저자는 아직 승인이 없습니다.

1. 운모 및 슬라이드 준비 <ol><li> 장소 번호 1 ½ (0.17 mm)는 염색 랙에 커버 슬립. </li><li> 60 ° C에서 2 % 세제에 30 분 동안 초음파 처리 </li><li> 탈 이온수로 20 회 반복한다. </li><li> 집게를 사용하여 슬라이드를 제거하고 압축 공기 또는 질소를 사용하여 건조한다. </li><li> 운모 시트를 가위 또는 면도날을 사용하여 10 × 10mm 사각형 조각으로 잘라. </li><li> 면도날을 사용하여 2 ~ 3 얇은 전단지에 각 운모 조각을 잘라. 참고 :이 단계는 날카로운 칼날의 사용을 필요로합니다. </li></ol> 2. 운모 조립 및 장착 상공 회의소 <ol><li> 에탄올로 청소 현미경 유리 슬라이드. </li><li> 광학 접착제를 사용하여 유리 슬라이드에 단계 1.6 절단 운모의 접착제 전단지. 낮은 점도의 접착제가 더 강하를 확산하고 운모를 접착하는 것이 좋습니다. </li><li> , UV 램프에서 10 분을 치료. NOTE : 접착제는 350 ~ 380 ㎚의 범위에서 최대 흡수 및 requ의 추천 에너지로 UV 광에 의해 경화된다전체 치료를위한 IRED 4.5 J / ㎠이다. 그러나, 다른 광원 (접착제 공급 업체에서 제공하는 자료를 참조)이 단계를 사용할 수 있습니다. </li><li> 스카치 테이프로 처음 몇 층을 제거하여 깨끗한 운모 표면을 노출. </li><li> 운모 표면에 광학 접착제의 작은 방울 (~ 20 μm의)를 배치합니다. 참고 :이 단계에서, 낮은 형광도 수준 높은 점성 접착제를 권장합니다. 우리의 경험은 낮은 (2.2 단계) 고점도 접착제의 조합을 사용하는 운모 분할 상당히 효과 (단계 2.7)을 증가 시킨다는 것을 보여 주었다. </li><li> 조심스럽게 기포를 방지, 접착제의 하락에 갓 청소 커버 슬립을 배치, 1 분에 정착 할 수 있습니다. </li><li> , UV 램프에서 10 분을 치료. NOTE :이 단계 후에, 유리 슬라이드, 운모 및 커버 슬립의 샌드위치 (몇 주까지) 시간의 비교적 긴 기간 동안 저장 될 수있다. 운모 표면이 신선한 확인하기 위해, 단지 실제 SLB 준비하기 전에 다음 단계로 진행합니다. 참고: 접착제가 380 ㎚ ~ 350의 범위에서 최대 흡수와 완전 경화에 필요한 에너지를 추천 UV 광에 의해 경화된다 4.5J/cm는 2이다. 그러나, 다른 광원 (접착제 공급 업체에서 제공하는 자료를 참조)이 단계를 사용할 수 있습니다. </li><li> 비디오 같이 exacto 칼을 사용하여 부드럽게 측 유리 슬라이드에서 커버 슬립을 분리. 대부분의 경우에, 운모의 얇고 편평한 층은 커버 슬립에 연결된 상태로 유지된다. (단계 2.4에서 반복) 재사용 할 수 운모 유리 슬라이드에 부착. </li><li> 운모 층이 여전히 커버 슬립에 접착되는 단계 2.8 분할시 완전히 제거되지 않았 음을 확인하기 위해, 육안으로 또는 해부 현미경으로 표면 품질을 확인합니다. 집게에 작은 상처를 만들거나 바늘을 해부하는 것은 운모에서 검출 가능하게 다른 일관성이있는 접착제를 구별하는 데 도움이 될 것입니다. </li><li> 1.5 ㎖의 유리 병 뚜껑의 고무 패킹을 제거하고 광을 사용하여 표면에 거꾸로 뚜껑을 접착제접착제 또는 매니큐어 및 UV 램프를 치료, 또는 공기는 각각 10 분을 건조 할 수 있습니다. 참고 : 샘플 AFM 이미징과 동시에 광 (TIRFM 또는 공 초점)을 준비하는 경우, 1.5 ㎖의 유리 병 뚜껑을 현미경 무대에 AFM 헤드 마운트 너무 작은 수 있습니다. 그 경우, 캡 AFM 헤드 장착에 적합한 큰 직경을 갖는 임의의 플라스틱 O-링에 의해 치환 될 수있다. 실험 운모 표면을 손상시키지 않고, 캡을 제거 요구의 경우, 실리콘 그리스 대신 접착제 또는 매니큐어 사용될 수있다. </li></ol> 3. 지원되는 지질 이중층 (SLB) 형성 <ol><li> 면에 실에 새로 제조 된 리포좀 솔루션을 놓습니다. SLB 형성에 필요한 최소 볼륨 ~ 30 μL입니다. 참고 : 리포좀 및 SLB 형성에 대한 자세한 내용은, 예컨대 가방 등 한 출판 프로토콜을 참조 2014 년 15.. </li><li> 원하는 프로토콜을 사용하여 SLB 형성을 진행합니다. 부화 중이미징, 챔버는 열 블록 또는 용융 온도 이상으로 사용되는 지질을 유지하는 데 필요한 온도를 유지하기 위해 가열 현미경 단에 배치 될 수있다. </li></ol>

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저자가 공개하는 게 없다.

이 프로토콜은 지질 이중층 증착 및 고해상 이미징 부드럽고 얇은 운모 표면의 제조 방법을 설명한다. 기술은 주로 고품질 운모 표면을 얻기위한 중요한 유리 운모 - 유리 샌드위치 (단계 2.8)의 세세한 제외에 한정되는 최소한의 수동 기술을 필요로한다. 운모는 클 리빙없이 광학 접착제에서 분리하는 것이 가능하기 때문에 갓 절단 운모의 검사는 항상 광학 접착제의 노출 된 영역을 남기고,이 시?...

본 프로토콜의 전반적인 목표는 원자 힘과 결합 될 수있는 광학 전반사 형광 현미경 (TIRFM) 또는 공 초점 현미경을 이용한 운모지지 지질 이중층 (SLB 수)의 고해상도 영상화 운모 표면의 제조 방법을 도시하는 것이다 현미경 (AFM). 유동화 증권 지질 클러스터링, 상 분리, 펩티드, 단백질 또는 다른 화합물 1-5 이중층 구성 요소 또는 상호 작용의 역학의 수많은 연구를위한 널리 사용되는 모델입니다. 다른 기판은 SLB 형성 연구 4,6-8의 특성에 따라 (즉, 유리, 운모, 이산화 규소, 중합체)에 사용될 수있다. 분리막 연구는 TIRFM 및 AFM 같은 현미경을 기반 영상 기술에 의존하고 있습니다. 유리는 투명하기 때문에 이에 TIRFM 이미징, 유리 표면은 일반적인 선택이다. 유리의 제조가 비교적 용이하고, 결과의 품질은 주로이전에 지질 소포의 증착 청소 철저한 표면에 의해 결정된다. 높은 축 방향의 해상도에 의한 AFM은 운모 표면을 필요로한다. 운모는 완벽한 기초 분열 부근으로, 규산염 광물이다. 따라서, 갓 절단 운모 심지어 서브 - 나노 미터 스케일 (9)에 막 고저차의 관찰을 허용, 원자 적으로 평면이다. 이러한 형광 상관 분광법 (FCS), 추적 단일 분자 (SMT) 및 (FRAP)을 photobleaching에 후 형광 복구와 같은 방법을 사용하여 확산 연구는 지질 막 역학들이 퇴적되는에 표면의 유형, 이에 유리에 크게 의존하지만 보여 주었다 운모는 매우 다양한 결과 10, 11를 줄 수 있습니다. 이러한 차이는 멤브레인 프로브의 확산 계수뿐만 아니라, 서로 다른 속도로 확산 입자의 분리 된 집단의 검출 및 가능한 다른 상태 사이의 전환뿐만 있습니다. 따라서,동일한 표면 (이 경우 운모에) 사용되지 않는 TIRFM 및 AFM 기술을 사용하여 얻어진 결과의 직접적인 비교가 종종 문제가된다. TIRFM 및 AFM 이중층 이미징이 같은 운모 표면 (12, 13)에 실시 된 일부 연구가 있지만, 운모는 거의 대부분 때문에 처리 문제, 광학 현미경에 사용되지 않습니다. 운모 준비는 광학 접착제 (12)를 사용하여 커버 슬립에 붙어있다 얇은 전단지,에 손으로 절단을 필요로한다. 그러나이 방법은 만족스러운 결과를 달성하기 위해 연습이 필요합니다. 더욱이, 얻어진 표면은 그들 어려운 낮은 작동 거리, 높은 개구 수 렌즈를 사용할 수 있도록, 흔히 물결 두껍다. 이 프로토콜에 기술 된 바와 같이 제조 된 운모의 표면은 매우 얇은 (170 μM의 coverslip에 두께를 포함하여 ~ 220 μm의) 매우 평면, 성공 고해상도 이미징을위한 중요하다 &quot;파상&quot;를 피하는. 그들은 사용될 수있다TIRFM 또는 공 촛점 설정하십시오. 또한, 동일한 시료를 AFM에 전달 될 수 있고, 심지어 TIRFM / 촛점과 AFM과 동시에 이미지화. 이러한 두 가지 기술을 결합하면 이중층 막 구조물 (14)과 확산 거동의 직접적인 상관 관계를 허용한다. 운모 표면이 갓 절단되어 있기 때문에, 그들은 깨끗하고 (유리 청소 프로토콜은 일반적으로 같은 피라니아 솔루션, 황산, 나트륨 / 수산화 칼륨 등 화학 물질을 포함한다) 시간이 많이, 제대로 재현하고, 잠재적으로 위험한 청소 절차가 필요하지 않습니다. 또한이 프로토콜에 설명 작은 챔버의 설치​​, 50 미만 μL에 효과적인 이중층의 형성에 필요한 소포의 양을 줄일 수 있습니다. 마지막으로, 표면 어셈블리의 전체 프로세스는 시간 소모적 종래의 운모 벽개 및 부착을 마찬가지로, (제조 미만 30 분 소요), 및 서, 고도의 기술을 필요로하지 않는 것이다.

<table border="0" cellpadding="0" cellspacing="0" width="710">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Name of Material/ Equipment</td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Bath Sonicator</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">FB15051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Coverslips 24 x 50mm - No H1.5</td>
			<td style="width:135px;">Marienfeld</td>
			<td style="width:119px;">102222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">DOPC</td>
			<td style="width:135px;">Avanti Polar Lipids</td>
			<td style="width:119px;">850357</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Hellmanex III (detergent)</td>
			<td style="width:135px;">Hellma Analytics</td>
			<td style="width:119px;">320.003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Mica V-1 Grade</td>
			<td style="width:135px;">SPI Suppliers</td>
			<td style="width:119px;">1872-CA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Optical Adhesive (high viscosity)</td>
			<td style="width:135px;">Norland Products</td>
			<td style="width:119px;">NOA63</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Optical Adhesive (low viscosity)</td>
			<td style="width:135px;">Norland Products</td>
			<td style="width:119px;">NOA60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sphingomyelin-ATTO647N</td>
			<td style="width:135px;">AttoTec</td>
			<td>AD 647N-171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">UV lamp</td>
			<td style="width:135px;">Synoptics Ltd.</td>
			<td style="width:119px;">GelVue GVM20</td>
			<td>The lamp was set to 100% power</td>
		</tr>
	</tbody>
</table>

preparation of mica supported lipid bilayers for high resolution optical microscopy imaging

지원되는 지질 이중층 (SLB 수)가 널리 막 특성 (상 분리, 클러스터링, 역학)과 같은 약물이나 펩티드 등의 다른 화합물과의 상호 작용을 연구하기위한 모델로 사용된다. 그러나 SLB 특성 사용 지원에 따라 다르다. SLB 이미징 및 측정에 일반적으로 사용되는 기술들은 단일 분자 형광 현미경, FCS 및 원자 힘 현미경 (AFM)이다. AFM은 매우 평탄한 표면 (일반적으로 운모)를 필요로하는 동안 대부분의 광학 이미징 연구가 유리 지지체 상에 실시되기 때문에 이들 재료의 충전 및 평활성 속성 강하게 확산 영향 때문에, 이러한 기술의 결과는, 직접 비교 될 수 없다. 불행하게도, 유리 슬라이드에 절단 및 운모의 얇은 조각을 접착제로 붙이기에 필요한 손재주의 높은 수준의 SLB 준비를위한 운모의 일상적인 사용에 장애물을 선물한다. 이 선택의 방법, 이러한 준비 운모 것입니다 있지만표면은 종종 특히 작은 작동 거리, 높은 수치 조리개 렌즈로, 요철 (물결 모양) 및 이미지에 어려운 끝나게. 여기에서 우리는 지질 소포 증착 및 SLB의 제조에 대해 얇고 편평한 운모 표면을 제조하기위한 간단하고 재현 가능한 방법을 제시한다. 또한, 우리의 사용자 정의 만든 챔버는 SLB 형성 소포 만 아주 작은 볼륨을 필요로합니다. AFM 연구에서 사용 된 것과 직접 필적 고품질 지질 이중층 표면의 효율적인 간단하고 저렴한 생산 전체적인 절차 결과.

SLB 수있는 지질 형광 프로브의 확산 동작은 기판에 따라 다르다. SMT 기술과 결합 된 TIRFM는 입자의 움직임을 시각화하고 그들의 확산 계수를 추출하기위한 중요한 방법이다. DOPC 유리와 운모 지원 (1,2 - dioleoyl-SN-글리세로 -3 - 포스 포 콜린) 이중층에 확산 핑고 마이 엘린 - ATTO647N 프로브의 단일 분자 신호는 첨부 된 애니메이션 그림에 표시됩니다. 운모 표면은 여기에 제시된 프로토콜에 따라...

Watch this Scientific Journal Video about Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging at JoVE.com

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

우리는 고해상도 현미경 운모지지 지질 이중층을 제조하는 방법을 제시한다. 운모는 원자 규모에서 투명하고 평면이지만, 거의 때문에 어려움을 처리하는 영상에서 사용되지; 우리의 준비는 운모 시트도 증착 결과 및 이중층의 제조에 사용되는 물질을 감소시킨다.

운모의 준비 고해상도 광학 현미경 이미징 지질 이중층 지원

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

preparation-mica-supported-lipid-bilayers-for-high-resolution-optical

Research

JoVE Journal

Bioengineering

13.3K 조회수.  Nanyang Technological University.  우리는 고해상도 현미경 운모지지 지질 이중층을 제조하는 방법을 제시한다. 운모는 원자 규모에서 투명하고 평면이지만, 거의 때문에 어려움을 처리하는 영상에서 사용되지; 우리의 준비는 운모 시트도 증착 결과 및 이중층의 제조에 사용되는 물질을 감소시킨다. 

비디오: Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

고해상도 광섬유 Microendoscopy에 대한 현장에서 세포 이미징

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

연구

생체공학

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

입체 광학 해상도 Photoacoustic 현미경

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. 
In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

나이프 - 에지 스캐닝 현미경에 대한 표본 준비, 이미징 및 분석 프로토콜

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

단일 분자 형광 현미경에 의한 수용체 역학의 고해상도 시공간 분석

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

위한 도구로 리포좀 캡슐화 된 근적외선 형광의 형광 담금질<em&gt; 생체</em&gt; 광학 이미징

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

원자 힘 현미경 이미징 및 지원 지질 이중층의 힘 분광학

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

평면에 단일 분자 형광 현미경은 이중층 지원

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

지원되는 지질 이중층에 리간드 나노 클러스터의 배열

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 &#181;m with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 &#181;m with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,&#160;in vivo&#160;small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

전환 가능한 음향 및 광학 해상도 Photoacoustic Microscopy for<em&gt; In Vivo</em&gt; 소 동물 혈관 조영술

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the ...

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.

운모의 준비 고해상도 광학 현미경 이미징 지질 이중층 지원

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