This project was supported by grants NIH NIGMS 8P41 GM103540-28 and P50-GM076516

1 샘플 준비 <ol><li> 10 % 소 태아 혈청 및 페니실린 스트렙토 마이신 50 ㎍ / ml의 100 IU / ㎖로 보충 된 DMEM을 사용하여 조직 배양 플라스크에 CHOK1 세포를 유지한다. 37 ° C에서 5 % CO 2 가습 인큐베이터에있는 세포를 품어. </li><li> 수확 후 0.16 mm의면 두께 14mm 직경 마이크로 웰에 CHOK1 세포를 플레이트. 60~70% 자랄 때 주변의 이미징을위한 최적의 밀도에 대한 종자 세포. </li><li> 37 ° C에서 하룻밤 세포를 품어, 5 % CO 2. 녹색과 튜 불린 추적기 녹색의 150 nm의 Lysotracker DND26의 50 nm의를 포함하는 용액에 세포를 HBSS (행크 버퍼 식염수)의 셀을 세 번 세척하고, 부화. 37 ° C에서 1 시간 동안 세포를 품어. 선택 단계 : 부화하기 전에, 미토콘드리아 매트릭스 염색 염료 (25 nM의)와 추가로 얼룩을 수행합니다. </li><li> 어떤 언 바운드 염료를 제거 HBSS에있는 세포를 세 번 씻으십시오. IMAG 이전에 신선한 DMEM 성장 매체를 추가세포를 보내고. </li></ol> 2 현미경 구성 비디오 프로토콜에 기재된 입자 추적 현미경 시판 도립 현미경 (도 4)의 프레임에 조립된다. 3D 궤도 입자 추적을위한 그러나 상용 모듈을 사용할 수 있습니다. <ol><li> 690 nm의 1,040 nm의 사이에 조정 가능한 출력 파장 범위, 사 펨토초 레이저의 여기 광원 : 코 히어 런트 카멜레온 - 울트라 II 티를 사용합니다. </li><li> 레이저 빔이 IR 코팅 거울을 사용하여 현미경의 후면 포트에 정렬되어 있는지 확인합니다. 레이저 빔은 방해석 선형 편광자 다음 회전 반 파장 판을 이용하여 감쇠된다. 샘플에서의 평균 전력은 0.5과 2 mW의 사이가되도록, 빔을 감쇠. 주 : 레이저 빔이 시료면에 집속 빔의 위치 및 궤적의 제어를 허용 검류계 미러 구동 모터의 쌍에 의해 반사된다. 에서짧은 패스 다이크로 익 미러에 반사 한 후 현미경 대물의 후방에 들어가기 전에, 빔 익스팬더 통과 갈바 노 미러의 출사 평행 레이저 빔 확장 전형적인 구성 (10X). </li><li> 광로에 높은 개구 물 대물 (60X, NA 1.2)를 배치하여 샘플에서 형광 광을 수집한다. 하나 또는 두 개의 채널 구성에 원하는 발광 파장에 의한 형광 필터 큐브를 선택. 상기 방출 된 형광 스펙트럼의 범위를 선택하는 발광 대역 통과 필터를 사용한다. </li><li> 전동 무대에 샘플을 놓고 객관적 압전 컨트롤러를 사용하여 현미경 목표의 미세한 움직임을 조정합니다. </li><li> 신호 판별 및 디지털 I / O 데이터 수집 카드로 전송되는 광전자 증 배관 튜브에 필터 정육면체로부터의 광을 직접. NOTE : 컴퓨터 파형은 I / O 카드의 아날로그 출력 및 제공하기 아르 생성스캐너에 에드는 전자를 제어 할 수 있습니다. 광전자 증 배관 및 스캐너로 출력 신호에서 광자의 카운트 입력은 측정 된 형광 SimFCS 역학 소프트웨어를위한 실험실에 의해 I / O 카드를 통해 제어된다. </li></ol> 3 이미징 <ol><li> 무대에서 세포를 놓고 투과광 조명을 사용하여 초점을 맞 춥니 다. </li><li> 염료를 혼입 한 세포를 식별하고 기본 미세 소관을 시각화하는 래스터 스캔 영상으로 전환. 소포 운동이 존재하는 초기 영역을 식별합니다. </li><li> 고립 된 소포가 확인되면, 궤도 추적에 대해 다음 매개 변수를 설정합니다 : <ol><li> 입자의 크기에 따른 추적되는 원형 주사의 크기를 정의하기 위해 궤도 반경을 선택한다. 포인트 이미 터, 감도 및 응답을 최적화 여기 빔의 점 확산 함수 (PSF)의 허리 동일한 궤도의 반경을 설정한다. </li><li> 축 설정축 방향을 따라 파티클 위치 국산화 수행이 궤도 사이의 거리를 정의하는 거리. 1.5-3 배 PSF 허리에 축 방향의 거리를 설정합니다. </li><li> 또한 광 표백 속도를 결정할 것이다 궤도의 각 시점에 소비되는 시간을 설정하는 입자의 밝기에 따라 체류 시간을 정의한다. 10 ~ 100 마이크로 초 사이의 체류 시간을 사용합니다. </li><li> 4-32 밀리의 순서 궤도주기를 산출하고, 입자의 위치를​​ 결정하기위한 높은 시간 해상도를 제공하기 위해 64 또는 128로 각각의 궤도에있는 포인트의 수를 설정한다. </li></ol></li><li> DC보기 래스터 스캔 이미지에서 커서 선택을 통해 그래픽 사용자 인터페이스를 통해 입자에 빔 중앙 위해 거울에 전달 상쇄 신호를 변경한다. </li><li> 궤도 검사에 래스터 이미지에서 현미경 모드를 전환하여 추적을 시작합니다. </li><li> 피드백 및 데이터 수집을 활성화합니다. </li></ol> 4 Traject모리 분석 <ol><li> 궤도와 함께 &#39;강도 카펫&#39;의 형태로 각 시점의 각 시점에서 수집 된 형광을 표시하도록 소프트웨어를 사용한다. 카펫을 따라 수집 된 강도는 다른 밝은 물체로 입자의 상호 작용에 대한 정보를 제공합니다. 물론 시계열의 형태로 시간에 걸쳐 광전자 증 배관으로부터 수집 형광 강도 (x, y 스캐너 및 Z의 -piezo의 시간 동안 DC 변위 즉) 궤적 정보를 모두 표시하도록 소프트웨어를 사용한다. </li><li> 궤도에 대한 관심 만 지역을 선택하는 시계열 표현하고 &#39;강도 카펫의 정보를 사용합니다. </li><li> 입자 궤적의 선택된 부분의 2D 투영을 표시 할 소프트웨어를 사용합니다. 입자의 형광 강도에 따라 색상 코드로 궤도를 적절한 컨트롤을 선택합니다. </li><li> 페이지를 표시하는 옵션을 선택합니다형광 강도에 따라 3D로 문서 궤도, 색상 코드 그것. 미세 소관을 따라 리소좀 운동의 특징을 시각화하기 위해 컨트롤을 사용하여 3D의 궤도를 돌립니다. </li></ol>

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The authors have nothing to disclose.

높은 시간 분해능으로 3 차원 형광 입자의 궤적을 달성 지난 년 동안 형광 현미경 기술과 계장의 엄청난 진행됨에도​​ 불구 분야에서 도전을 유지하고있다. 높은 시간 해상도가 이차원 추적 입자를 달성 한 경우, 축 방향에 확장은 전형적으로 시스템 (10)의 주파수 응답에서의 급격한 감소를 가져온다. 이 동영상 프로토콜에서는 이광자 현미경을 사용하여 높은 시?...

방법 중 다수의 현미경을 사용 입체적으로 형광 발색 입자를 추적하는 날짜로 개발되어왔다. 대부분의 접근은 일반적으로 이상적인 축 방향으로 트래킹을 달성하기 위해 현미경 출사 광학계의 정의 수정과 조합이 차원에서 추적하기 적합한 고속 카메라의 사용에 의존한다. 이 프로세스가 적절한 시간 해상도 (10Hz의)는 일반적으로 시간 소모적이며, 얻을 수 있지만, 종래의 입자가있다 추적되는 경우에만, Z-스택의 시계열을 수행하여 형광체 입자를 추적 할 수있다 (촛점 또는 두 개의 광자 중) 레이저 주사 현미경 액티브 피드백 메커니즘 (2)에 의해 작은 래스터 촬상 영역의 중심에 있었다. 록킹에서 입자로의 개념은 궤도 추적 방법의 기초이다. 대신 래스터의 원형 궤도가 형광 입자의 주위에 수행되는 검사합니다. 따라서 형광의 강도궤도 정확하게 입자의 총수 4 편재. 입자의 국부적 위치는 현미경 스캐너 재 중심 위치에 입자 궤도를 작동하는 데 사용될 수있다. 현미경의 갈바 노 스캐너는 아날로그 전압에 의해 구동된다. 이 전압의 AC 성분은 사인 및 원 궤도를 수행 할 수 있도록 X 및 Y 스캐닝 미러에 적용 코사인 파 즉, 집속 레이저 빔의 궤도를 수행 할 수 있습니다. 신호의 DC 오프셋은 궤도의 중심 위치를 변경하는 것을 용이. 궤도주기가 결정되며, AC 신호 파형이 구성되면, 피드백 시스템은 신호의 단지 DC 성분을 업데이트 할 필요가있다. 검출기로부터 수집 된 형광 신호를 판독 할 수 소프트웨어는 DC 전원을, 스캐너 및 검출기를 제어하기 위해 입자의 위치를​​ 계산하고 갱신 할 필요설정합니다. 궤도 파라미터 (크기 및 타이밍)의 신중한 선택을 묘화 성공적인 피드백에 필요한 이들 파라미터는 추적 할 필요가 형광 물질의 특성에 기초하여 조절 될 필요가있다. 기술의 물리적 및 수학적 기초는 2D 및 3D 애플리케이션 모두에 대해 과거 4,5에 기재 하였다. 이 프로토콜에서는 간단히 셋업의 주요 하드웨어 구성 요소를 설명하고, 더욱 구체적으로 파라미터의 선택과 시료 전처리는 살아있는 세포 내의 높은 시간적 해상도 리소좀의 변위 다음 우릴 있도록 일반적인 실험에 필요한.

<table border="0" cellpadding="0" cellspacing="0" width="525">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:197px;">Name</td>
			<td style="width:160px;">Company</td>
			<td style="width:168px;">Model</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lysotracker DND 26</td>
			<td>Life technologies</td>
			<td>L-7526</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tubuline tracker Green</td>
			<td>Life technologies</td>
			<td>T34075</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mitotracker RED</td>
			<td>Life technologies</td>
			<td>M7512</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coherent Chamelon- ultra II TI</td>
			<td>Coherent</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcite polarize</td>
			<td>Melles Griot Glan&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Galvanometer-motor mirror</td>
			<td>Cambridge technologies</td>
			<td>M 6350</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dichroic mirror</td>
			<td>Chroma technologies</td>
			<td>700 DCSPXR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Motorized stage</td>
			<td>ASI</td>
			<td>MS2000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Piezo&#160;</td>
			<td>PI</td>
			<td>P721-LLQ</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Photomultiplier tube</td>
			<td>Hamamatsu</td>
			<td>H7422P-40</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Data acquisition card</td>
			<td>IO tech</td>
			<td>PCI 1128-4000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaging software</td>
			<td>LFD</td>
			<td>Global for images-SimFCS</td>
		</tr>
	</tbody>
</table>

3d orbital tracking in a modified two-photon microscope: an application to the tracking of intracellular vesicles

이 동영상 프로토콜의 목적은 변형 된 이광자 현미경을 사용하여 1 입체 형광 입자 궤도 추적 실험을 수행하고 분석하는 방법을 설명하는 것이다. 종래의 접근 방식 (래스터 스캔 또는 프레임들의 스택에 기초하여 넓은 시야)에 대향 된 바와 같이, 3D 궤도 추적 지역화 및 높은 공간 (10 nm의 정확도) 및 시간 해상도 (50 Hz에서의 주파수 응답)의 3 차원 변위에 따라 허용 마이크론이 수백의 길이 비늘에 이동 형광 입자. 이 방법은 추적 된 대상물 주위에 원형 궤도를 수행하기 위해 두 개의 광자 레이저 스캐닝 현미경의 하드웨어를 제어하는​​ 피드백 알고리즘을 기반으로한다 : 피드백기구의 변위를 제어하여 중심에 형광 물체를 유지 주사 빔 3-5. 이 기술의 장점을 설명하기 위해, 우리는 알 내에서 빠르게 움직이는 세포 기관, 리소좀을 따라iving 셀 6,7. 세포는 표준 프로토콜에 따른 도금 및 상업적 리소좀 염료를 사용하여 염색 하였다. 우리는 살아있는 세포의 내부 3D 궤도 추적 실험을 수행하기 위해, 간단히 하드웨어 구성과보다 상세히 제어 소프트웨어를 논의한다. 우리는 상세히 주사 현미경을 제어하고 입자 주위 닫힌 궤도 빔의 움직임을 사용하기 위해 필요한 파라미터들을 논의한다. 우리는이 방법이 효과적으로 생균 내에 3 차원 미세 소관을 따라 표시된 리소좀의 빠른 움직임을 추적 할 수있는 방법을 보여줌으로써 결론. 리소좀은 일반적으로 미세 소관 네트워크 8 따라 모션을 지시 표시, 0.4-0.5 μM / 초 범위의 속도로 이동할 수있다.

이 프로토콜에 따라 고속 3D 단일 입자 추적은 형광 표지 리소좀의 변위를 추적하는 개질 이광자 현미경을 이용하여 살아있는 세포 내부에서 수행 될 수있다. 실험 수행 엔도 좀의 성숙 과정 9 후 셀 내부 이동 절연 리소좀 추적 이루어져있다. 리소좀은 녹색 형광 염료를 사용하여 염색하고 2 광자 여기 악용 930 nm에서 여기시켰다. 우리의 데이타는 32 밀리 초 (도 1A)의 시간 해상...

Watch this Scientific Journal Video about 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles at JoVE.com

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

수정 된 두 개의 광자 현미경에서 3D 궤도 추적 : 세포 내 소포의 추적에 응용

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

3d-orbital-tracking-modified-two-photon-microscope-an-application-to

Research

JoVE Journal

Bioengineering

10.1K 조회수.  University of California, Irvine. In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope. 

비디오: 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3 and agarose4, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5 which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact &beta;-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80&deg; C as many 150 &mu;L aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL. 
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1&alpha; for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.

A method for photo-encapsulation of cells in a crosslinked PEG hydrogel is described. Hypoxic signaling within encapsulated murine insulinoma (MIN6) aggregates is tracked using a fluorescent marker system. This system allows serial examination of cells within a hydrogel scaffold and correlation of hypoxic signaling with changes in cell phenotype.

캡슐화된 세포 집계 내에서 추적 Hypoxic 신호

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

연구

생체공학

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

입자 추적 microrheology은을 사용하여 3D 종양 모델에서 세포 외 매트릭스 강성의 경도 측정

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

표면 형광을 가진 조직 매트릭스 구성 요소 및 두 개의 광자 현미경의 장기의 intravital 면역 형광 이미징

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

입자 추적 Microrheology에 의한 바이오 필름의 기계적 성질의 제자리 매핑에서

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

입자의 공간 추적을위한 멀티 플렉스 전자 감지와 미세 유체 플랫폼

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

마이크로 웰 어레이 플랫폼 내 미생물 공동체 개발의 구성 및 추적

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

실시간 3D 단일 입자 추적에 대 한 프로토콜

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

균일 한 골 재를 생산 하 오 모양의 혈관에 포유류 세포의 문화를 떨고는 궤도

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

메카노생물학적 연구를 위한 궤도 셰이커에 6웰 플레이트에서 내피 세포의 분할 성장

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.