시오. </li></ol> 간 2. 관류 고정 <ol><li> 37 ºC - 35 따뜻한 관류 버퍼와 정착액. 방혈 버퍼와 정착의 정확한 온도는 조직의 무결성을 보장하는 데 도움이됩니다. </li><li> 50 밀리그램 / kg 케타민, 5 밀리그램 / kg 자일 라진 단일 복강 내 주사하여 동물을 마취. </li><li> 동물하에되면뒷다리의 철수와 꼬리 핀치에 의해 평가 깊은 마취는, Y 절개 간 포털 정맥을 노출 동물의 복부에 무딘 종료 가위로 이루어집니다. 참고 :이 장비는 쉽게 현미경에서 볼 수됩니다 미생물 및 일반 쓰레기와 시료의 오염을 방지하기 위해 깨끗해야합니다. </li><li> 다른 더 원심 간으로, 간으로 포털 정맥 주위에 한 근위 두 개의 매우 느슨한 봉합을 묶어. </li><li> 캐 뉼러를 고정 .Tighten 봉합사 (쥐 래트 18 G 22 G) 적절한 크기의 IV 캐 뉼러와 문맥을 Cannulate. </li><li> 인산염 완충 식염수로 관류를 시작 37 ºC로 가열하고, H 2 0 10 cm의 압력. PBS 5 내지 20㎖의 보통 간에게서 피를 뽑다 것이 요구된다. 참고 : 기포들이 색전술과 압력 차 유물을 유도으로 정맥에 연결된 튜브를 통해 간을 입력 할 수 있도록하지 마십시오 간세포 손상, SI관류 압이 너무 높으면 nusoids 및 LSEC의 fenestrations 발생한다. </li><li> 복부 및 흉부 대정맥은 버퍼가 간에서 자유롭게 종료 할 수 있도록 절단. 이 LSECs 높은 배압 손상을 방지 할 수 있습니다. </li><li> 간은 혈액의 무료되면, EM 정착액으로 PBS를 교체하고 간 경화 매우 창백한 될 때까지 약 5 분 동안 관류. </li><li> 관류를 중단하고 1로 고정 간을 잘라 - 날카로운 메스를 사용하여 2 mm의 3 블록. </li><li> 4 ºC에서 72 시간 - 24 EM 정착액의 포스트 수정 조직. 참고 : 일반적으로, underfixation는 overfixation보다 더 많은 유물이 발생합니다. </li><li> 4 ºC에서 저장을위한 0.1 M 나트륨 cacodylate 버퍼 후 수정 기간 다음 정착액 변경합니다. 참고 :이 방법으로 저장 시편 (최대 12 개월) 시간의 상당한 기간, 그러나 적절한 보조 고정 보존되며, 시험은 최상의 결과를 권장합니다. </li></ol> 3. 니들 Perfusion 참고 : 니들 고정 직접 생검 간 조직에 정착의 주입을 포함 관류 고정의 변종이다. 정착액 그들을에게서 피를 뽑다 및 정착액하는 조직의 모든 블록을 노출하기 위해 혈관을 플러시하는 목적이다. 케어는 압력 부상 (18, 19)을 피하기 위해 낮은 주입 압력을 유지하기 위해주의해야합니다. <ol><li> 동물이나 피사체 간의 작은 조각을 제거하고 혈액과 이물질을 제거하기 위해 식염수에 헹군다. </li><li> 미세 게이지 바늘 (일반적으로 인슐린 주사기)를 장착 주사기로 생리 식염수를 그립니다. </li><li> 혈액이 조직에서 플러시 될 때까지 간으로 매우 천천히 식염수를 주입한다. 여러 주사가 필요할 수 있습니다. </li><li> 미세 게이지 바늘 (일반적으로 인슐린 주사기)를 장착 다른 주사기로 EM 정착액을 그립니다. </li><li> 이 단단하고 색 황갈색이 될 때까지 간으로 매우 천천히 정착액을 주입한다. 다중 분사S가 필요할 수 있습니다. </li><li> 날카로운 메스를 사용하여 2 mm의 3 블록 - (1)에 고정 간을 잘라. 72 시간 4 ° C의 - 24 EM 정착액에 품어. 차 고정을 시작하기 전에 0.1 M 나트륨 cacodylate 버퍼에 3 회 반복한다. </li></ol> 주사 전자 현미경 4. 준비 <ol><li> 워시 샘플 0.1 M 나트륨 cacodylate 버퍼에 3 회. 지질를 해결하려면 포스트는 2 시간 동안 0.1 M 나트륨 cacodylate 버퍼에 2 %의 산화 오스뮴의 샘플을 수정합니다. 참고 : 글루 타르 알데히드와 오스뮴 테트 록 사이드 크로스가 반응 SEM에 인공물을 생성 완전한 세척은 매우 중요합니다. </li><li> 탈수 개시 에탄올 농도 증가에 샘플을 헹군다. 5 분 동안 50 % 에탄올에서 우선 린스; 70 % 에탄올로 한 후 5 분 동안 3 회; 90 % 에탄올로 5 분간 3 회 씻어; 100 %의 에탄올을 5 분 동안 2 회 씻어; 최종적으로 100 % 에탄올 (분 자체)에서 10 분 동안 2 회 헹군다. </li><li> 샘플에서 모든 에탄올을 제거<html> 및 fumehood에 헥사 메틸 디 실라 잔 (HMDS)로 교체하고 10 분 동안 둡니다. 조직 샘플에서 HMDS를 제거하고 증발 할 수 있습니다. 주 : 따라서 최종 탈수 공정에 사용하기 전에 RT에 도달하도록 냉간시 HMDS 높은 흡습성이다. </li><li> 선택적으로, 즉시 샘플을 장착하거나 SEM 마운트 할 준비가 될 때까지 데시 케이 터에 배치합니다. </li></ol> 5. 장착 참고 : 올바른 표본 장착이 현미경에서 분석 할 수 명확하게 묘사 사인 곡선의 수를 최대화합니다. <ol><li> 금속 현미경 스텁의 기본 레이블과 스텁의 상단에 양면 전도성 카본 테이프를 적용합니다. </li><li> 후속 SEM위한 최선 정현파와 표면을 식별하기 위해 해부 현미경을 사용하여 샘​​플을 시각화. 그루터기의 탄소 테이프 표면에 위쪽으로 그 표면을 놓습니다. </li><li> 스틱 표본 단단히 그루터기에, 표본 지금 스퍼터 코팅에 대한 준비가되어 있습니다. </li></ol>ove_title &quot;&gt; 6. 코팅 참고 : 스퍼터 코터 경내에서 표본을 전도성 금속 (금 또는 백금)의 벌금 필름 시편 코팅 및 전자 빔의 손상으로부터 보호한다. 코팅이 경우 관심 너무 두꺼운 구조 모호 할 수있다. <ol><li> 자동 미세 스퍼터 코터의 진공 챔버에 배치 할 스텁은 45 초 동안 회전 모드와 자동 스퍼터 코팅을 설정합니다. 그러나 금도 적합, 미세한 디테일에 대한 백금 코팅을 사용합니다. </li><li> 마운트 된 간 표본의 외부 표면에 탄소 페인트의 얇은 층을 적용 코트가 아닌 사인 표면을 돌보는. </li><li> 시험편을 백금으로 코팅 탄소 페인트가 건조되면 그들은 현미경에서 관찰을위한 준비가되어 있습니다. 샘플은 데시 케이 터에 저장 될 수있다. </li></ol> 7. 스캐닝 전자 현미경을 사용하여 <ol><li> 장소는 주 사형 전자 현미경으로 표본을 현미경으로 진공을 반환. &lt;/ 리&gt; <li> 낮은 배율에서 시작하여 시험편의 시각화를위한 현미경 표준 작동 절차를 사용하여, 서서히 배율을 증가시킨다. </li><li> 현미경 거리, 장소의 크기와 난시를 작업에 맞게 조정되어 있는지 확인합니다. </li><li> 고정 무결성을 확인합니다. LSEC이 틈에서 대부분 무료이며, 30 측정 fenestrations 포함되어있는 경우 - 250 nm의 직경이 일반적으로 그 시편 준비는 성공적이었다 나타냅니다. </li><li> 정현파는 간극의 전체 또는 fenestrations없는 경우 표본 준비 성공되지 않았거나 중요한 병리학있다. 이 경우, 매우 미세한 면도날 샘플을 슬라이스하고, 절단면을 재 도장 성공적 고정 정현파 분석. </li><li> 약 15,000-20,000 × 배율 좋은 고정 및 fenestrations 명확하게 (볼 수 있습니다 경우 일반적으로 적어도 10 fenestrations를 포함와 파편의 무료 평면 LSEC 표면을 선택). </li><li> 간 당 적어도 두 개의 블록을 사용하여, 블록의 다른 영역에서, 간 당 적어도 10 정현파 이미지를 저장한다. 15,000에서 10 현미경을 샘플링 - fenestrations의 정량은 전체 간을 대표하는 동물 20,000 × 배율은 일반적으로 충분합니다. </li></ol> 8. 분석 참고 : NIH로부터 다운로드 할 수없는 ImageJ에 소프트웨어는 창호 직경 및 주파수 (정량화하기 위해 이용된다 <a href="http://imagej.nih.gov/ij">www.imagej.nih.gov/ij을</a> ). <ol><li> 이미지 열기 J 및 사진 (화면 1 샷)에 포함 된 스케일 막대를 사용하여 스케일을 설정합니다. </li><li> 유창하고 defenestrated 지역을 포함 LSEC의 전체 평면 주변 다각형 ImageJ에있는 도구, 추적을 사용하여. 이 거짓 기공을 팽창하므로 ​​바로 체 플레이트를 추적하지 마십시오. obscuri있을 세포 표면에있는 임의의 부스러기가 큰 조각을 제외NG의 fenestrations. </li><li> , 창호 직경을 측정하는 줄 도구를 선택하여 각 창호의 가장 긴 직경을 통해 라인을 추적하고, 라인을 측정하기 위해 &quot;M&quot;을 누르고, &quot;D&quot;를 영구적으로 사진에 선을 그릴 (그릴). 이 라인은 창호 직경으로서 정의된다. 줄의 길이는 지금 ImageJ에 결과 상자 (화면 2 샷)에 사용할 수 있습니다. </li><li> 큰 250 nm를 초과 LSEC 세포질 구멍뿐만 아니라 fenestrations 모든 갭을 측정한다. 간격은 종종 격차는 질병과 독성의 결과로 발생할 수 있지만 가난한 관류 또는 고정을 반영하는 유물이다. 간격에 대한 데이터는 나중에 분석 창호 파라미터 계산에서 제외된다. </li><li> 원형하지 않은 갭 영역은 간극 주위 추적 및 ImageJ에 그들의 면적을 계산에 의해 산출한다. </li><li> furthe에 대한 Excel 스프레드 시트로 ImageJ에의 결과 상자에서 데이터를 붙여 넣기R 분석. </li></ol> 9. 계산 <ol><li> (간격이 ≥ 250 nm의 아르 격차, 포함하지 않음) 모든 창호 직경의 평균 창호 직경 = 평균. </li><li> R, 반경, 개별 fenestrations 직경 (R = D / 2)을 포함하지 않는 갭으로부터 계산된다 창호 면적 = πr 2. </li><li> 다공성 (%) = (Σ (πr 2) / 분석 전체 면적 - 갭의 Σ (영역 =)) (μm의 2)) × 100. </li><li> 창호 주파수 = fenestrations의 총 수 / (전체 면적 (갭 영역) (μm의 2) - Σ 분석했다. </li></ol> 창호 데이터의 프리젠 테이션 (10) 주 : 창호 데이터 정량화를 포함한 가능한 한 간행물은 다음과 같은 정보를 포함해야 <ol><li> 사용 경계 직경은 일반적으로 (fenestrations을 정의하는 것을 확인하는 문 창호 직경,창호 주파수 (수 / μm의 2) 및 다공성 (%)) 나노 50-250 사이. </li><li> 갭이 분석 창호 직경 간, 블록, 이미지 및 fenestrations 수의 도수 분포 그래프 분석에 포함되어야에 포함되었는지 여부를 확인하는 문. </li></ol>

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The authors have nothing to disclose.

정확하고 재현성 간 정현파 내피의 상태를 측정하는 기능이 고도로 전문화 된 세포의 생물학을 이해하는데 중요한 단계이다. 구조화 조명 현미경 (32), 원자력 현미경 (33) 및 D-STORM (직접 확률론 광학 Reconstrucion 현미경) (34) 같은 새로운 기술은 시험 관내에서 이러한 세포의 형태에 관한 중요한 정보를 부여하지만 SEM 시각화하고 자신의 구조를 측정하는 주 방?...

간 사인 내피 세포 (LSECs)는 매우 간 사인 곡선의 벽을 일렬로 차별화 된 내피 세포 수 있습니다. LSECs가 아닌 diaphragmed 있습니다 fenestrations으로 천공되어, 세포 횡단는 직경이 50 ~ 250 nm의 모공. LSECs의 표면의 20 %까지 체 플레이트 1-3 (도 1)라고 수십 내지 수백 그룹 보통 fenestrations 의해 덮여있다. Fenestrations는 고효율 한외 여과 시스템을 구축, 및 혈장 혈액 간세포 nanosubstrates 사이의 전송을 허용한다. Fenestrations 동적 구조입니다 - 크기 및 / 또는 번호가 모두 다양한 생리 학적 상태, 약물, 질병에 대한 응답으로 변경 될 수 있습니다. 예를 들어, fenestrations는 공급 상태 4보다 금식에 큰; 세포 노화와 여러 질병 상태 7-13에서 발생 당 5, 6 및 크기의 감소 및 fenestrations 수 2 디 iodoamphetamine는 창호 수를 증가 </sup&gt;. fenestrations의 크기 및 수의 정확한 측정 LSEC 모폴로지는 다양한 질환, 독성, 및 생리적 상태에 의해 영향을받는 방법을 이해하는 것이 중요하다; 간 기능에 미치는 영향; 개발에 대한 치료 적 개입 1 창호를 변조. fenestrations의 연구는 어렵다. 그대로 간 조직 또는 배양 LSECs 모두 이전에는 관찰하여 전자 현미경이 가능했습니다 있도록 fenestrations의 직경은, 기존의 광학 현미경의 해상도 아래에 자리 잡고 있습니다. 주사 전자 현미경 (SEM)을 자주, SEM은 수천 내피 표면 및 측정의 큰 영역의 관찰이 가능하기 때문에 창호의 크기, 주파수 및 공극률 (fenestrations 의해 천공 LSEC 막의 백분율)를 연구하기 위해 사용되어왔다 하지 수만 fenestrations 수천의 경우. 그 유틸리티에도 불구하고,에서보고 된 결과에 대한 연구를 SEM을 기반이러한 창호 크기, 수, 빈도와 같은 다공성 LSEC 파라미터는 문헌 (표 1)에서 광범위하게 다양하다. Fenestrations 및 체 플레이트 계약, 휴식 팽창 또는 표본 준비 중 합체 깨지기 쉬운 구조입니다, 따라서 신중한 처리를 자신의 무결성을 유지하기 위해 필요합니다. 상승 관류 압 (14); 정착 및 버퍼 (15)의 잘못된 삼투압; 부적절한 고정 또는 고정 시간; 및 사후 고정 탈수 및 건조 속도는 미세 구조의 보존을 방해 유물 (그림 2)을 생성 할 수 현미경에 대한 처리의 모든 영역입니다. fenestrations 손실 ( &#39;defenestration&#39;) 및 창호 수축 저감 창호 직경 셀 다공성 결과 불량한 고정의 결과로서 발생할 수있다. SEM 분석을위한 시료의 보존을 개선하기위한 방법은 앞에서 설명 15-17되었고 논의 될여기에 표본 보존을 개선하는 방법에 대한 팁. 시험편 보존 주요 목표는 LSEC의 표면이 가시화 될 수 있도록 정현파에서 혈액을 제거하고, 고압 또는 지연 고정 하나에서 LSEC 손상을 방지한다. 포털 정맥을 통해 정착의 항목 간 관류 간 고정을 위해 선호하는 방법입니다. 상세하게 설명한 바와 같이 다른 곳에서 16, 18 관류가 낮은 압력에서 수행되어야한다 (예를 들면 H 2 0 10cm) LSEC에 압력 관련 관류 유물과 손상을 방지하려면, 일반적으로 세포막에 내 큰 격차를 나타내. 다른 19 상세히 설명하지만, 적절한 정착 종종, 사람과 동물에서 간 생검 바늘 관류를 사용하여 얻을 수있다. 혈액 샘플에서 플러시 될 때까지이 방법은 조직으로 직접 주사를 포함 고정액과 조직은 견고하고 고정된다. 전자 현미경 샘플의 고정이 반환 한 할 필요가간은 매우 신속한자가 분해 공정의 결과로서 발생하는 미세 구조적 변화를 방지하는 혈류의 중단 다음 최대한 빨리 ORMED. 또한 인공물의 포함을 최소화하고 fenestrations의 측정을 표준화 이미지 분석의 방법을 제시한다. 다공성 및 창호 주파수 현미경에 대한 사인 곡선의 선택의 변화, 인공물의 이미지 분석, 셀 면적의 측정은 발표 결과에 큰 차이를 주도했다. 평가 및 fenestrations 및 데이터 프리젠 테이션에 대한 최소 요구 사항을 측정하는 표준화 된 접근 방식은 분명히 이전 4,10,20-31 문헌에 언급되지 않았다.

<table border="0" cellpadding="0" cellspacing="0" width="658">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Name of material</td>
			<td style="width:100px;">Company</td>
			<td style="width:93px;">Catalogue Number</td>
			<td style="width:311px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">EM grade Glutaraldehyde</td>
			<td style="width:100px;">ProSciTech</td>
			<td style="width:93px;">C001</td>
			<td style="width:311px;">Store stock at -20 C until needed, avoid refreeze</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">Paraformaldehyde powder</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td>158127</td>
			<td style="width:311px;">Always prepare Paraformaldehyde fresh</td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:155px;">Sodium Cacodylate powder</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:93px;">C0250</td>
			<td style="width:311px;">Prepare 0.2 M stock, pH 7.4 by dissolving powder in dH2O, used mostly at 0.1 M by preparing 1:2 dilution</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:155px;">Calcium Chloride</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:93px;">C1016</td>
			<td style="width:311px;">Prepare 1 M CaCl2by dissolving powder in dH2O</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:155px;">Osmium tretroxide</td>
			<td style="width:100px;">ProSciTech</td>
			<td style="width:93px;">C011</td>
			<td style="width:311px;">Wash ampoules in weak acid prior to use to avoid contamination. Prepare 2 % stock in glass bottle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">Ethanol- Absolute</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td>459836&#160;</td>
			<td style="width:311px;">100 % Ethanol must be high grade and stored with Molecular Sieve</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Other grades of Ethanol</td>
			<td style="width:100px;">Labtech</td>
			<td style="width:93px;">EL5</td>
			<td style="width:311px;">Prepare graded Ethanols with dH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:155px;">Hexamethyldisilazane</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td>52619&#160;</td>
			<td style="width:311px;">Allow to reach room temperature before use</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:155px;">Cannulas</td>
			<td style="width:100px;">Terumo</td>
			<td style="width:93px;">TSROX1832C, TSROX2225C, TSROX2419C</td>
			<td style="width:311px;">18 G is suitable for most rats, 22 G is suitable for most mice, but it is good to have a few 24 G on hand in case of very small mice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">Conductive Carbon tape</td>
			<td style="width:100px;">ProSciTech</td>
			<td>IA0201</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:155px;">Carbon Paint</td>
			<td style="width:100px;">ProSciTech</td>
			<td>I003</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">Ketamine</td>
			<td style="width:100px;"></td>
			<td style="width:93px;"></td>
			<td style="width:311px;">Must be optained under licence</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">Xylazine</td>
			<td style="width:100px;"></td>
			<td style="width:93px;"></td>
			<td style="width:311px;">Must be obtained under licence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:155px;">Molecular Sieve</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:93px;">208647</td>
			<td style="width:311px;">Removes water from the 100 % Ethanol</td>
		</tr>
	</tbody>
</table>

a standardized method for the analysis of liver sinusoidal endothelial cells and their fenestrations by scanning electron microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

주 사형 전자 현미경 저배율에서의 초기 시각화는 많은 간 큰 용기 및 정현파 (도 1a)을 관찰하기에 충분히 큰 노출 영역 간 시편의 평평한 표면을 보여준다. 장착 스텁에 올바른 간 블록 배치를 보장하는 것은 이러한 이유 때문에 (그림 1B)를 위해 피해야한다 사인 곡선과 간 Glisson의 캡슐의 선명한 이미지를 얻기 위해 필수적이다. 배율을 높이면 간 조밀 맥관계의 가까이 ?...

Watch this Scientific Journal Video about A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy at JoVE.com

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

주사 전자 현미경으로 간 사인 내피 세포의 분석 및 그들의 Fenestrations위한 표준화 된 방법

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

a-standardized-method-for-analysis-liver-sinusoidal-endothelial-cells

Research

JoVE Journal

Biology

14.3K 조회수.  University of Sydney and Concord Hospital. The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

비디오: A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

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

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

연구

생물학

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

쥐과 밸브 내피 세포의 분리

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

침연 (macerated) 조직의 전자 현미경을 스캔하면 세포 외 매트릭스를 시각화하기

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

Studying the Supramolecular Organization of Photosynthetic Membranes within Freeze-fractured Leaf Tissues by Cryo-scanning Electron Microscopy

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we describe a technique for studying the supramolecular organization of photosynthetic (thylakoid) membranes within leaf samples. This is achieved by high-pressure freezing of leaf tissues, freeze-fracturing, double-layer coating and finally cryo-SEM imaging. Use of the double-layer coating method allows acquiring high magnification (&#62;100,000X) images with minimal beam damage to the frozen-hydrated samples as well as minimal charging effects. Using the described procedures we investigated the alterations in supramolecular distribution of photosystem and light-harvesting antenna protein complexes that take place during dehydration of the resurrection plant Craterostigma pumilum, in situ.

Here we describe a procedure for studying freeze-fractured plant tissues. High-pressure frozen leaf samples are freeze-fractured and double-layer coated, yielding well preserved frozen-hydrated samples that are imaged using the cryo-scanning electron microscope at high magnifications with minimal beam damage.

알아내는 주사 전자 현미경에 의해 동결 골절 잎 조직 내에서 광합성 멤브레인의 초분자 조직을 공부

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM.
Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells.
The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here.

문제있는 공장, 난균 강 및 곰팡이 샘플에 대한 전자 현미경 (SEM) 프로토콜을 스캔

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

애기 씨의 높은 처리 살 균에 대 한 표준화 된 방법

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

스캐닝 전자 현미경 (SEM)에서 형태소 분석에 대 한 화학 건조를 사용 하 여 간결한 진 핵 유기 체의 준비

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

고압 냉동, 전자 레인지를 이용한 콘트라스트 향상, 그리고 볼륨 이미지에 대 한 포함 하는 최소한의 수 지에 의해 생물학 견본 준비

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

초구조적으로 보존된 세포 내 막 단백질 국소화의 전자 현미경 분석을 위한 CryoAPEX 방법

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

We describe the preparation of ribbons of serial sections and their collection on large transfer support for use as Array Tomography samples, along with automated imaging procedures in a scanning electron microscope. The protocol allows screening, retrieval, and targeted imaging of local, rare events, and the acquisition of large data volumes.

스캐닝 전자 현미경 을 사용하여 대량 정보의 표적 수집을위한 배열 단층 학워크

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...