이 작품은 호프 학술 상 국방 시대의학과 (제 W81XWH - 05-0396)과 에드워드 B. 브라운 III에 의생명 과학 수상의 퓨 학술 의해 투자되었다.

1. 광학을 맞춥니다. MP - FRAP 실험을위한 주요 장비는 다음과 같습니다 모드 잠금 레이저 소스, Pockels 셀 (빔 변조에 대한), 펄스 발생기, 이색성, 객관적인 렌즈, 형광 방출 필터, 게이 티드 photomultiplier 튜브 및 데이터 기록 시스템 (광자 카운터 그리고 멀티채널 스케일러). 2. 안전 모니터 권한을 확인합니다. <ol><li> 샘플 내에서 형광 창출을위한 낮은지만, 합리적인, 전원에 레이저를 감쇠. </li><li> 샘플 내에 중점을두고 있습니다. </li><li> 더 이상 기대 형광 복구보다 시간 척도를 통해 통합하는 광자 카운터를 설정합니다. 초점 볼륨 (우리 현미경 소프트웨어이이 지점 검사에 의해 이루어집니다) 샘플 내에 정지 개최와 함께, 광자 개수 데이터의 합리적인 숫자를 기록합니다. </li><li> 힘을 증가하고 광자 카운트 데이터의 또 다른 시리즈를 가져가라. 광자 카운트의 증가는 전력 증가와 관련하여 현저하게 감소로 나타날 때까지 반복합니다. </li><li> 로그 (전원)의 함수로 플롯 로그 (광자 카운트). 두 아이의 기울기로부터 편차는 모니터 기간 동안 표백 나타냅니다. 모니터의 전원 아래에 상처를 해제하는 전원을 선택하지만, 노이즈 비율 합리적인 신호를 허용하는. </li></ol> 3. 펄스 발생기와 다중 스케일러의 입력 매개 변수를 확인합니다. <ol><li> 확인 다시 - 오브 - 더 - 봉투 문학 및 / 또는 스톡 - 아인슈타인 방정식과 확산 방정식에 따라 확산 계수 및 복구 시간을 추정하고 있습니다. </li><li> 펄스 발생기 및 스케일러에 필요한 매개 변수 값을 설정합니다. <ol class="lalpha"><li> 펄스 발생기에서 사전 표백제 및 표백 시간을 설정합니다. 엄지의 규칙의 좋은 세트는 미리 표백제는 기대 반 복구 시간보다 1-2 배 큰되어야하고, 표백제 시간은 예상 하프 복구 시간 한 열번째해야합니다. </li><li> 스케일러에서 약 절반 표백제 기간에 빈의 너비를 설정하고 기록 당 방식의 숫자를 설정 선택한 빈 폭 및 레코드 당 방식의 제품보다 크거나 예상 완전히 회복 플러스 미리 약 같다 그러한 - 표백과 표백 시간. </li><li> 펄스 발생기로 돌아가 빈 너비 시간 당 기록 방식의 수를 제품의 반전보다 단지 작은 값을 동일 pre-bleach/bleach/monitor 순서의 빈도를 설정합니다. * </li><li> 마지막 레코드의 수를 설정 / 선택한 모니터의 전원에서 신호 강도에 따라 스케일러를 검사합니다. * 그것은 펄스 발생기에 설정된 완벽한 pre-bleach/bleach/monitor 순서 (1/frequency)의 시간이 스케일러에 설정된 데이터 수집 시간 (빈 폭 X 방식 / 기록)보다 큰 것이 매우 중요합니다. 이 기준이 충족되지 않을 경우, 여러 표백제 트리거는 스케일러에서 단일 레코드 내에 나타날 수 있습니다. </li></ol></li><li> 이전에 결정 수용 수준으로 모니터의 전원을 설정합니다. 모니터 표백 테스트 중에 결정 표백 컷오프 위 200 MW 정도로 표백제 전원을 설정합니다. 이러한 파워는 PC의 결정에 걸쳐 서로 다른 전압으로 설정 둘 수 있습니다. </li><li> , 불을 끄기 PMT 켜고, 현미경 소프트웨어에 지점 검사를 시작 현미경을 봉쇄, 다음 펄스 발생기 및 스케일러를 시작합니다. </li><li> 1) 사전 표백제 형광, 2) 형광 즉시 포스트 표백제 및 데이터 세트의 끝에 3) 형광 : 곡선을 세우고있는 세 가지 중요한 사항을 표시하여 결과 복구 곡선 분석. 형광 값 사전 즉시 더 반 복구 시간을 추정 후 표백제.를 사용하여 </li><li> 하프 복구 시간이 예상과 함께, 사전 표백제, 표백에 대한 새로운 가치를 결정하기 이전에 설명한 엄지 손가락의 규칙, 그리고 빈 폭 기간이를 사용합니다. 또한,보다 완전한 회복을 달성하는 데 필요한 시간을 추정하기 위해 설정 데이터의 끝에 사전 표백제 형광 및 형광을 사용합니다. 엄지의 규칙으로, 데이터는 데이터의 후반에 대해보고해야하는 전체 복구를 위해 허용하는 기간 동안 수집해야합니다. </li><li> 귀하의 곡선은 강력한 표백 및 원활한 복구를 전시 때까지 펄스 발생기 및 스케일러에 매개 변수를 조정 매개 변수를 새로운 곡선을 그리고 조정을 계속합니다. 가능하다면, 표백제 기간 수는 점을 명심하고, 엄지의 규칙 아래에 감소한다. 너무 약하거나 너무 깊이 표백제를 얻을 경우 표백 파워도 조정할 수 있습니다. </li></ol> 4. 여기 채도에 대한 테스트합니다. <ol><li> 이전에 결정 적절한 모니터 및 표백 능력을 사용하여 MP - FRAP 곡선 일련의 가져가라. 적절한 수학적 형식과 비선형 최소 제곱 피팅 알고리즘을 (아래의 &quot;데이터 분석&quot;참조)를 사용하여, 사전 표백제 형광으로 표준화된 각각의 복구를 분석할 수 있습니다. 표백제 깊이 매개 변수의 장착 가치를 기록합니다.</li><li> 표백 파워의 가치를 증가에서 MP - FRAP 곡선의​​ 연속 시리즈 복용 분석 진행합니다. </li><li> 로그 (전원)의 함수로 플롯 로그 (표백 깊이 매개 변수). 두 아이의 기울기로부터 편차는 여기 채도를 나타냅니다. 강한 표백제를 산출 표백 전원을 선택하지만, 채도를 발생하지 않습니다. </li></ol> 5. MP - FRAP 곡선을 복용 계속합니다. <ol><li> 위와 같이 모든 매개 변수가 제대로 설정됩니다 곡선을 복용 계속합니다. </li></ol> 6. 데이터 분석. <ol><li> 형광 데이터에서 미리 표백제 및 표백 데이터를 같은 시간 데이터를 설정 및 조정을 제거하는 것이 바로 사후 표백제 형광 값 t = 0에 해당합니다. </li><li> 평균 미리 표백제 형광와 관련하여 발생하는 형광 복구를 정상화. </li><li> 적절한 수학적 모델과 같은 MATLAB에서 lsqcurvefit 함수로 비선형 최소 제곱 피팅 알고리즘을 사용하여 표준 곡선 맞추기. 여기에 제시 모델 확산 및 대류 흐름 모두에 의해 영향을 형광 recoveries 계정에 대한 : <img src="/files/ftp_upload/1636/1636eq.jpg" alt="방정식" /> F O가 미리 표백제 평균 형광 어디에, β는 표백 깊이 매개 변수이며, τ D의 특성 잘 퍼지는 복구 시간, R은의 요골 (W R) 넓이로 축의 제곱의 비율 (W Z)입니다 두 광자 초점 볼륨, τ VX가 = W R / V X, τ VY = W R / V Y는 τ VZ = w Z / V Z와 V 2 = V는 x 2 + V Y 2 + V Z 2입니다 대류 흐름의 속도. 이미징 비행기에 갇혀 흐름의 경우, τ VZ → ∞ 및 1 / τ VX + 1 / τ VY 1 / τ VR로 교체할 수 있습니다. 대류 흐름의 부재에서 분자 두 exponentials 1로 이동하여 표현은 크게 두 피팅 파라미터 β와 τ D로 줄어 듭니다. 확산 계수는 쉽게 D = W R 2 / 8τ D로 계산됩니다. </li></ol> 7. 대표 결과. <img src="/files/ftp_upload/1636/1636fig1.jpg" alt="그림 1" /> 그림 1. 대표 표백제와 FITC - BSA에 대한 복구 곡선. 좋은 복구 곡선은 데이터의 후반에 대해보고하는 전체 복구에서 형광과 함께 강력한 표백 및 원활한 복구를 전시하고 있습니다. <img src="/files/ftp_upload/1636/1636fig2.jpg" alt="그림 2" /> 그림 2. FITC - BSA를위한 표준화 복구 곡선 (적색)와 관련된 최소 제곱 적합 (블랙). 이 적합 문헌과 일치 52.9 um2 / s의 확산 계수를 산출.

<ol>
	<li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Biophys J. 60, 73-76 (1990).</li><li>Brown, E. B., Wu, E. S., Zipfel, W., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+molecular+diffusion+in+solution+by+multiphoton+fluorescence+photobleaching+recovery.">Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery.</a> Biophys J. 77, 2837-2849 (1999).</li><li>Sullivan, K. D., Sipprell, W. H. B. r. o. w. n., Jr, E. B., Brown, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+model+of+fluorescence+recovery+expands+the+application+of+multiphoton+fluorescence+recover+after+photobleaching+in+vivo.">Improved model of fluorescence recovery expands the application of multiphoton fluorescence recover after photobleaching in vivo.</a> Biophys J. 96, 5082-5094 (2009).</li></ol>

photobleaching 후 다중 광자 형광 회복의 능력은 3D 해상도 두꺼운 샘플을 탐사하는 기능에있다. 1990 년대에 개발 이후, MP - FRAP은 세포 기관, 전직 생체내 두꺼운 조직 슬라이스하고, 생체내 조직과 interstitium에의 확산 계수 (또는 유사한 전송 매개 변수)를 결정하는 데 사용되었습니다. 이 문서에서는, 우리는 실험 매개 변수를 설정 데이터를 복용하고, 복구 곡선을 분석, MP - FRAP 실...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Ti:sapphire laser</td>
<td>&nbsp;</td>
<td>Spectra Physics</td>
<td>Mai Tai</td>
<td>&nbsp;</td>
<tr>
<td>Pockels Cell</td>
<td>&nbsp;</td>
<td>Conoptics</td>
<td>350-80</td>
<td>&nbsp;</td>
<tr>
<td>Laser Scanning Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>Fluoview</td>
<td>&nbsp;</td>
<tr>
<td>Short pass dichroic mirror</td>
<td>&nbsp;</td>
<td>Chroma Technologies</td>
<td>670 DCSX-2P</td>
<td>&nbsp;</td>
<tr>
<td>Photomultiplier tube</td>
<td>&nbsp;</td>
<td>Hamamatsu</td>
<td>HC125-02</td>
<td>&nbsp;</td>
<tr>
<td>Photon counter</td>
<td>&nbsp;</td>
<td>Stanford Research Systems</td>
<td>SR 400</td>
<td>&nbsp;</td>
<tr>
<td>Multi-channel scaler/averager</td>
<td>&nbsp;</td>
<td>Stanford Research Systems</td>
<td>SR 430</td>
<td>&nbsp;</td>
<tr>
<td>Pulse Generator</td>
<td>&nbsp;</td>
<td>Stanford Research Systems</td>
<td>DG 535</td>
<td>&nbsp;</td>
<tr>
<td>FITC-BSA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>--</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

measuring diffusion coefficients via two-photon fluorescence recovery after photobleaching

photobleaching 후 다중 형광 복구 macromolecules의 확산 계수 (또는 유사한 전송 매개 변수)을 측정하는 데 사용되는 현미경 기술이며, 모두에 적용할 수있는<em&gt; 체외에서</em&gt;과<em&gt; 생체내에</em&gt; 생물 학적 시스템. photobleaching 후 다중 형광 복구 후 빔을 attenuating하고 photobleached 분자를 교체에 관심 확산의 영역 밖에서 아직도 형광 분자로 형광을 모니터링, 강렬한 레이저 플래시를 사용하여 형광 샘플 내에서 관심 영역을 photobleaching에 의해 수행됩니다 . 우리는 Pockels 전지 (레이저 변조기)를 통해 및 샘플에 레이저 스캔 상자와 객관적인 렌즈를 통해 광학 경로를 따라 레이저 빔을 정렬하여 데모를 시작합니다. 단순화하기 위해, 우리는 수성 형광 염료의 샘플을 사용합니다. 그런 다음 적절한 실험을 포함하여 샘플에 대한 매개 변수를 모니터와 표백 능력, 표백 기간, 빈 너비 (광자 집계) 및 형광 복구 시간을 결정합니다. 다음, 우리는 복구 곡선, 크게 향상된 처리량을위한 LabVIEW (내쇼날 인 스트 루먼트, 오스틴, TX)를 통해 자동으로 사용할 수있는 프로세스를 주셔서 절차를 설명합니다. 마지막으로, 확산 계수는 같은 MATLAB (Mathworks, 내틱, MA)와 같은 소프트웨어를 사용하여 쉽게 프로그래밍 최소 제곱 피팅 알고리즘을 사용하여 적절한 수학적 모델 복구 데이터를 피팅에 의해 결정됩니다.

Watch this Scientific Journal Video about Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching at JoVE.com

Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching

이 문서에서 우리는 photobleaching 후 다중 광자 형광 복구를 사용하여 확산 계수를 측정하기위한 절차를 설명합니다. 우리는 샘플에 대한 광학 경로를 따라 레이저를 정렬하고 적절한 실험 매개 변수를 결정함으로써 시작되며, 다음 발생을 계속하고 마지막으로 피팅 형광 복구 곡선.

Photobleaching 후 두 광자 형광 복구를 통해 확산 계수 측정

Multi-fluorescence recovery after photobleaching is a microscopy technique used to measure the diffusion coefficient (or analogous transport parameters) ...

measuring-diffusion-coefficients-via-two-photon-fluorescence-recovery

Research

JoVE Journal

Biology

11.1K 조회수.  University of Rochester. 이 문서에서 우리는 photobleaching 후 다중 광자 형광 복구를 사용하여 확산 계수를 측정하기위한 절차를 설명합니다. 우리는 샘플에 대한 광학 경로를 따라 레이저를 정렬하고 적절한 실험 매개 변수를 결정함으로써 시작되며, 다음 발생을 계속하고 마지막으로 피팅 형광 복구 곡선.

비디오: Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

라이브 zebrafish의 배아에서 두 광자의 axotomy하고 시간이 경과 공촛점 영상

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

연구

생물학

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

siRNA Transfection 후의 유사 분열의 시간 저속 영상

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

라이브 Zebrafish 태아에서 두 광자 기반 Photoactivati​​on

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

Spectrally 해결된 두 광자 현미경을 사용하여 G 단백질 결합 수용체 상호 작용의 생체내 부량에서

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

배아 줄기 세포 생활에서 염색질 단백질 역학을 측정하는 Assays (FRAP 및 플립)를 Photobleaching

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

다중 타겟 추적 (MTT)에 의해 플라즈마 막의 분자 확산을 매핑

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

전 · Cryo 키트를 사용하여 마우스 정자 Cryopreservation 및 복구

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)1&#160;was developed to enable routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely available code to measure diffusion coefficients of proteins. kICS calculates a time correlation function from a fluorescence microscopy image stack after Fourier transformation of each image to reciprocal (k-) space. Subsequently, circular averaging, natural logarithm transform and linear fits to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS.
First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope. Then, a region of interest (ROI) avoiding intracellular organelles, moving vesicles or protruding membrane regions is selected. The ROI stack is imported into a freely available code and several defined parameters (see Method section) are set for kICS analysis. The program then generates a &#34;slope of slopes&#34; plot from the k-space time correlation functions, and the diffusion coefficient is calculated from the slope of the plot. Below is a step-by-step kICS procedure to measure the diffusion coefficient of a membrane protein using the renal water channel aquaporin-3 tagged with EGFP as a canonical example.

This paper provides a step by step guide to the fluctuation analysis technique k-Space Image Correlation Spectroscopy (kICS) for measuring diffusion coefficients of fluorescently labeled plasma membrane proteins in live mammalian cells.

K-공간 이미지의 상관 관계 분광학을 사용하여 EGFP-태그 플라즈마 막 단백질의 확산 계수의 간편한 측정

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

높은 처리량 응용 프로그램에 대한 성인 Zebrafish의 마음의 복구

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

세포 내 칼슘의 두 광자 이미징<sup&gt; 2 +</sup&gt; 고립 된 쥐 대동맥의 취급 및 내피 세포에서 산화 질소 생산과 부드러운 근육 세포

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...