우리는 가비 마틴이 작업을 시작하고 피지와 ImageJ에 프로젝트 참여자의 모든 EMBO2010 3D 발달 이미징 워크숍의 주최자에게 감사의 말씀을 전합니다.

1. 4D 시간 경과 이미징 실험 화상 획득을 위해 사용되는 설정은 사용되는 장비에 따라 달라질 것이다. 여기, 핀홀 크기, 대물의 개구, 시료의 굴절률과 시료가 내장 된 매체의 파장 : 광학 부 샘플 공 초점 현미경의 능력은 여러 가지 요인에 의존한다. 선택된 공 촛점 핀홀 사이즈 채집 광학 부재의 두께를 결정한다. 작은 핀홀이 얇은 광학적 부분 z 축 해상도를 증가하지만 캡처 빛의 양을 감소를 일으킬 것이다. 큰 핀홀이 Z 축 해상도를 감소하지만 캡처되는 광량을 증가 광학 부재의 두께를 증가시킬 것이다. 이전의 교정에 4D 데이터를 수집하는 동안 고려해야 할 추가 요소는 다음과 같습니다 : <ol><li> D를 제거하거나 제한 표류 스캔 속도를 최적화단일 시간 지점의 캡처를 uring. 따라서 이미지 수집을위한 시간은 시간 점 사이의 간격의 작은 분획이어야한다. </li><li> 그것은 필요한 보정을 확인할 수는 없습니다로 완벽하게 반복 구조, 또는 그리드, 등록을위한 구조로서 적합하지 않다. 무작위로 배포 구슬 또는 고르지 구조는 모호 등록을 허용합니다. </li><li> 드리프트가 예상되는 경우, 그 영역은 화상 스택 내에 남아 있도록하기 위해 스캐닝 영역과 상하 초점 한계를 증가시킨다. </li><li> 확보에 더하여, 그 구조를 해결 연구되고 동적 이벤트의 시간 해상도를 제공하기 위해 샘플링 레이트를 설정하기위한 적절한 공간 해상도가있다. 참고 : 이미지의 시간 점 사이의 시간 따라서 정기적으로 반복 이벤트 사이의 최소 절반 시간 간격하고 불규칙한 이벤트를 감소해야한다. </li></ol> 2. 공 촛점 D 열기ataset 오픈 소스 패키지 피지 현미경 실험에서 수집 된 데이터를 다수의 프로세스를 수행하기위한 사전 설치된 플러그인을 포함하는 ImageJ에 프로그램의 분포입니다. 소프트웨어는 쉽게 플러그인 업데이트 아키텍처를 제공하고,이 프로토콜에 사용되는 정확한 3D 드리프트 플러그인의 복사본을 포함한다. 이 소프트웨어는 오픈 현미경 환경의 바이오 형식의 가져 오기 플러그인의 사용을 통해 독점 현미경 이미지 포맷의 광대 한 배열의 가져 오기를 지원합니다. <ol><li> 피지 프로그램을 설치합니다 (http://fiji.sc/Downloads). </li><li> 궤적 바이오 형식 가져 오기 플러그인을 사용하여 획득 한 데이터 집합을로드합니다. </li><li> 데이터 세트는 프로그램에 할당 된 메모리보다 큰 경우, 메모리 관리부 내에 &quot;사용하는 가상 스택 옵션&quot;을 선택한다. </li></ol> 3. 후 처리에 3D 개체의 드리프트 보정 오랜 시간이 경과하는 과정에서임베디드 때에도 샘플 이동할 수 실험. 어떤 움직임을 보정하고 묘화 이주 이벤트를 분석 할 수 있도록, 화상의 후 처리가 수행 될 수있다. 모든 이미지 후 처리는 명확하게이 작품에서 파생 된 분석의 방법에서 기술되어야한다. <ol><li> 데이터 집합이로드되면, 올바른 3D 드리프트 플러그인을 실행합니다. </li><li> 여러 콘텐츠 채널이있는 경우, 이미지를 등록하는 데 사용되는 채널을 선택한다. 이 이상적으로 오히려 어떤 철새 나 모바일 요소보다 샘플 내에서 정적 구조를 표현한다. 이것이 가능하지 않은 경우, 최소한의 움직임 채널 선택해야합니다. </li><li> 이 분석에 사용되는 시스템에서 사용 가능한 RAM 원래 데이터 세트의 적은 배 이상의 크기 인 경우, 사용하는 가상 스택 옵션을 선택한다. 이는 오히려 RAM으로 파일을 저장하는 것보다 화상 시퀀스로서 등록 hyperstack를 저장할 것이다. <ol><li> 출력 각각에 플러그인의 폴더를 선택합니다 - 지금 수정GES 파일. 별도의 이미지 파일은 각 Z 위치에서 각 채널에 대해 생성됩니다. </li></ol></li><li> 플러그인은 다음 데이터 집합에 적용되는 필요한 보정을 결정하기 위해 각 시간 점 사이의 쌍대 위상 상관 관계 분석을 실시한다. </li></ol>

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저자는 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

오랜 시간 경과 현미경 실험에서 파생 된 데이터 세트의 샘플 드리프트를 해결하기 위해 후 처리 소프트웨어를 사용하는 우리의 능력은 요인에 의해 제한됩니다. 샘플의 철새 이동 대 드리프트를 식별 할 수있는 기능은 사용 된 세포 마커에 따라 달라집니다. 어느 널리 샘플 내에서 표현이나 이미지 수집하는 동안 철새 이벤트에 관련되지 않은 세포 마커 드리프트 보정을위한 최고의 소스를 제공...

공 촛점 이미징 널리 형태의 세포의 움직임과 변화를 따라 세포 및 발달 생물학에 사용됩니다. 다른 초점면에 광학 섹션의 일련 캡처 허용 후 3D 데이터 세트의 시간 경과 시리즈를 생성하여 네 차원 (4D)으로 확장 될 수있는 샘플의 3 차원 (3D) 모델의 생성. 4D 데이터 세트의 생성은 세포의 움직임과 행동의 상세한 측정을 할 수 있습니다. 장기간의 시간 경과 실험에서는 샘플의 움직임을 관찰하는 것이 일반적입니다. 이는 하드웨어 제어 단계와 초점 위치에서 약간의 부정확으로 인해 발생할 수 있습니다. 다른 사람의 경우, 드리프트 샘플 장착 미디어 내에서 샘플 성장과 유연성에 의해 유도 된 운동의 결과이지만. 방법은 하드웨어를 중심으로 시스템을 개선하고 설치 매체의 증가 강성 등의 이러한 움직임을 보상하거나 제한하기 위해 존재한다. 그러나, 이러한 접근법에 의한 촬상으로의 많은 경우에 적용 할 수 없다등 최대 샘플 유지 보수 및 성장에 적합한 조건을 제공해야합니다. 오픈 소스 소프트웨어 솔루션은 ImageJ에 또는 피지 StackReg 및 TurboReg (http://bigwww.epfl.ch/thevenaz/stackreg/) 5 플러그인을 사용하여, 시간이 지남에 따라 2 차원 운동의 보정을 위해 존재하지만, 이들은 수 없습니다 4D 데이터 세트에 적용 할. 샘플의 드리프트를 교정하기 위해 우리는 오픈 소스 영상 처리 플랫폼, 피지 1을 활용하는 플러그인 (올바른 3D 드리프트)를 개발했습니다. 우리의 플러그인은 입체 시간 경과 실험 샘플 드리프트의 결과로서 발생하는 움직임을 보정하기 위해 위상 상관 등록을 수행 할 수있다. 위상 상관 관계 (6) 이미지 사이의 변환을 결정하는 계산 효율적인 방법입니다. 여기에 설명 플러그인 Preibisch 외. (7)에 의해 개발 된 위상 - 상관 라이브러리를 이용한다. 멀티 채널 실험에서, 플러그인은 determi에 하나의 채널을 이용필요한 보정 NE. 이 보정은 다음 4D 데이터 세트의 등록 결과 추가적인 채널에 적용된다. 제브라 피쉬 모델 시스템에서 많은 시간, 심지어 며칠 8의 기간 동안 시간 경과 촬상을 행할 수있다. 제브라 피쉬를 장착하기위한 일반적인 방법은 운동 9-11을 제한, 저 융점 아가 로스 (0.8 ~ 1.5 %)에서 마취 라이브 배아를 포함하는 것입니다. 이동이 규제되는 동안 샘플의 성장이 정지 한 위치를 이동 시야 내의 세포의 결과 발생한다. 배아에서 세포의 이동을 수행하기 위해 먼저 전체 샘플의 움직임을 보정 할 필요가있다. 이 프로토콜은 제브라 시험편으로 개발되었고, 화상 체절 개발 (12)에 이용되었지만 어떠한 4D 촛점 집합에 적용될 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="406">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A5040</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Low gelling temperature agarose</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9414-25G</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Dumont #4 Forceps</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">0208-4-PO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable 3mL graduated</td>
			<td rowspan="2" style="width:71px;">Samco</td>
			<td rowspan="2" style="width:119px;">212</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Polyethylene transfer pipette</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">9cm bacterial grade Petri dishes</td>
			<td style="width:71px;">Greiner Bio One</td>
			<td style="width:119px;">632180</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fluorinated ethylene propylene (FEP) tubing</td>
			<td style="width:71px;">Bola</td>
			<td style="width:119px;">S1815-04</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Zeiss LSM-710 Confocal microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">W Plan-Apochromat 20x/1.0 DIC Objective</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">421452-9600-000</td>
		</tr>
	</tbody>
</table>

sample drift correction following 4d confocal time-lapse imaging

사차원 (4D) 공 초점 데이터 세트의 생성; 시간이 지남에 따라 3D 이미지 시퀀스로 구성된; 발달 과정에 관여 세포의 동작을 캡처 할 수있는 훌륭한 방법을 제공합니다. 세포의 움직임을 추적하고, 수행하는 기능은 화상 획득 동안에 샘플의 드리프트 또는 어떤 경우에는 증가로 인해 발생하는 샘플의 움직임에 의해 제한된다. 드리프트 및 / 또는 성장에 의해 영향을받는 데이터 세트에서 추적 셀은 셀 위치의 분석으로이 운동을 통합 할 것입니다. 이 샘플 내에서 정적 구조의 명백한 움직임의 원인이 될 수 있습니다. 따라서 이전의 세포 추적에 대한 모든 샘플 드리프트 수정해야합니다. ImageJ에 2,3의 오픈 소스 피지 분배 (1)과 통합 궤적 도구 4를 사용하여, 우리는 공 초점 데이터 세트에서 잘못된 샘플의 움직임을 제거 할 수있는 올바른 3D 드리프트 플러그인을 개발했다. 이 프로토콜은 효율적으로 초점 포에 샘플 번역 또는 변경에 대한 보상연장 된 시간 경과 실험을 통해 세포의 움직임을 시각화하고 측정하는 능력을 유지하면서 사차원 촛점 데이터 세트마다 포인트를 등록하는 단계 상관을 이용함으로써 구성비.

13 - 개발 제브라 피쉬에서 빠른 근육 세포는 19시간 후 수정 (체절 단계 20)에서 다핵 섬유로 융합. 핵의 움직임과 우리가 근육의 모든 레이블을하는 골격 α-액틴 프로모터의 통제하에 녹색 형광 단백질 (GFP)을 발현하는 형질 전환 균주를 사용하여 4D 공 촛점 시간 경과 이미징을 실시 근육 세포의 융합을 시각화하기 위해 전지 (14)과 mCherry이 핵을 레이블, 핵 이행 순서로 태그 적색 ?...

Watch this Scientific Journal Video about Sample Drift Correction Following 4D Confocal Time-lapse Imaging at JoVE.com

Sample Drift Correction Following 4D Confocal Time-lapse Imaging

시간 경과 현미경은 개발 프로세스의 시각화를 할 수 있습니다. 화상 취득 동안에 성장 또는 샘플의 드리프트가 정확하게 따르고 개발 중에 세포의 움직임을 측정하는 능력을 감소시킨다. 우리는 시간에 따른 입체 샘플 드리프트 보정하기 위해 오픈 소스 이미지 처리 소프트웨어를 사용하는 방법을 설명한다.

4D 공 촛점 시간 경과 이미징을 다음 샘플 편차 수정

The generation of four-dimensional (4D) confocal datasets; consisting of 3D image sequences over time; provides an excellent methodology to capture ...

sample-drift-correction-following-4d-confocal-time-lapse-imaging

Research

JoVE Journal

Bioengineering

16.3K 조회수.  Monash University.  시간 경과 현미경은 개발 프로세스의 시각화를 할 수 있습니다. 화상 취득 동안에 성장 또는 샘플의 드리프트가 정확하게 따르고 개발 중에 세포의 움직임을 측정하는 능력을 감소시킨다. 우리는 시간에 따른 입체 샘플 드리프트 보정하기 위해 오픈 소스 이미지 처리 소프트웨어를 사용하는 방법을 설명한다. 

비디오: Sample Drift Correction Following 4D Confocal Time-lapse Imaging

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

엔지니어링된 심장 조직 이식 다음과 같은 면역 반응의 평가 (EHT)에 대한 Bioluminescence 이미징

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

연구

생체공학

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

비디오 - 속도 검색 공촛점 현미경과 Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

RootChip를 사용하여 루트 환경의 신속한 조작과 Arabidopsis 루트 성장의 시간 저속 형광 이미징

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

3 차원 세포 배양 모델의 질량 분석 이미징을위한 준비 전략 샘플

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

ICPMS 샘플 소개를위한 미세 유체 칩

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

양적 Confocal 반사 현미경을 통해 3D 매트릭스에 포유류 세포 분열

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

장기 나노 물질 노출 다음 체외 물질 독성 테스트를 위한 고급 3D 간 모델

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

시간 경과 현미경 검사를 사용하여 대장균에서 생물학적 소음의 지속적인 측정

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

광섬유 공초점 레이저 미세 내시경 검사를 사용하여 안구 약물 전달 시스템의 생체 내 측두 체 이미징

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such ...

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

We report a stage-top, flexible environmental chamber for time-lapse imaging of live cells using upright stimulated Raman scattering microscopy with transmitted signal detection. Lipid droplets were imaged in SKOV3 cells treated with oleic acid for up to 24 h with a 3 min time interval.

자극 라만 산란 현미경을 사용한 타임랩스 라이브 셀 이미징을 위한 유연한 챔버

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many ...