This work is funded by the Cure for Cancer Foundation, Dutch Technology Foundation (STW) and The Netherlands Organisation for Health Research and Development (ZonMw).

제시된 절차는 대학 의료 센터 암스테르담, 등록 번호 NL41985.018의 임상 시험 심사위원회의 승인을 연구 프로토콜에서 발생한다. 서면 동의서는 모든 참가자가 필요합니다. 1. 시스템 <ol><li> 이 실험을 위해, 1,280-1,350 nm 파장 대역 (13)에서 동작하는, 푸리에 도메인의 OCT 시스템을 사용한다. 푸리에 도메인 저 코 히어 런스 간섭계는 제 세대 시간 영역의 OCT 시스템에 비해 데이터 획득 속도를 증가 연속 스캔을 허용한다. 주 : OCT 시스템은 90 ° 각도로 나선형 ~, 광섬유 탐침을 스캔 인터페이스된다. 그것은 2.7F의 외경 (0.9 mm)와 135cm의 삽입 길이를 가지고있다. 프로브는 54mm의 철수 범위와 구동 모터 및 광 컨트롤러 (설치 독)를 통해 OCT의 콘솔에 연결합니다. 인수의 OCT 데이터 세트는 541 단면 이미지 (B-스캔) w 구성15 ㎛, 축 해상도 i 번째 (도 1, 2). </li><li> 정확하고 재현성있는 감쇠 측정을 보장하기 위해, 측정함으로써 교정 μ 10월 Kodach 등. (14) (15)에 의해 전술 한 바와 같이 (예 Intralipid) 지방 에멀젼의 중량 퍼센트에 기초하여 증가하는 농도에 대한. 한마디로 : <ol><li> 0.125, 0.250, 0.5, 1.0, 2.0, 4.0, 10, 15 및 20 (주) 퍼센트의 농도를 달성하기 위해 탈염 H 2 O와 20 %의 지방 유제의 표준 배치를 희석. <ol><li> 지방 에멀젼 혼합물 200ml에 프로브의 OCT를 놓고 측정의 OCT를 획득. </li><li> 상호 참조 문헌에 알려진 값 μ 10월 값을 추출 하였다. </li></ol></li></ol></li></ol> 2. 타임 아웃 및 환자 위치 <ol><li> 절차를 시작하기에 앞서, 이름, 생년월일, 절차, 홍보의 날짜를 확인 &quot;시간을&quot;수행ocedural 측, 항응고제 사용, 알레르기. </li><li> 종양의 위치에 따라 하나 또는 경향 측와위에서 환자를 배치했다. 적절한 지원과 함께 환자를 제공하고 그 / 그녀가 20 ~ 40 분 동안의이 위치에 편안하게 기대하는 경우 확인합니다. </li><li> 초음파 (US) (16)를 사용하여, 종양 지역화 영구 잉크로 피부에 바늘 엔트리 포인트를 표시한다. 참고 : 컴퓨터 단층 촬영 (CT)를 사용하는 경우, 액세스 바늘의 원하는 위치를 국산화 유연한 바늘 안내 템플릿을 사용합니다. </li></ol> 3. 소독과 멸균 드레이핑 <ol><li> 외과 모자와 입을 커버를 착용 할 것. </li><li> (2.3 단계) 이전에 배치 바늘 항목 표시를 제거하지 않도록주의하면서, 클로르헥시딘 / 알코올 용액을 사용하여 천자 사이트 주위의 피부를 청소합니다. 넓은 지역을 소독하는 것은 예기치 않은 액세스 바늘 위치 변경시 추가 청소의 필요성을 방지 할 수 있습니다. </li><li> R로10 ML의 주사기, 무딘 흡인 바늘, 21 G 주사 바늘, 메스, 15 G 동축의 도입 니들, 18 G 투관침 바늘 및 16 : 멸균 내용의 egard가 포함 된 경피적 천자 세트를 엽니 다 G 코어 생검 총. </li><li> 이후 손 소독제를 적용, 철저하게 손을 씻으십시오. 수술 가운 및 멸균 장갑을 착용 할 것. </li><li> 멸균 커튼에서 환자를 커버. </li><li> 초음파 프로브 주위에 살균 커버를 적용하고 장소에 봉침을 수정합니다. </li></ol> 4. 10월 준비 <ol><li> OCT의 콘솔을 시작하고 콘솔 인터페이스를 사용하여 환자 ID 표시된 필드, 성, 이름과 생년월일 (생년월일)에서 환자 정보를 입력합니다. </li><li> 멸균 내용과 관련하여, 간섭 단층 조사, 멸균 장착 독 커버하고, 5 ml의 루어 잠금 주사기를 포함 OCT의 패키지 압축을 풉니 다. </li><li> OCT의 콘솔 설치 독 멸균 커버를 적용합니다. 비 멸균 장착 독을 필요 안내조수의 도움이됩니다. </li><li> 0.9 % NaCl로 5 ML의 주사기를 입력하고 플러싱 포트에 연결합니다. 물이 프로브 커버의 말단 부분에 나타날 때까지 OCT의 프로브를 세척합니다. </li><li> 장착 도크에 OCT의 프로브를로드합니다. 회전과 적절한 기능을 확인 붉은 빛을 방출한다 프로브를로드 한 후. 세척 및 손상의 위험을 최소화하기 위해로드시의 보호 커버에 프로브를 남겨주세요. </li><li> 표지에서 OCT의 프로브를 제거합니다. 딱딱한 표면에 프로브를 놓고 끝을 단축하기 위해 메스를 사용합니다. 광섬유와 프리즘에 압력을 최소화하기 위해 절단시 프로브의 선단부를 고정한다. 방향을 방출 (적색) 빛을 사용하여, 프리즘에서 5mm의 말단을 잘라. </li></ol> 5. 펑크 <ol><li> 피부와 2 % 리도카인을 사용하여 깊은 층 (20 ㎎ / ㎖)을 마취. 을 적용하려면 리도카인을 허용 몇 분 정도 기다립니다. 통증이있는​​ 경우 환자에게 문의하십시오. </li><li> 봉침을 사용하여, 배치촬상 통해 위치 확인 15 G 동축의 도입 니들. 배치가 만족 스러우면, 밀폐 장치 (날카로운 바늘 코어)을 제거한다. </li><li> 종양 피어싱, 도입 니들을 통해 18 G 투관침 바늘을 놓습니다. 다시 촬상와 바늘의 위치를​​ 확인한다. 배치가 만족스러운 경우 폐색을 제거합니다. </li><li> 저항 느낌까지 투관침 바늘까지 OCT의 프로브를 공급. </li><li> OCT의 프로브를 고정하고, 종양 조직으로의 OCT 프로브 노출 투관침 바늘 후퇴. 종양 내 투관침 바늘의 끝을 유지하는 것은 호흡 사이클 동안 OCT의 프로브의 꼬임을 최소화한다. 이것은 프로브 손상의 위험을 낮춘다. </li><li> 10월 검색 : <ol><li> 데이터 세트 당 541 B-검사에서 설정 한 콘솔,의 OCT 검사를 수행합니다. 여기에 사용 된 시스템의 OCT 특정 파라미터 조정이 필요없는 5.4 cm의 길이에 걸쳐 자동 철수를 수행한다. </li><li> (품질, 유물에 대한 스캔 및 고체 조직의 모양을 확인도 1a). 공예품은 가장 일반적으로 원형 띠가 정상의 OCT 패턴 (그림 1B)에서 밖으로 서 나타납니다. </li><li> 유물을 다시 검사 후 지속되면 프로브를 교체합니다. </li></ol></li><li> 10월 3일 데이터 세트의 최소 때까지 단계를 반복 5.6 취득. </li><li> 현재 위치에서 도입 니들을 떠나, OCT의 프로브와 투관침 바늘을 제거합니다. </li><li> 핵심 생검 총을 팔과 영상의 위치를​​ 확인, 도입 니들을 통해 놓습니다. </li><li> 위치 결정이 양호하면, 생검 총을 발사. </li><li> 병리과 프로토콜에 따라 용기에 조직 검사 재료를 놓습니다. 여기에, 종이 속지 배양 접시에 장소 생검은 충분히 0.9 % NaCl로 포화. </li><li> 핵심 조직 검사 품질을 확인하고 충분한 자료를 얻을 때까지 단계 5.9와 5.10를 반복합니다. </li></ol>

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The authors of this article have nothing to disclose.

이 책에서 우리는 신장, 10월 기반 경피적 바늘의 가능성에보고한다. 이것은 &quot;광 조직 검사&quot;로 불리는 종양의 분화에 대한 임상 적용 기술에의 OCT 개발에 필수적인 첫 번째 단계입니다. 우리의 첫 번째 25 명의 환자는 쉽고 안전한 절차로 경피적 OCT를 보여 주었다. 광학 생검은 기존의 핵심 조직 검사를 통해 두 가지 장점이있다. 먼저, OCT 데이터의 실시간 수집 및 분석은 종래 병리학 처리 시...

지난 수십 신장 대중 1, 2의 발생률이 꾸준히 증가를 보여 주었다. 지금까지 신장 질량 치료 결정은 주로 MRI 및 CT 결상 특성, 나이 및 동반 질환에 기초하여 이루어지고있다. 그러나 이러한 진단 방법과 임상 매개 변수는 진정으로 신장 대량의 악성 가능성을 감지하는 기교가 부족하다. 핵심 조직 검사 또는 병리학 적 평가 (진단)에 대한 충분한 조직과 미세 침 흡인는 95~100% 3의 범위에서 민감도와 특이도 모두 객관적인 종양의 분화를 제공합니다. 따라서 조직 검사는 의심 신장 대중 4,5의 평가에 동의를 얻고있다. 그러나, 충분한 조직없이 생검 지연, 진단 또는 신장 실질 (비 - 진단) 정상에 전체 10 내지 20 %의 비율로 발생하고, 심지어 작은 신장 질량의 30 % 이하로 (&lt;4cm, SRM들)을 수립 추가적인 대한 필요성으로 인해 자주 진단 과정조직 검사 절차 3,5. 광 결맞음 단층 영상 OCT (는) 신장 질량 분화에서 상기 장애물을 극복 할 수있는 잠재력을 갖는 신규 한 영상 기법이다. 근적외선 빛의 후방 산란에 기초하여, OCT를 2~3mm의 효과적인 조직 침투에 15㎛의 축 해상도 이미지를 제공한다 (도 1, 2). 조직 침투, 조직 특이 광산란 얻어진의 밀리미터 당 신호 세기의 손실 감쇠 계수로 표현된다 (μ OCT를 mm -1). 페이버 등 (6)에 의해 설명 된 바와 같이. 조직 학적 특성은 조직의 분화 (그림 3)에 대한 정량적 매개 변수를 제공 간섭 단층 값을 μ 상관 관계가 될 수있다. 발암 동안, 악성 세포는 수가 증가와 함께 더 크고 불규칙적 인 모양을 표시 핵<html> 더 높은 굴절률 및 더 활성 미토콘드리아. 인해 세포 성분이 과발현, μ OCT의 변화에 영향을받지 양성 종양 조직 또는 악성 종양 (7)에 비교할 때 예상 할 수있다. 최근에 우리는 양성과 악성 신 대중 8,9 구별하는 표면의 OCT 능력을 공부했다. 16 명의 환자에서 종양 조직의 수술 중의 OCT 측정은 외부 배치의 OCT 프로브를 사용하여 수득 하였다. 동일한 환자의 조직의 영향을받지 않고 측정의 OCT 구성된 제어 암. 정상 조직은 종양의 OCT 분화 잠재력을 확인하는 악성 조직에 비해 상당히 낮은 중앙값 감쇠 계수를 나타내었다. 이러한 정량 분석은 이러한 urothelial 암종 10,11 외음부 상피 종양 분화 12 악성 조직의 다른 유형의 등급에 유사한 방식으로 적용되었다. ENT는 &quot;&gt; 우리는 실시간 이미징에 현장 종양의 분화와 결합. 본 연구의 목적은 기반 경피적 바늘을 설명하는 것입니다 제공, 광학 생검으로 OCT를 개발하는 것을 목표로, 환자의 10월 방법은 진단 고체 강화 신장 질량.이 방법의 설명은 우리가 알고있는, 최초의 신장 종양의 바늘의 가능성을 기반으로 OCT를 평가합니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>15G / 7.5cm Co-Axial Introducer Needle</td>
			<td>Angiotech, Gainesville, USA</td>
			<td>MCXS1612SX</td>
			<td></td>
		</tr>
		<tr>
			<td>18G / 20cm Trocar Needle</td>
			<td>Cook medical, Bloomington, USA</td>
			<td>DTN-18-20.0-U</td>
			<td></td>
		</tr>
		<tr>
			<td>16G / 20cm Quick-Core Biopsy Gun</td>
			<td>Cook Medical, Bloomington, USA</td>
			<td>G07827</td>
			<td></td>
		</tr>
		<tr>
			<td>Ilumien Optis PCI Optimization System (OCT &#38; FFR)</td>
			<td>St. Jude medical, St. Paul, USA</td>
			<td>C408650</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>Dragonfly Duo Imaging Catheter</td>
			<td>LightLab Imaging, Westford, USA</td>
			<td>C408644</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>Sterile Dock Cover</td>
			<td>CFI Med. Solutions, Fenton, USA</td>
			<td>200-700-00</td>
			<td>Part of Dragonfly Kit. 
			St. Jude medical, St. Paul, USA. (C4088643)</td>
		</tr>
		<tr>
			<td>5ml Luer-lock Syringe</td>
			<td>Merit Med. Syst., South Jordan, USA</td>
			<td>C408647</td>
			<td></td>
		</tr>
		<tr>
			<td>10ml Syringe</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>300912</td>
			<td></td>
		</tr>
		<tr>
			<td>18G Blunt Fill Needle</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>305180</td>
			<td></td>
		</tr>
		<tr>
			<td>21G Injection Needle</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>301155</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scalpel</td>
			<td>BD, Franklin Lakes, USA</td>
			<td>372611</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 0,9% solution</td>
			<td>Braun, Melsungen AG, Germany</td>
			<td>222434</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidoca&#239;ne HCl 2% (20mg/ml) solution</td>
			<td>Braun, Melsungen AG, Germany</td>
			<td>3624480</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Ultrasound Gel, Aquasonic 100</td>
			<td>Parker Lab. Inc., Fairfield, USA</td>
			<td>GE424609</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Ultrasound Cover</td>
			<td>Microtek Med., Alpharetta, USA</td>
			<td>PC1289EU</td>
			<td></td>
		</tr>
		<tr>
			<td>Pathology Container</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AMIRA software package</td>
			<td colspan="2">FEI Visualization Sciences Group, Hillsboro, USA</td>
			<td>Software platform for 3D data analysis</td>
		</tr>
		<tr>
			<td>FIJI software package (open source)</td>
			<td>Open source, http://fiji.sc/Fiji</td>
			<td></td>
			<td>Open source image processing software</td>
		</tr>
	</tbody>
</table>

in vivo, percutaneous, needle based, optical coherence tomography of renal masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real time images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm. Within the OCT images the loss of signal intensity per millimeter of tissue penetration, the attenuation coefficient, is calculated. The attenuation coefficient is a tissue specific property, providing a quantitative parameter for tissue differentiation. 
Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these parameters and diagnostic methods lack the finesse to truly detect the malignant potential of a renal mass. A successful core biopsy or fine needle aspiration provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100%. However, a non-diagnostic rate of 10-20% overall, and even up to 30% in SRMs, is to be expected, delaying the diagnostic process due to the frequent necessity for additional biopsy procedures. 
We aim to develop OCT into an optical biopsy, providing real-time imaging combined with on-the-spot tumor differentiation. This publication provides a detailed step-by-step approach for percutaneous, needle based, OCT of renal masses.

제 25 종양 (23 명) 중 24의 OCT 성공적인 절차 총 수행 하였다. 하나의 경우 프로브 고장의 OCT 검사를 취득 할 수 없다는되었다. 설명 부분에서 상세히 설명된다 (AE)가 발생한 두 부작용. 일반 환자의 특성은 표 1에 있습니다. OCT를 콘솔 획득 한 데이터 세트의 즉각적인 질적 분석을 위해 리얼 타임 10 월 이미지를 제공하는 소프트웨어를 미리 설치했다. 추가 분석 및 ?...

Watch this Scientific Journal Video about In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses at JoVE.com

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

비보, 경피적, 니들 기반, 신장 질량의 빛 간섭 단층 촬영에서

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...

in-vivo-percutaneous-needle-based-optical-coherence-tomography-renal

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JoVE Journal

Medicine

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

8.8K 조회수.  Academic Medical Center. Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

비디오: In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

망막 순환 도플러 광 결맞음 단층 촬영 장치

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

연구

의학

Near Infrared Optical Projection Tomography for Assessments of &beta;-cell Mass Distribution in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen's (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)2; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction2 and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations3. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.

Near Infrared Optical Projection Tomography for Assessments of &#946;-cell Mass Distribution in Diabetes Research

We describe the adaptation of optical projection tomography (OPT)1 to imaging in the near infrared spectrum, and the implementation of a number of computational tools. These protocols enable assessments of pancreatic &beta;-cell mass (BCM) in larger specimens, increase the multichannel capacity of the technique and increase the quality of OPT data.

당뇨병 연구에서 β-세포 질량 분포의 평가를위한 적외선 광학 프로젝션 단층 촬영 부근

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

생성 및 마우스의 동맥 병변의 3 차원 정량 광학 프로젝션 단층 촬영을 사용하여

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

세포 추적을위한 생체 19 F MRI에서

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

경피적 바늘 생검에 의해 획득 골격근 조직에서 고립 된 미토콘드리아의 준비 및 호흡계 평가

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

인간 종양 이종 이식에서 마우스 세포의 고갈은 크게 대상 세포의 다운 스트림 분석을 향상

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

초고 해상도 마우스 광학 일관성 단층 촬영 망막 유전자 치료 연구에서 안 구내 주사를 돕기 위해

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

Vivo에서 스펙트럼-도메인 광학 일관성 단층 촬영 스캐닝 레이저 Ophthalmoscopy와 Cx3cr1gfp/gfp 기자 쥐의 이미징

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

신장 기능 사 질병 마우스 모델에서의 평가

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis (NASH) Resolution

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and assessment of improvement in disease endpoints, often reflected as a percent of each treatment arm that improved, worsened or remained unchanged. Traditional preclinical rodent studies for putative NASH therapies are often limited by not knowing the level of liver disease/NASH present at the start of therapeutic intervention, instead of randomizing treatment groups on easily measurable endpoints such as body weight, metabolic status or similar. Here, we describe a liver biopsy technique in a diet-induced NASH mouse model, for the assessment of baseline liver disease in order to exclude mice that do not exhibit fibrosis and to equally distribute animals with similar fibrosis between treatment groups. These levels can then be compared to the terminal, post-intervention levels for a truer understanding of in vivo pharmacological effects and thus more accurately reflect clinical trial design strategies. The mouse is properly anesthetized and prepared for the surgery using sterile conditions. A small incision is made in the upper abdomen and the left lateral lobe of the liver is exposed. A wedge of the liver is surgically removed, and a similar-sized piece of absorbable gelatin is put in its place to stop any bleeding. The mouse is surgically sutured and stapled closed and will recover back to normal within 1 day. The entire process takes 5-10 min per mouse. Here we exemplify the utility of this procedure by leveraging the pre-study biopsy to assess the impact of the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide on NASH endpoints in mice.

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis NASH Resolution

In clinical settings, a liver biopsy is used to assess stages of non-alcoholic steatohepatitis (steatosis, inflammation, hepatocyte ballooning, and fibrosis). Here, we illustrate a survivable liver biopsy in mice which can be used for histological analyses to enable assessment of therapeutic agents in a manner more aligned with clinical trials.

무알코올 Steatohepatitis (NASH) 해상도 평가 하기 위해 쥐에 지속 가능 간 생 검 절차의

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and ...

비보, 경피적, 니들 기반, 신장 질량의 빛 간섭 단층 촬영에서

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