이 작품은 NIH 그랜트 어이 14990에 의해 지원되었다.

1 부 : 해부 및 바이폴라 셀 절연 <ol><li> 표 2에 나열된 솔루션을 준비, 링거스 &#39;의 산도는 (외부) 솔루션은 NaOH와 7.4으로 조정되어야하며 내부 솔루션의 산도는 7.2 CsOH로 조정해야합니다. 알루미늄 호일과 빛으로부터 내부 솔루션을 보호하고 4를 유지 ° C를 사용까지; </li><li> 해부하기 전에 적어도 30 분 금붕어를 어두운 적응; </li><li> (; 시그마, 세인트 루이스, MO 낮은 칼슘 2 + 링거스 입력 V의 hyaluronidase, 1100 단위 / ML)과 L - 시스테인 (0.5 MG / ML 10 ML 동물이 어두운 적응하지만, hyaluronidase 5 ML 준비 낮은 칼슘 2 + 링거스) 솔루션과 papain (동결 건조된 분말, 40 단위 / ML 무게, 소화 솔루션의 5 ML 위해를 시그마, 세인트 루이스, MO); </li><li> 수술 가위로 빠르게 잘린하여 금붕어를 안락사와 11 # 메스 블레이드와 두뇌 및 척수를 파괴; </li><li> # 7 곡선 더몬트 집게의 도움으로 추가 - 안구 근육을 파괴하고 홍채 가위로 시신경을 절단하여 눈을 제거; </li><li> 필터 종이 찔린 11 # 메스 블레이드의 팁과 scleral limbus의 조각에 올려 놓으 아이 전구; </li><li> 구멍 전체가 내부 vannas의 가위의 칼날을 소개하고 거리 전체 앞쪽에 세그먼트를 잘라; </li><li> 나머지 광학 컵 위에 종이 필터의 작은 조각을 놓고 종이가 유리 유머와 흠뻑 젖어있는 권한을 부여하기 위해 압력 좀 넣으; </li><li> 그것에 연결된 망막과 필터 종이를 들고 vannas 가위 파와 시신경을 잘라; </li><li> # 7 더몬트 핀셋의 도움으로 여과지에서 망막에서 hyaluronidase 솔루션과 껍질이있는 35mm 플라스틱 문화 접시에있는 망막을 포함하는 필터 용지를 놓습니다; </li><li> 산업 소강 단일 깨끗 블레이드의 절반과 4-6 조각으로 망막 컷과 20 분 동안 hyaluronidase 용액에 앉아 보자; </li><li> 을 적용하려면 hyaluronidase를 기다리는 동안, papain으로 L - 시스테인 솔루션의 5 ML을 추가하고 액체가 투명하게 될 때까지 (약 50~10분) 앉아 보자; </li><li> 낮은 칼슘 2 + 링거스에 배 망막의 조각을 씻고 그들 30~35분에 대한 papain 솔루션에 앉히지; </li><li> 낮은 칼슘 2 + 링거스에 배 망막의 조각을 씻어 4에서 사용하기 전까지 그들을 저장할 ° 낮은 칼슘 2 포함 35mm 플라스틱 문화 접시에있는 C + 링거스; </li><li> 세포를 떼어 놓다하려면, 낮은 칼슘 2 + 링어의 500 ML를 포함하는 microcentrifuge 관에서 망막의 한 조각을 넣고 천천히 모든 공기 방울을 생산하지 않는 신중하게, 유리 분리 피펫으로 위아래로 pipetting하여 망막을 씹다. 분리의 pipettes은 알콜 램프와 유리 파스퇴르 피펫의 팁을 가열하고 약간 해부 집게의 도움으로 그것을 절곡하여 가공되며 </li><li> 이전에 적은 칼슘 2 + 링거스 2 ML 가득한 집에서 만든 녹화 실로 망막 정지 한 방울을 추가하여 플레이트 분리된 세포를. 챔버는 중앙에 원형 전체와 35 mm 플라스틱 문화 요리와 실리콘 엘라스토머 (Sylgard 184와 함께 바닥에 붙어 1.78 굴절률 유리 (PlanOptik, 독일)의 원형 coverslip의 아래쪽 절반으로 구성되어, 다우 코닝 , 미들랜드, MI). </li></ol> 2 부 : 바이폴라 셀 로딩과 와시 아웃 시냅스 vesicles의 TIRF 이미지는 최상의 매우 높은 NA의 객관적이고 민감한 카메라 객관적인 타입 TIRFM 현미경을 사용하여 수행됩니다. 실험을 위해, 우리는 EMCCD (캐스케이드 512B, 로퍼 과학, 투싼,​​ AZ)로 1.65 NA 목표 (APO x100 O HR, NA 1.65, 올림푸스, 일본)를 사용하여 선택합니다. 매우 높은 NA의 목적의 사용은 높은 굴절률 유리 coverslips 및 침지 유체 (유황과 디 - 이오도 메탄)의 사용을 요구. 우리의 조건에서, 여기 라이트는 약 50 nm의의 일정한 길이가 기하 급수적으로 부패 분야로 제한됩니다. <ol><li> 현미경 목표 높은 굴절률 액체 (M 시리즈, 굴절률 = 1.7800, Cargille 연구소 시더 그로브, NJ)의 한 방울을 추가합니다; </li><li> 현미경 목표 위에 조심스럽게 기록 챔버를 놓고 신중하게 접지 전극과 실로 superfusion 출구 파이프를 탑재; </li><li> 챔버는 세포가 싱크대와 바닥을 준수하도록 10~20분에 대한 현미경에 앉아 보자; </li><li> 그 동안에, 1mM trolox ® ((±)- 6 - 히드록시 -2 ,5,7,8 - tetramethylchromane - 2 - 카르복실산 5 ML 준비, 시그마, 세인트 루이스, 높은 K + 링거스에 MO) 솔루션 . 녹아까지 Sonicate; </li><li> 낮은 칼슘 2 + 링거스 1 MM ADVASEP - 7 (시그마, 세인트 루이스, MO) : ADVASEP - 7 세척 솔루션 15 ML을 준비합니다. ADVASEP - 7 사용 선택하고 원하는 경우 생략할 수 있습니다; </li><li> superfusion의 리튬을 정화NES 및 추가 ADVASEP - 7 세척 솔루션, 낮은 칼슘 2 + 링거스 및 제어 대타가 superfusion 시스템의; </li><li> 얇은 벽으로 둘러싸인 borosilicate 유리 (; WPI, 사라소타, 플로리다 퀵 - Fil ® TW150 - 3)에서 pipettes를 로드할 당겨. 퍼퍼 피펫 resistances는 1.5-2.5 MΩ 범위에; </li><li> 솔루션, FM1 -​​ 43 ® (Invitrogen, 칼스 배드, CA - - (4 (dibutylamino) styryl) pyridinium dibromide, &quot;특별한 포장을&quot;N - (3 - triethylammoniumpropyl) -4) 준비. 첫째, FM1 -​​ 43 중 하나를 유리병 (100 MG) ® 160 μL 증류수를 추가하여 1 ㎜의 주식을합니다. 이 주식은 최대 일주일 4 ° C에 보관하실 수 있습니다. 그런 다음, FM1 - 43 5 μL를 추가 ® 1 ML 높은 K + 링거스 + 1mM trolox ® 수 있습니다. 알루미늄 호일과 빛으로부터 보호 솔루션을 4에서 보관 ° C를 사용까지; </li><li> 에 대한 현미경 명시야 조명을 켜고 그대로 바이폴라 세포에 대한 검색합니다. 약간 신경이 단단히 챔버 하단에 첨부되어 있는지 확인하기 위해 현미경 탭; </li><li> 가까운 관심 세포에 superfusion 펜을 위치하고 지속적으로 낮은 칼슘 2 + 링거스으로 준비를 perfuse; </li><li> 객실 조명을 끄고 빨간색 긴 패스 필터 (즉, RG630, 쇼트, 독일)을 추가 FM 염료 여기를 최소화하기 위해 광 경로; </li><li> FM 염료 용액 10 μL와 로딩 피펫을 채우기 micromanipulator에 피펫을 탑재하고 당신이 부하하려는 바이폴라 세포와 같은 초점 비행기에까지 압력없이 준비에 피펫을 낮추십시오. 당신은 적어도 두 전극 홀더를 확인하십시오 : FM 염색에 사용되는 한 그것은 세포 용액을 오염시킬 수 있습니다 다른 패치 클램핑 사용하거나, 수 없습니다; </li><li> 축삭 터미널에서 약 10 μm의의 거리에서 퍼퍼 오픈 위치, superfusion 시스템을 해제 및 피펫 과압을 설정하여 10 초 동안 염료 솔루션을 퍼프; </li><li> 30 초 동안 기다, 피펫을 움직이지 않고, 압력을 끄고; </li><li> 에 superfusion을 켜고 5 분 ADVASEP - 7 솔루션 챔버 목욕. 그 동안에, 목욕탕에서 호흡기의 피펫을 제거; </li><li> 5 분 후에, 낮은 칼슘 2 + 링거스로 전환하고 초과 염료의 제거를 허용하도록 25~30분 위해 챔버를 perfuse. </li></ol> 3 부 : 패치 클램핑 및 TIRFM 이미징 <ol><li> 준비가 세척하는 동안 두꺼운 벽의 borosilicate 유리 (; 서터 악기 회사 노바토, CA B150 - 86 - 10)에서 패치 pipettes를 가져옵니다. 패치 피펫 resistances은 80-10 MΩ 범위에; </li><li> 씻어가 완료되면,보기의 필드의 중앙에 축삭 터미널 장소; </li><li> 그리고 micromanipulator에 장착, 내부 솔루션 7μL와 패치 피펫을 채우기, 공기 방울, 코트 용융 치과 왁스 (커 주식 회사, 오렌지, CA 끈적 왁스)와 피펫을 없애기 위해 피펫을 탭; </li><li> 에 피펫 과압을 켜고 준비에 서서히 피펫을 낮추십시오. 앰프의 피펫 저항 정확한 피펫의 오프셋과 각 앰프 컨트롤과 함께 용량을 확인하십시오; </li><li> 링거스를 제어할 수 superfusion 스위치. 피펫과 세포 사이 gigaseal를 만들려면, 약간 세포 기관에 대한 전극의 끝부분을 터치하고 부드럽게 전극에 부정적인 압력을 적용하는 동안 피펫 과압을 해제; </li><li> 봉인 반면, 앰프에서 &quot;전체 셀&quot;모드를 선택하고 -60 MV하는 잠재력을 가지고있는 세포를 설정; </li><li> , 전체 축삭 터미널을 포함한 이미징 소프트웨어와 관심 영역을 선택 명시야 조명을 끄고, 짧게 488 nm의 레이저로 (30 MS) 터미널을 노출하여 TIRF 영상에 맞는 초점 비행기를 찾으면; </li><li> 피펫에 약간 부정적인 압력을 적용하는 동안 앰프의 &quot;기력&quot;명령을 사용하여 세포에 침투; </li><li> 셀 커패시턴스와 직렬 저항에 대한 정정하고있는 동안 관심의 전압 프로토콜을 적용 시냅스 vesicles의 영상 움직임. 이것은 전압 프로토콜 카메라 프레임 속도에 동기가이 단계에서 중요하다. 우리의 경우, 각 프레임은 30 MS 길이, 전압 변경이 값 (즉, 모든 300 MS 또는 10 프레임)의 배수에서 발생하므로, </li><li> 복구 수 있도록 재판을 사이에 최소한 40 초 정도 기다리십시오; </li><li> 에서 561 nm의 레이저를 돌려 동안 시냅스 리본의 위치를​​ 확인하려면, 사진을 찍는. </li></ol><img src="/files/ftp_upload/1305/fig1.jpg" alt="그림 1" /> 그림 1 : 실험 설정. 488 nm의 레이저 (파란색)은 목표의 뒤에 초점 비행기의 주변에 집중하고 유리 수성 매체 인터페이스에 도달하면 총 내부 반사를 앓고있다. 반사 빔에 의해 생성된 전자기장은 봇 가장 가까운 시냅스 vesicles에 로드된 형광단을 자극다음 (녹색) 형광 유리 챔버 톰. 형광등은 다음 관찰자의 눈 (묘사) 또는 CCD 카메라로 안내합니다. 몇 군데 세포의 막 잠재력은 그들을 패치 - 클램핑하여 동시 제어됩니다. 이러한 접근 방식은 수신 신호 (막 전압)와 출력의 연결 (exocytosis) 사이의 관계의 연구를 수 있습니다. <img src="/files/ftp_upload/1305/fig2.jpg" alt="그림 2" /> 그림 2 : 일반적인 결과입니다. 왼쪽 절연 금붕어 바이폴라 세포의 명시야 이미지. 오른쪽 상단 : 바이폴라 세포 축삭 터미널에서 시냅스 vesicles의 TIRF 이미지가 1-43 FM 탑재 ® 및 488 nm의 레이저 (FM 염색)와 몇 군데. 오른쪽 하단 : 561 NM 레이저와 패치 세포 및 이미징 축삭 터미널 후 동일한 터미널 이미지. 시냅스 리본은 rhodamine 기반 RIBEYE 바인딩 펩타이드 (Rpep_rhod)에 의해 표시됩니다. 표 1 : 특정 시약 및 장비. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 이름 </td><td> 유형 </td><td> 제조 업체 </td><td> 목록 </td><td> 논평 </td></tr><tr><td> - </td><td> 에어 테이블 </td><td> 뉴 포트 주식 회사 </td><td> - </td><td> - </td></tr><tr><td> IX70 </td><td> 거꾸로 현미경 </td><td> 올림푸스 </td><td> - </td><td> 명시야의 텅스텐 램프와 TIRF위한 측면 오프닝 포트가 장착된 </td></tr><tr><td> TH4 - 100 </td><td> 램프 전원 </td><td> 올림푸스 </td><td> - </td><td> - </td></tr><tr><td> FF498_581 - Di01 </td><td> 이색성 필터 </td><td> Semrock </td><td> - </td><td> - </td></tr><tr><td> NF01 - 405_488_568 </td><td> 배출 필터 </td><td> Semrock </td><td> - </td><td> - </td></tr><tr><td> APO x100 O HR </td><td> 목표 </td><td> 올림푸스 </td><td> - </td><td> NA 1.65 </td></tr><tr><td> RG630 </td><td> 레드 글래스 필터 </td><td> 쇼트 </td><td> - </td><td> - </td></tr><tr><td> - </td><td> 488 NM 레이저 </td><td> 응집 성의 </td><td> - </td><td> 최소 전력으로 사용 </td></tr><tr><td> - </td><td> 셔터 </td><td> Uniblitz </td><td> - </td><td> - </td></tr><tr><td> VMM - D1 </td><td> 셔터 드라이버 </td><td> Uniblitz </td><td> - </td><td> - </td></tr><tr><td> - </td><td> 561 NM 레이저 </td><td> Melles Griot </td><td> - </td><td> - </td></tr><tr><td> - </td><td> 셔터 </td><td> Uniblitz </td><td> - </td><td> - </td></tr><tr><td> VMM - D3 </td><td> 셔터 드라이버 </td><td> Uniblitz </td><td> - </td><td> - </td></tr><tr><td> 재관류 압력 키트 </td><td> Superfusion 시스템 </td><td> 과학 자동화 </td><td> 09-04 </td><td> - </td></tr><tr><td> 재관류 펜 </td><td> Superfusion 시스템 </td><td> 과학 자동화 </td><td> - </td><td> - </td></tr><tr><td> Valvelink 8 </td><td> Superfusion 시스템 컨트롤러 </td><td> 과학 자동화 </td><td> - </td><td> - </td></tr><tr><td> 캐스케이드 512B </td><td> EM CCD 카메라 </td><td> 로퍼 과학 </td><td> - </td><td> - </td></tr><tr><td> Metamorph 7.1 </td><td> 이미징 소프트웨어 </td><td> 분자 장치 </td><td> - </td><td> - </td></tr><tr><td> EPC - 9 </td><td> 패치 클램프 증폭기 </td><td> HEKA Elektronik </td><td> - </td><td> - </td></tr><tr><td> 펄스 </td><td> 앰프 소프트웨어 </td><td> HEKA Elektronik </td><td> - </td><td> - </td></tr><tr><td> MP - 285 </td><td> Micromanipulator </td><td> 서터 악기 </td><td> - </td><td> - </td></tr><tr><td> - </td><td> 전극 홀더 </td><td> HEKA Elektronik </td><td> - </td><td> 1.5 mm OD 유리, 2 단위 </td></tr><tr><td> 퀵 - Fil ® TW150 - 3 </td><td> Borosilicate 유리 모세관 </td><td> WPI </td><td> - </td><td> 필라멘트없이 </td></tr><tr><td> B150 - 86-10 </td><td> Borosilicate 유리 모세관 </td><td> 서터 악기 </td><td> - </td><td> 필라멘트와 함께 </td></tr><tr><td> P - 97 </td><td> Microelectrode의 풀러 </td><td> 서터 악기 </td><td> - </td><td> 3x3 상자 필라멘트, 환경 챔버가 장착된 </td></tr><tr><td> - </td><td> 압력 진공 공기 펌프 </td><td> 토마스 과학 </td><td> 7893B05 </td><td> pipettes을위한 챔버 및 압력에서 액체를 제거하기 위해 진공을 만듭니다 </td></tr><tr><td> MatLab R2008a </td><td> 분석 소프트웨어 </td><td> MathWorks </td><td> - </td><td> - </td></tr><tr><td> 353001 </td><td> 35mm 플라스틱 문화 요리 </td><td> 매 </td><td> - </td><td> - </td></tr><tr><td> - </td><td> 높은 굴절률 유리 </td><td> PlanOptik </td><td> - </td><td> 굴절률 488 NM = 1.78 </td></tr><tr><td> 시리즈 M </td><td> 높은 굴절률의 액체 </td><td> Cargille 실험실 </td><td> - </td><td> 굴절률 = 1.78 </td></tr><tr><td> Sylgard 184 </td><td> 실리콘 엘라스토머 키트 </td><td> 다우 코닝 </td><td> - </td><td> - </td></tr><tr><td> 글루타티온 </td><td> Tripeptide </td><td> EMD 화학 </td><td></td><td> 무료 radica내 청소 </td></tr><tr><td> Hyaluronidase </td><td> 효소 </td><td> 시그마 </td><td> H6254 </td><td> 유형 V </td></tr><tr><td> L - 시스테인 </td><td> 아미노산 </td><td> Fluka </td><td> 30,090 </td><td> papain을 활성화 </td></tr><tr><td> Papain </td><td> 효소 </td><td> Fluka </td><td> 76,220 </td><td> Carica 파파야에서 </td></tr><tr><td> Trolox ® </td><td> 수용성 비타민 E </td><td> 시그마 </td><td> 56,510 </td><td> 자유 라디칼 스캐빈저 </td></tr><tr><td> ADVASEP - 7 </td><td> Sulfonated B - 사이클로 덱스트린 유도체 </td><td> 시그마 </td><td> A3723 </td><td> FM 1-43 감소 ® 배경 형광 </td></tr><tr><td> FM 1-43 ® </td><td> 형광 염료 </td><td> Invitrogen </td><td> T35356 </td><td> &quot;특수 포장&quot; </td></tr><tr><td> 스티키 왁스 </td><td> 피펫 코팅 에이전트 </td><td> 커 주식 회사 </td><td> - </td><td> 피펫 커패시턴스를 감소 </td></tr></tbody></table> 표 2 :이 연구에서 사용 생리 솔루션을 제공합니다. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 물질 </td><td> 낮은 칼슘 2 + 링거스 </td><td> 제어 링거스 </td><td> 하이 K + 링거스 </td><td> 내부 솔루션 </td></tr><tr><td> NaCl </td><td> 120 MM </td><td> 120 MM </td><td> 97.5 MM </td><td> - </td></tr><tr><td> KCl </td><td> 2.5 MM </td><td> 2.5 MM </td><td> 25 MM </td><td> - </td></tr><tr><td> MgCl 2 </td><td> 1 ㎜ </td><td> 1 ㎜ </td><td> 1 ㎜ </td><td> 4 MM </td></tr><tr><td> CaCl 2 </td><td> 0.5 MM </td><td> 2.5 MM </td><td> 2.5 MM </td><td> - </td></tr><tr><td> HEPES </td><td> 10 MM </td><td> 10 MM </td><td> 10 MM </td><td> 10 MM </td></tr><tr><td> EGTA </td><td> 0.75 MM </td><td> - </td><td> - </td><td> 0.5 MM </td></tr><tr><td> 포도당 </td><td> 10 MM </td><td> 10 MM </td><td> - </td><td> - </td></tr><tr><td> 글루타티온 </td><td> 2 MM </td><td> 2 MM </td><td> - </td><td> 1 ㎜ </td></tr><tr><td> CH 3 CSO 3 S * </td><td> - </td><td> - </td><td> - </td><td> 100 MM </td></tr><tr><td> TEACl </td><td> - </td><td> - </td><td> - </td><td> 10 MM </td></tr><tr><td> ATP - MG </td><td> - </td><td> - </td><td> - </td><td> 10 MM </td></tr><tr><td> GTP - 리 </td><td> - </td><td> - </td><td> - </td><td> 1 ㎜ </td></tr><tr><td> Rpep -로드 &#39;** </td><td> - </td><td> - </td><td> - </td><td> 5 MM </td></tr><tr><td> 음량 </td><td> 200 ML </td><td> 100 ML </td><td> 5 μL </td><td> 100 μL </td></tr></tbody></table> * 세슘 methanesulfonate. ** RIBEYE 바인딩 펩타이드 : rhodamine + EQTVPVDLSVARDR - cooh (MW 1997.75).

<ol>
	<li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+internal+reflection+fluorescence+microscopy+in+cell+biology.">Total internal reflection fluorescence microscopy in cell biology.</a> Traffic. 2, 764-774 (2001).</li><li>Zenisek, D., Steyer, J. A., Almers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport,+capture+and+exocytosis+of+single+synaptic+vesicles+at+active+zones.">Transport, capture and exocytosis of single synaptic vesicles at active zones.</a> Nature. 406, 849-854 (2000).</li><li>Zenisek, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle+association+and+exocytosis+at+ribbon+and+extraribbon+sites+in+retinal+bipolar+cell+presynaptic+terminals.">Vesicle association and exocytosis at ribbon and extraribbon sites in retinal bipolar cell presynaptic terminals.</a> Proc. Natl. Acad. Sci. U. S. A. 105, 4922-4927 (2008).</li><li>Zenisek, D., Horst, N. K., Merrifield, C., Sterling, P., Matthews, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+synaptic+ribbons+in+the+living+cell.">Visualizing synaptic ribbons in the living cell.</a> J. Neurosci. 24, 9752-9759 (2004).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+imaging+of+surface+fluorescence+with+very+high+aperture+microscope+objectives.">Selective imaging of surface fluorescence with very high aperture microscope objectives.</a> J. Biomed. Opt. 6, 6-13 (2001).</li><li>Rouze, N. C., Schwartz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continous+and+transient+vesicle+cycling+at+a+ribbon+synapse.">Continous and transient vesicle cycling at a ribbon synapse.</a> J. Neurosci. 18, 8614-8624 (1998).</li></ol>

빛이 거리와 함께 기하 급수적으로 방울 년부터 2) 목적 형 TIRF 현미경의 장점은 1) 그함으로써 빛을 - 오브 - 초점 밖으로 최소화 목적의 초점 평면 내의 좁은 지역에 여기 광을 제한함으로써 우수한 광학 sectioning을 제공한다는 점입니다 , 수직 방향으로 움직임은 형광 강도의 변화로 모니터링할 수 있으며, 높은 수치 조리개 목적 1.5을 통해 3) 효율적인 조명 컬렉션. 기술의 주요 단점은 그것이 전자 ...

imaging exocytosis in retinal bipolar cells with tirf microscopy

총 내부 반사율 형광 (TIRF) 현미경은 유리 같은 높은 굴절률 물질에 가까운 형광 분자의 선택적 이미징에 의해 세포막에서 일어나는 사건의 연구를 수있게 해주는 기술입니다<sup&gt; 1</sup&gt;. 이 문서에서는, 우리는 금붕어 망막으로부터 격리 망막 바이폴라 세포의 시냅스 vesicles의 이미지 exocytosis이 기술을 적용할 수 있습니다. 이 뉴런들은 대형 축삭 터미널로 인해 이런 종류의 연구에 매우 적합합니다. 양극성 세포를 클램핑 동시에 패치함으로써, 사전 시냅스 전압과 시냅스 릴리스 사이의 관계를 조사 수 있습니다<sup&gt; 2,3</sup&gt;. 양극성 세포 터미널 내부 시냅스 vesicles가에 의해 형광 염료 (FM 1-43 ®)와 함께 로드된 공동 피고 높은 K를 포함하는 염료와 닮은 솔루션<sup&gt; +</sup시냅스 터미널에&gt; 집중. 이것은 세포를 depolarizes하고 glutamatergic vesicles에 endocytosis와 결과의 염료 이해를 촉진. 약 30 분 거리에 여분의 염료를 세척 후, 전지는 패치가 고정되어와 488 nm의 레이저와 동시에 몇 군데 것에 대해 준비가되어 있습니다. 패치 피펫 솔루션은 시냅스 리본 단백질 RIBEYE을 선택 바인딩 rhodamine 기반의 펩티드를 포함<sup&gt; 4</sup&gt; 단말기가 561 nm의 레이저로 몇 군데있을 때이를 구체적으로 리본을 라벨. 이것은 활성 영역의 정확한 현지화 및 추가 - 시냅스 이벤트에서 시냅스의 분리 수 있습니다.

Watch this Scientific Journal Video about Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy at JoVE.com

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

이 비디오에서는, 우리는 총 내부 반사율의 형광 (TIRF) 현미경을 사용하여 금붕어 망막 바이폴라 세포에서 단일 시냅스 소포의 exocytosis와 인​​신 매매를 라벨 및 시각화하는 방법을 보여줍니다.

TIRF 현미경과 망막 바이폴라 세포 이미징 Exocytosis

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

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Research

JoVE Journal

Biology

11.6K 조회수.  Yale University School of Medicine. 이 비디오에서는, 우리는 총 내부 반사율의 형광 (TIRF) 현미경을 사용하여 금붕어 망막 바이폴라 세포에서 단일 시냅스 소포의 exocytosis와 인​​신 매매를 라벨 및 시각화하는 방법을 보여줍니다.

비디오: Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

FM 레이블을 사용하여 뉴런의 Exocytosis 측정

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

연구

생물학

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

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

siRNA Transfection 후의 유사 분열의 시간 저속 영상

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

라이브 세포의 저장소로 작동하는 칼슘 항목의 형광 기반의 측정 : 교양 암 세포에서 골격근 섬유에

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

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) ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

하나 나노 미터의 정확도와 형광 이미징 (피오나)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

TIRF 현미경을 통해 단일 ​​이벤트 해상도에서 G 단백질 결합 수용체의 Clathrin 매개 엔도 시토 시스를 시각화

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

동적 세포 돌출부에서 소프트웨어를 이용한 단백질 농도 추적을위한 그래픽 사용자 인터페이스

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

이미징 FITC-dextran 규제 Exocytosis 위한 기자로 서

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

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 ...