Name: long-term high-resolution intravital microscopy in the lung with a vacuum stabilized imaging window
Uploaded: 2016-10-06T15:00:00.000+00:00
Duration: 7 min 19 s
Description: Watch this Scientific Journal Video about Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window at JoVE.com

This research was supported by NIH-CA100324, Einstein National Cancer Institute's cancer center support grant P30CA013330, R01CA172451 to JWP and the Integrated Imaging Program. This technology was developed in the Gruss-Lipper Biophotonics Center and the Integrated Imaging Program at the Albert Einstein College of Medicine. We acknowledge the support of these Centers in this work. The authors thank Mike Rottenkolber, Ricardo Ibagon and Anthony Leggiadro of the Einstein machine shop for their skilled and timely craftsmanship, the laboratory of Matthew Krummel for generously sharing their window design drawings, Kevin Elicieri and Jeremy Bredfeldt for their expertise in microscopy and their amplifier recommendations and Allison Harney and Bojana Gligorijevic for informative discussions.

The authors have nothing to disclose.

단백질 및 항체로 형광 표지 기능 태그와 함께 생체 광학 영상에서 높은 해상도는 전이성 캐스케이드에 대한 우리의 이해를 증가 극적으로했다. 이는 직접적인 시각화 및 단일 셀 및 종양 세포의 서브 세포 매개 숙주 세포 및 미세 정량화있게되었다. 기본 종양 내에서이 영상은 성장 침입 또는 보급 6,7 중 하나의지지 이산 미세 환경의 발견, 예를 들어, 주도하고있다. 침략의 ...

고해상도 생체 내에 광학 영상은 단일 셀 및 하위 세포 매개 변수 측정 및 정량화 될 수 있도록 다수의 생물학적 과정을 이해하는데 중요한 것으로 입증되었다. 암 연구에서, 종양 및 기질 세포의 생체 내에 영상은 그대로 동물에만 존재하는 많은 미세 환경의 상호 작용 6-11의 발견하게되었다. 생체 내에서 단일 세포 해상도 광학 영상을 이용하여 유방암 intravasation 및 종양 세포의 보급과 관련된 미세 환경에 대한 발견은 심지어 예후 유방암 환자의 12-16 치료에 대한 반응에 대한 신규 마커하게되었다. 그대로 내부 중요한 장기 내에서 깊은 볼에 사용할 수있는 최고의 영상 기술은 전체 기관의 뛰어난 전망을 제공하고 임상 증상을 생산하기도 전에 병리을 표시 할 수있는 임상 양상 (MRI, PET, CT)입니다. 그들은 수없는, 시간owever 때문에 단일 셀 해상도의 부족으로 종양의 진행을 구동 전이의 초기 단계와 세포 메커니즘을 공개합니다. 그 때까지는 폐 전이가이 양식에서 볼 수 있습니다, 그들은 잘 설립 및 확산된다. 그들은 훨씬 이전에 이미 도달 생존의 초기 단계 촬상 19 예상보다 도착 파종 성 종양의 폐에 도달하거나 17 생존하지 않거나 초기 휴지 18 남아 세포 및 관찰의 90 %가 중요하게되는 예상 주어 원거리 사이트에 시딩 전이성 종양 성장의 반복 과정을 이해. 폐에서 이러한 관측을 수행하면 그러나 매우 어려운 입증했다; 영상화 연구의 대부분은 단지 하나의 시점에서 폐로보기를 제공 체외 이식편 또는 제제 (20-23)을 이용했다. 이러한 준비는 유용한 정보를 제공 할 수 있지만rmation, 그들은 미세 환경의 다양한 구성 요소 사이에 발생하는 상호 작용, 원인과 결과 관계, 역학의 완전한 이해를 제공하지 않습니다. 적절한 순환 시스템 (및 항상성 수반 불균형)과 신체의 면역 시스템의 나머지 부분에서 단절의 부족은 내리기이 제제는 생체 내에서 그대로 조직에서 생성 결론을 확인 할 수 있습니다. 많은 그룹은 Wearn과 독일은 흉막 층 (24)을 노출 외과 적 먼저 테리 이식 이미징 창 (25)을 활용하는 첫번째 인에 그대로 폐 2,4,5,24-33의 생체 내에 이미징을 수행했습니다. 폐의 고해상도 영상은 크게 폐의 지속적인 움직임에 의해 방해되고, 몇몇 기술은 이러한 한계를 극복하기 위해 개발되었다. 바그너와 Filley 27 개 폐의 자연적인 움직임을 연구바그너는 조직 (28)를 고정하기 위해 자신의 창 수술 준비에 진공을 이용하면서 상대적으로 고정 된 영역에 걸쳐 이식 창을 찾기 위해 자신의 수술 프로토콜을 설계했습니다. 기관지 클램프, 순차적 인 가사와 게이트 이미징, 오버 샘플링 수집, 폐 엽 진공 (34)의 접착 : 그 이후, 다양한 기술은 이미지에 포함 폐를 활용하고있다. 이러한 각각의 장단점을 갖고 어떤 하나의 기술은 서로 34 우량 등장하지 않았다. 예를 들어, 기관지 클램핑 순차적 무호흡증은 폐에서 가스의 정상적인 교환을 변경하고 무기폐의 원인이 될 수 있습니다. 정문 이미징 및 오버 샘플링 된 인수는 이러한 단점을하지만 고속 또는 전문 영상 장비 널리 액세스 할 수 없습니다 필요하지 않습니다. 결국 폐 모두 접착제 및 진공 기술은 상술 한 문제점을 모두 피할 수 있지만,주의가 탁하지 않은 경우 전단력에 의한 손상을 나타낼 수있다엉. 최근, 진공 창 소형화되었고 공 초점 및 다 광자 현미경 4,5,33 우수한 고해상도 화상을 사용하여 마우스에서 사용되도록 획득 된이. 표 1은 이러한 오랜 역사를 요약 및 신규를 설명 그 종이를 강조 생체 내에 폐 촬상 창 사용의 발전. 이 프로토콜은 가능한 가장 높은 세포 내 해상도 라이브, 그대로 폐의 이미지 전이에 장시간 경과 광자 생체 내에 현미경의 사용을 설명합니다. 이미지는 고 개구 수 대물 렌즈 복수 광전자 증 배관 (PMT) 검출기를 구비 한 다 광자 현미경을 사용하여 최대 12 시간 동안 획득된다. 형질 전환 마우스 모델은 형광 고 분자량 덱스 트란과 형광 단백질 형질 전환 된 종양 세포 (혈관과 종양 세포가 respectivel 레이블을 함께 형광 라벨 기본 식세포에 사용된다와이). 형광 표지 된 세포의이 선택은 종양 세포 내피 세포 - 대 식세포 상호 작용과 역학의 시각화 할 수 있지만,이 프로토콜은 형광 또는 비 형광 마우스의 변형을 위해 작동합니다. 인수 후, 잔류 드리프트 운동 (있는 경우) 레이블이없는 순환 혈액 세포에 의한 점멸 제거하기 위해 35, 36 및 사용자 지정 매크로 시간 평균에게 혈관 채널을 플러그인 피지를 사용하여 제거된다. 이 프로토콜은 촬상 전이에 초점을 맞추고 있지만, 기술은 폐 고해상도 단일 셀 이미징 관찰 다른 여러 생물학적 과정에 적용될 수있다.

<table><tbody><tr><td>Nickel-Plated Brass Vacuum Regulator 1/8 NPT Female, w/ Gauge, 0 - 20&quot; Hg Vacuum</td><td>McMaster Carr</td><td>4172K12&amp;nbsp;</td><td>Vacuum Regulator</td></tr><tr><td>Brass Barbed Hose Fitting Adapter for 1/4&quot; Hose ID X 1/8&quot; NPTF Male Pipe</td><td>McMaster Carr</td><td>5346K13</td><td>Vacuum Regulator Hose Adapter</td></tr><tr><td>Pyrex Brand Filtering Flasks with Tubulation; Neck tooled for rubber stopper No. 4; Capacity: 50 ml</td><td>Corning Life Sciences Glass</td><td>5360-50</td><td>Vacuum Flask</td></tr><tr><td>Round Glass Coverslips Thickness #1.5, 0.16 - 0.19 mm 10 mm dia.&amp;nbsp;</td><td>Ted Pella, Inc.</td><td>260368</td><td>Cover slips</td></tr><tr><td>Exel International Disposable Safelet I.V. Catheters; 22 g x 1 in.&amp;nbsp;</td><td>Exel International</td><td>26746</td><td>Tracheal Catheter</td></tr><tr><td>PERMA-HAND Black Braided Silk Sutures, ETHICON LIGAPAK Dispensing Reel Size 2-0</td><td>VWR</td><td>95056-992</td><td>String</td></tr><tr><td>Liquid Super Glue, Clear, 0.14 oz</td><td>Hendel Corp.</td><td>LOC1647358</td><td>Cyano-acrylate Glue</td></tr><tr><td>Tetramethylrhodamine isothiocyanate&amp;ndash;Dextran</td><td>Sigma-Aldrich</td><td>T1287-500MG</td><td>155 kD Dextran</td></tr><tr><td>Laboratory Clear Tygon PVC Tubing, 1/16&quot; ID, 1/8&quot; OD, 1/32&quot; Wall Thickness, 25 ft. Length</td><td>McMaster Carr</td><td>5155T12</td><td>Thin Tubing &amp;amp; Tubing for Luer</td></tr><tr><td>Crack-Resistant Polyethylene Tubing, 1/8&quot; ID, 1/4&quot; OD, 1/16&quot; Wall Thickness, White, 50 ft. Length&amp;nbsp;</td><td>McMaster Carr</td><td>5181K24&amp;nbsp;</td><td>Thick Tubing</td></tr><tr><td>Depillatory Lotion</td><td>Nair</td><td>-</td><td></td></tr><tr><td>Micro Medical Tubing 95 Durometer LDPE</td><td>Scientific Commodities Inc.</td><td>BB31695-PE/1</td><td>Tubing for tail vein catheter</td></tr><tr><td>30 G x 1 in. BD PrecisionGlide Needle</td><td>BD</td><td>305128</td><td>Needles for tail vein catheter</td></tr><tr><td>Puritan Nonsterile Cotton-Tipped Swabs&amp;nbsp;</td><td>Fisher Scientific</td><td>867WCNOGLUE</td><td></td></tr><tr><td>Clear Polycarbonate Barbed Tube Fitting, Reducing Straight for 3/32&quot; x 1/16&quot; Tube ID</td><td>McMaster Carr</td><td>5117k51</td><td>Connectors between tubes</td></tr><tr><td>One-Hole Rubber Stoppers</td><td>Fisher Scientific</td><td>14-135F</td><td>Stopper for Vacuum Flask</td></tr><tr><td>SHARP Precision Barrier Tips, For P-100, 100 &amp;micro;l</td><td>Denville Scientific Inc.</td><td>P1125</td><td>Pipette Tip</td></tr><tr><td>Laboratory tape</td><td>Fisher Scientific</td><td>159015R</td><td></td></tr><tr><td>Puralube</td><td>Henry Schein Animal Health</td><td>008897</td><td>Opthalmic Ointment</td></tr><tr><td>Gemini Cautery Kit</td><td>Harvard Apparatus</td><td>726067</td><td>Cautery Pen</td></tr><tr><td>Graefe Micro Dissecting Forceps; Serrated; Slight Curve; 0.8 mm Tip Width; 4&quot; Length</td><td>Roboz Surgical</td><td>RS-5135&amp;nbsp;</td><td>Forceps</td></tr><tr><td>Extra Fine Micro Dissecting Scissors 4&quot; Straight Sharp/Sharp 24 mm</td><td>Roboz Surgical</td><td>RS-5912</td><td>Sharp Scissors</td></tr><tr><td>Micro Dissecting Scissors 4&quot; Straight Blunt/Blunt</td><td>Roboz Surgical</td><td>RS-5980</td><td>Blunt Scissors</td></tr><tr><td>Wipes</td><td>Fisher Scientific</td><td>06-666-A&amp;nbsp;</td><td>Harness</td></tr><tr><td>PhysioSuite System</td><td>Kent Scientific</td><td>PhysioSuite</td><td>Vitals Monitor</td></tr><tr><td>1 ml Syringe, Tuberculin Slip Tip</td><td>BD</td><td>309659</td><td>Syringe</td></tr><tr><td>Cyano acrylate</td><td>Staples</td><td>LOC1647358</td><td>Cover Slip Adhesive</td></tr><tr><td>Petroleum Jelly</td><td>Fisher Scientific</td><td>19-086291</td><td>Water Barrier</td></tr><tr><td>Adapter Luer Cannulla 1.5 - 2.2 mm</td><td>Harvard Apparatus</td><td>734118</td><td>Catheter Connector</td></tr><tr><td>PhysioSuite MouseSTAT pulse oximeter</td><td>Kent Scientific</td><td></td><td>Pulse Oximeter</td></tr><tr><td>Isoethesia (isoflurane)</td><td>Henry Schein Animal Health</td><td>50033</td><td>250 ml</td></tr><tr><td>Oxygen</td><td>TechAir</td><td>OX TM</td><td></td></tr><tr><td>1x PBS</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>PVC Ball Valve, Push to Connect, 1/4 In</td><td>Grainger</td><td>3CGJ7</td><td>Vacuum Valve</td></tr><tr><td>Small Animal Ventilator</td><td>Harvard Apparatus</td><td>683</td><td>Alternative is available from Kent Scientific: MouseVent</td></tr><tr><td>OptiMEM Reduced Serum Medium</td><td>ThermoFisher Scientific</td><td>31985062&amp;nbsp;</td><td></td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>ThermoFisher Scientific</td><td>11668019</td><td></td></tr><tr><td>MacBlue Tg(Csf1r*-GAL4/VP16,UAS-ECFP)1Hume/J Mice</td><td>Jackson Laboratory</td><td>026051&amp;nbsp;</td><td></td></tr><tr><td>Multiphoton Microscope</td><td>Olympus</td><td>Fluoview FV1000</td><td>Alternative to custom built scope</td></tr><tr><td>Environmental Enclosure</td><td>Precision Plastics</td><td>Chamber for FV1000</td><td>Alternative to custom built enclosure</td></tr><tr><td>Phosphate Buffered Saline</td><td>ThermoFisher Scientific</td><td>14190136</td><td></td></tr><tr><td>Laser Power Meter</td><td>Coherent</td><td>FieldMaxIITOP</td><td></td></tr><tr><td>Laser Power Meter Head</td><td>Coherent</td><td>PM10</td><td></td></tr><tr><td>pcDNA3-Clover Fluorescent Protein Vector</td><td>Addgene</td><td>40259</td><td></td></tr><tr><td>G418 Sulfate Selective Antibiotic</td><td>ThermoFisher Scientific</td><td>10131027</td><td></td></tr><tr><td>MoFlo Fluorescent-Activate Cell Sorter&amp;nbsp;</td><td>Beckman Coulter</td><td>XDP</td><td></td></tr><tr><td>Trypsin EDTA 1x</td><td>Corning</td><td>25-052-Cl</td><td></td></tr><tr><td>40 &amp;micro;m Mesh</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>96 Well Plate</td><td>Costar</td><td>3599</td><td></td></tr><tr><td>60 mm Culture Dish</td><td>Corning</td><td>430196</td><td></td></tr><tr><td>10 cm Culture Dish</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A4503</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline 1x</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>C57BL/6J Mouse</td><td>Jackson Laboratory</td><td>000664&amp;nbsp;</td><td></td></tr><tr><td>Kim Wipes</td><td>Fisher Scientific</td><td>06-666-A&amp;nbsp;</td><td></td></tr></tbody></table>

long-term high-resolution intravital microscopy in the lung with a vacuum stabilized imaging window

이러한 폐, 간, 뼈 등의 보조 사이트에 전이가 약 90 % 1의 사망률과 외상 이벤트입니다. 이러한 사이트 중 폐 인해 몸, 섬세한 자연과 적절한 생리 유지에 중요한 역할 내에서 밀폐 된 위치로 생체 내에 광학 영상을 사용하여 평가하기가 가장 어렵다. 임상 양상 (양전자 방출 단층 촬영 (PET), 자기 공명 영상 (MRI) 및 컴퓨터 단층 촬영 (CT)이)이 조직의 비침 이미지를 제공 할 수있는 반면, 한 번의 픽셀과 초기 시드 이벤트를 시각화하는데 필요한 해상도를 결여 거의 천 세포로 구성. 단지 종양 세포의 도착 후 이벤트가 생존과 이후의 성장에 결정적 전이성 폐 시드 가정의 현재 모델. 이 단일 세포 분석이 실시간으로 생체 내에 이미징 도구 시딩 CEL의 표현형을 정의하기 위해 필요하다는 것을 의미LS는이 모델을 테스트합니다. 폐의 고해상도 광학 영상은 다양한 생체 제제를 사용하여 수행되었지만, 이러한 실험은 전형적으로 단일 시간 시점 분석하고 (인해 대폭 변화된 환경 온도 풍부, 사이토 카인 등 아티팩트 가능한 잘못된 결론에 취약 ) 흉강 및 순환 시스템 3에서 제거한. 최근 작품은 진공 그러나 영상 창 2,4,5 안정화 사용 가능한 그대로 폐의 시간 경과 생체 내에 광학 영상입니다 보이고있다 일반적인 영상 시간은 약 6 시간으로 제한되어있다. 여기에서 우리는 12 시간의 기간에 걸쳐 이러한 윈도우를 이용한 폐의 장기간 생체 내에 촬영 된 영상을 수행하는 프로토콜을 기술한다. 이 방법을 사용하여 얻어진 시간 경과 이미지 시퀀스는 폐의 시각화 및 세포 - 세포 상호 작용의 정량, 막 역학과 혈관 관류 수 있습니다. 우리는 더 라폐의 미세 혈관의 전례 명확한 도면을 제공하는 영상 처리 기술을 방접원을 그리다.

이 방법으로 얻을 수있는 결과의 유형을 설명하기 위해, 수술 전에 시점 변화에 MacBlue 마우스 (44)의 꼬리 정맥에 형광 단백질 클로버 표지 E0771-LG 종양 세포 주입. 수술 후, 155 가와사끼 로다 민 표지 된 덱스 트란은 혈관과 시간 경과 영상이 수행 된 표시하는 IV를 주입 하였다. 쥐 24 시간 후 분사를 이미징 할 때, 하나?...

Watch this Scientific Journal Video about Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window at JoVE.com

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

진공 안정 이미징 창으로 폐의 장기 고해상도 생체 내에 현미경

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

long-term-high-resolution-intravital-microscopy-lung-with-vacuum

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

13.1K 조회수.  Albert Einstein College of Medicine. This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window. 

비디오: Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

전 비보 인간의 폐 종양에 거주 CD8 T 림프 톨의 이미징 조각 사용 하 여 Confocal 현미경 검사 법

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

연구

암 연구학

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

2 광자 현미경을 사용하여 감염된 폐의 Neutrophils에 의해 Phagocytosis와 NET - 형성의 직접 관측

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

면역학 및 감염병학

Longitudinal Intravital Imaging Through Clear Silicone Windows

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging windows is particularly powerful because it allows longitudinal observation of the same tissue over days to weeks. Typical imaging windows comprise a glass coverslip in a biocompatible metal frame sutured to the mouse&#8217;s skin. These windows can interfere with the free movement of the mice, elicit a strong inflammatory response, and fail due to broken glass or torn sutures, any of which may necessitate euthanasia. To address these issues, windows for long-term abdominal organ and mammary gland imaging&#160;were developed from a thin film of polydimethylsiloxane (PDMS), an optically clear silicone polymer previously used for cranial imaging windows. These windows can be glued directly to the tissues, reducing the time needed for insertion. PDMS is flexible, contributing to its durability in mice over time&#8212;up to 35 days have been tested. Longitudinal imaging is imaging of the same tissue region during separate sessions. A stainless-steel grid was embedded within the windows to localize the same region, allowing the visualization of dynamic processes (like mammary gland involution) at the same locations, days apart. This silicone window also allowed monitoring of single disseminated cancer cells developing into micro-metastases over time. The silicone windows used in this study are simpler to insert than metal-framed glass windows and cause limited inflammation of the imaged tissues. Moreover, embedded grids allow for straightforward tracking of the same tissue region in repeated imaging sessions.

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin.&#160;These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

투명한 실리콘 윈도우를 통한 종방향 생체 내 이미징

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging ...

A Permanent Window for Investigating Cancer Metastasis to the Lung

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as the bone, brain, and lung. Although extensively studied, the mechanistic details of this process remain poorly understood. While common imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), offer varying degrees of gross visualization, each lacks the temporal and spatial resolution necessary to detect the dynamics of individual tumor cells. To address this, numerous techniques have been described for intravital imaging of common metastatic sites. Of these sites, the lung has proven especially challenging to access for intravital imaging owing to its delicacy and critical role in sustaining life. Although several approaches have previously been described for single-cell intravital imaging of the intact lung, all involve highly invasive and terminal procedures, limiting the maximum possible imaging duration to 6-12 h. Described here is an improved technique for the permanent implantation of a minimally invasive thoracic optical Window for High-Resolution Imaging of the Lung (WHRIL). Combined with an adapted approach to microcartography, the innovative optical window facilitates serial intravital imaging of the intact lung at single-cell resolution across multiple imaging sessions and spanning multiple weeks. Given the unprecedented duration of time over which imaging data can be gathered, the WHRIL can facilitate the accelerated discovery of the dynamic mechanisms underlying metastatic progression and numerous additional biologic processes within the lung.

Here, we present a protocol for the surgical implantation of a permanently indwelling optical window for the murine thorax, which enables high-resolution, intravital imaging of the lung. The permanence of the window makes it well-suited to the study of dynamic cellular processes in the lung, especially those that are slowly evolving, such as metastatic progression of disseminated tumor cells.

폐에 암 전이를 조사하기위한 영구적 인 창

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Intravital 현미경 종양 관련 맥 관 구조를 사용 하 여 고급 등 유전자 변형 형광 생쥐 피부 집계 창 실

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

의학

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

고급 폐 유도 빛 시트 현미경 검사 법에 따라 알레르기 염증의 Vivo 모델에서 실험에서 인간 림프 톨의 이미징

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and other respiratory pathologies in vivo is difficult due to the limited accessibility and inherent motion artifacts of the lungs. Nonetheless, various approaches have been developed to overcome these challenges. This protocol describes a method for intravital fluorescence microscopy to study real-time leukocyte-endothelial interactions in the pulmonary microcirculation in an experimental model of ALI. An in vivo lung imaging system and 3-D printed intravital microscopy platform are used to secure the anesthetized mouse and stabilize the lung while minimizing confounding lung injury. Following preparation, widefield fluorescence microscopy is used to study leukocyte adhesion, leukocyte rolling, and capillary function. While the protocol presented here focuses on imaging in an acute model of inflammatory lung disease, it may also be adapted to study other pathological and physiological processes in the lung.

Intravital fluorescence microscopy can be utilized to study leukocyte-endothelial interactions and capillary perfusion in real-time. This protocol describes methods to image and quantify these parameters in the pulmonary microcirculation using a vacuum-stabilized lung imaging system.

진공 안정화 이미징 시스템을 이용한 실험적 급성 폐 손상에서의 폐 미세순환의 생체내 광역 형광 현미경 검사

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung ...

<meta charset="utf8"></head><body>생쥐 폐 이식에서 세포 역학의 실시간 모니터링

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

<meta charset="utf8"></head><body>저자 스포트라이트: 생체 내 이미징을 통한 폐 이식 면역학의 발전

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

이식된 마우스 폐의 생체 내 2광자 현미경

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...

진공 안정 이미징 창으로 폐의 장기 고해상도 생체 내에 현미경

요약

더 많은 비디오 탐색

이 비디오의 챕터

전 비보 인간의 폐 종양에 거주 CD8 T 림프 톨의 이미징 조각 사용 하 여 Confocal 현미경 검사 법

2 광자 현미경을 사용하여 감염된 폐의 Neutrophils에 의해 Phagocytosis와 NET - 형성의 직접 관측

투명한 실리콘 윈도우를 통한 종방향 생체 내 이미징

폐에 암 전이를 조사하기위한 영구적 인 창

Intravital 현미경 종양 관련 맥 관 구조를 사용 하 여 고급 등 유전자 변형 형광 생쥐 피부 집계 창 실

고급 폐 유도 빛 시트 현미경 검사 법에 따라 알레르기 염증의 Vivo 모델에서 실험에서 인간 림프 톨의 이미징

진공 안정화 이미징 시스템을 이용한 실험적 급성 폐 손상에서의 폐 미세순환의 생체내 광역 형광 현미경 검사

이식된 마우스 폐의 생체 내 2광자 현미경

이식된 마우스 폐의 생체 내 2광자 현미경

이식된 마우스 폐의 생체 내 2광자 현미경