저자는 H188 cAMP FRET 바이오 센서와 케니 트린 (사우스 앨라배마 대학)을 제공하는 데 도움이 우리의 사용자 정의 개발 프로그래밍 스크립트를 실행하는 데 걸린 시간을 줄이기 위해 우리에게 제공하는 박사 Kees Jalink (네덜란드 암 연구소 및 고급 현미경 검사법을위한 반 Leeuwenhoek 센터) 인정하고 싶습니다.
저자는 자금 출처를 인정하고 싶습니다: 미국 심장 협회 (16PRE2713004), 국립 과학 재단; (1725937) NIH, S100D020149, S10RR027535, R01HL058506, P01HL0666299).

<ol>
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리프슬리 박사와 리치 박사는 스펙트럼 이미징 기술을 상용화하기 위해 설립된 스타트업 회사인 SpectraCyte, LLC에 대한 재정적 관심을 공개합니다. 그러나 이 프로토콜에 설명된 모든 절차는 SPECtraCyte, LLC와 관련이 없는 상업적으로 이용 가능한 제품을 사용하여 수행되었습니다.

FRET 바이오센서의 개발은 단일 세포에서 순환 뉴클레오티드 신호의 측정 및 시각화를 허용하고, 세포 외 신호 이벤트13,22,37,38을시각화하기위한 큰 약속이있다. 그러나 FRET 바이오 센서의 사용은 많은 형광 단백질 계 FRET 리포터의 낮은 신호-잡음 특성 및 FRET 기자의 약한 트랜스퍼션 또는 발현 효율을 ...

순환 아데노신 모노인산염(cAMP)은 세포 분열, 칼슘 유입, 유전자 전사 및 신호 변환을 포함한 주요 세포 및 생리적 과정에 관여하는 두 번째 메신저입니다. 증거의 성장 몸은 신호 특이성을 달성하는 세포를 통해 cAMP 구획의 존재를 제안1,2,3,4,5,6,7. 최근까지, cAMP 구획화는 다른 G-결합 수용체작용제에의해 유도된 뚜렷한 생리적 또는 세포 효과에 기초하여 유추되었다8,9,10,11. 최근에는 FRET 기반 형광 이미징 프로브가 세포12,13,14에서cAMP 신호를 직접 측정 및 관찰하기 위한 새로운접근법을제공하고 있다.
Förster 공명 에너지 전달(FRET)은 분자가15,16에근접할 때 비방사적 방식으로 기증자와 수용자 분자 간에 에너지 전달이 발생하는 물리적 현상이다. FRET 기반 형광 지표의 발달과 함께, 이러한 물리적 현상은 단백질-단백질 상호 작용17,단백질 공동 국소화18,Ca+2 신호19,유전자 발현20,세포 부문21 및 순환 뉴클레오티드 신호화를 연구하기 위해 생물학적 응용 분야에서 사용되었습니다. FRET 기반 cAMP 지표는 일반적으로 cAMP 결합 도메인, 기증자 불소 호및 수용자불소호레(22)로구성됩니다. 예를 들어, 이 방법론에 사용되는 H188 cAMP센서(12)22는 에팍으로부터 얻은 cAMP 결합 도메인으로 구성되며, 청록색(기증자)과 금성(수용자) 형광 사이에 끼어 있다. 기저 조건(언바운드)에서 청록색과 금성은 형광류 사이에 FRET가 발생하는 방향으로 있습니다. 바인딩 도메인에 대한 cAMP의 바인딩시, 청록색과 금성이 떨어져 이동하도록 형성 변화가 발생하여 FRET가 감소합니다.
FRET 기반 이미징 접근 방식은 셀 내에서 cAMP 신호를 조사하고 시각화하기 위한 유망한 도구를 제공합니다. 그러나, 현재 FRET 기반 현미경 이미징 기술은 종종 세포 전 세포 공간 선명도로 FRET를 측정하기에 충분한 신호 강도를 달성하는 데 부분적으로만 성공적입니다. 이는 많은 FRET 기자의 제한된 신호 강도, FRET 효율의 변화를 정확하게 정량화하는 데 필요한 높은 수준의 정밀도, 세포 자동불화(23)및24와같은 혼란스러운 요인의 존재를 포함한 여러 가지 요인에 기인한다. 결과는 종종 약한 SNR에 의해 괴롭히는 FRET 이미지, FRET에 있는 세포 극변경의 시각화를 아주 어렵게 만듭니다. 또한, 공간적으로 국소화된 cAMP 신호에 대한 조사는 거의 2개의 공간 차원에서만 거의 독점적으로 수행되었으며 축 cAMP 분포는거의 25로간주되지 않습니다. 이는 SNR이 cAMP 그라데이션을 세 가지 공간 차원으로 측정하고 시각화하는 기능을 방해했기 때문일 수 있습니다. 낮은 SNR을 가진 FRET 센서를 사용하는 한계를 극복하기 위하여는, 우리는 단하나세포25,26,27에서FRET를 측정하기 위하여 하이퍼 스펙트럼 화상 진찰 및 분석 접근법을 구현했습니다.
하이퍼스펙트럼 이미징 접근법은 NASA가 위성 이미지28,29에존재하는 지상파 물체를 차별화하기 위해 개발되었다. 이러한 기술은 이후 형광 현미경 필드(30)로번역되었으며, 스펙트럼 검출기를 제공하는 여러 상용 공초점 현미경 시스템이 있습니다. 기존의 (비 스펙트럼) 형광 이미징에서, 샘플은 밴드 패스 필터 또는 레이저 라인을 사용하여 흥분하고, 방출은 종종 형광의 피크 방출 파장에 맞게 선택된 제2 밴드 패스 필터를 사용하여 수집된다. 대조적으로, hyperspectral 화상 진찰 접근은 특정 파장 간격에 형광 방출26,31,32 또는 여기33,34의 완전한 스펙트럼 단면도를 샘플링하는 것을 추구합니다. 우리의 이전 연구에서, 우리는 Hyperspectral 화상 진찰 및 분석 접근이 전통적인 필터 기지를 둔 FRET 화상 진찰 기술(26)에비교될 때 세포에 있는 FRET 신호의 향상한 정량화를 제공할 수 있다는 것을 보여주었습니다. 여기서는 4차원(x, y, z 및 λ) 하이퍼스펙트럼 FRET 이미징 및 분석을 수행하여 3개의 공간 차원에서 cAMP 분포를 측정하고 시각화하는 방법론을 제시합니다. 이러한 접근법은 단일세포(25)에서작용제 유도 된 cAMP 공간 그라데이션의 시각화를 허용하였다. 흥미롭게도, 고뇌에 따라, cAMP 그라데이션은 세포에서 명백할 수 있습니다. 여기에 제시된 방법론은 FRET 측정의 정확도를 향상시키기 위해 비 균일한 배경 및 세포 자동 형광의 스펙트럼 미혼을 사용합니다. 이 방법론은 cAMP FRET 바이오 센서를 사용하여 폐 미세 혈관 내피 세포 (PMVEC)에서 입증되지만, 방법론은 대체 FRET 기자 또는 대체 세포주와 함께 사용하기 위해 쉽게 수정 될 수 있습니다.

<table><tbody><tr><td>Attofluor Cell Chamber</td><td>Invitrogen</td><td>A7816</td><td>Attofluor contains steel cell chambers and a rubber O-ring. Cell chamber holds the coverslip and O-ring provides a lock in mechanism to hold the buffer in cell chamber with out leakage</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Fisher Scientific</td><td>BP231-100</td><td>Solvent used to prepare stock solution forskolin.</td></tr><tr><td>DRAQ5 Fluoroscent Probe Solution</td><td>Thermo Scientific</td><td>62252</td><td>Nuclear label</td></tr><tr><td>Dulbecco Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>11965-092</td><td>Contains nutrients and growth factors for the cells to grow and divide in the culture dishes.</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma</td><td>F6178</td><td>Growth factor suppliment that is added to culture medium, DMEM</td></tr><tr><td>Forskolin</td><td>Sigma</td><td>F3917</td><td>Adenyly cyclase activator.</td></tr><tr><td>H188 Cyclic AMP FRET biosensor</td><td>Netherland Cancer Institute, Dr. K. Jalink</td><td>Gift</td><td>Plasmid encoding Turquoise (donor fluorophore), Venus (acceptor fluorophore), and binding domain obtained from Epac.</td></tr><tr><td>Image J</td><td>image.net</td><td>Free download</td><td>Another image processing platform used to extact spectral information and image processing.</td></tr><tr><td>Integrating Sphere</td><td>Ocean Optics</td><td>FOIS-1</td><td>Used to measure illumination intensity of the laser line at different laser intensities (?).</td></tr><tr><td>Laminin Mouse Protein, Natural</td><td>Invitrogen</td><td>23017-015</td><td>Coverslips are coated with laminin and this helps in cell attachment, growth and motility of the cell.</td></tr><tr><td>Lipofectamine 3000 Transfection Kit</td><td>Invitrogen</td><td>L3000-015</td><td>Transfection reagent used to transfect cells with H188 FRET biosensor</td></tr><tr><td>MATLAB</td><td>Mathworks</td><td>R2019a</td><td>Image processing operations (linear unmixing and FRET efficiency calculations) are performed by writing custom programs in MATLAB programming environment</td></tr><tr><td>Nikon A1R confocal microscope</td><td>Nikon Instruments</td><td>Nikon A1R</td><td>Spectral image acquisition is performed using confocal microscope.</td></tr><tr><td>Nikon Elements Software</td><td>Nikon Instruments</td><td>Software dongle</td><td>used to export and handle nd2 image files (multidimensional image files) that are aquired using Nikon A1R</td></tr><tr><td>NIST-Traceable Calibration Lamp</td><td>Ocean Optics</td><td>LS-1-CAL-INT</td><td>A lamp with a known spectrum for use as a standard</td></tr><tr><td>PBS pH 7.4 (1X)</td><td>Gibco</td><td>10010-023</td><td>coomonly used buffer suring cell culture</td></tr><tr><td>Pulmonary Microvascular Endothelial Cells (PMVECs)</td><td>In house - Cell culture core, Univeristy of South Alabama</td><td>Isolated from Rat pulmonary microvasculature</td><td>PMVECs form inner lining of a blood vessel.</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Gibco</td><td>15140-122</td><td>antibiotics are added to culture medum to prevent contamination of the cells.</td></tr><tr><td>Pre-Cleaned Gold Seal Micro Slides</td><td>Clay Adams</td><td>3010</td><td>Microscope slides used for cell fixation</td></tr><tr><td>ProLong Diamond Antifade Mounting Media</td><td>Invitrogen</td><td>P36961</td><td>If samples are fixed using antifade mountant, then the later protects fluoroscent dyes and chromophores from fading.</td></tr><tr><td>Spectrometer</td><td>Ocean Optics</td><td>QE65000</td><td>Used to measure spectral response of the light source (?)</td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Gibco</td><td>25200-056</td><td>Digests the protein-protein bond between the cell and cell matrix and helps to disscociate and lift the cells during cell plating.</td></tr><tr><td>Tyrodes Buffer</td><td>Made in-house</td><td>Made in-house</td><td>Tyrodes buffer is used to make working solutions and to maintain cells in aqueous solution during image acquisition.</td></tr><tr><td>6 Well Cell Culture Plate</td><td>Corning</td><td>3506</td><td>Laminin coated coverslips are placed in 6-well culture dish (one coverlisps/well). Cells along with medium is added into each well.</td></tr><tr><td>25 mm Round Microscope Cover Slips</td><td>Fisher Scientific</td><td>12545102</td><td>Cells were grown on round glass coverslips</td></tr><tr><td>60X Ojective</td><td>Nikon Instruments</td><td>Plan Apo VC 60X/1.2 WI &amp;infin;/0.15-0.18 WD 0.27</td><td>water immersion and commonly used objective for cells</td></tr></tbody></table>

measurement of 3-dimensional camp distributions in living cells using 4-dimensional (x, y, z, and &lambda;) hyperspectral fret imaging and analysis

순환 AMP는 세포 및 생리 활동의 넓은 범위에 관여하는 두 번째 메신저입니다. 몇몇 연구 결과는 cAMP 신호가 구획화되고, 그 구획화가 cAMP 신호 통로 내의 신호 특이성에 기여한다는 것을 건의합니다. F렌스터 공명 에너지 전달(FRET) 기반 바이오 센서의 개발은 세포내 cAMP 신호를 측정하고 시각화하는 능력을 발전시켰습니다. 그러나 이러한 측정은 종종 두 개의 공간 차원으로 제한되어 데이터를 잘못 해석할 수 있습니다. 현재까지, 본질적으로 낮은 신호 대 잡음 비(SNR)를 나타내는 FRET 센서를 사용하는 기술적 한계로 인해 3개의 공간 차원(x, y 및 z)으로 cAMP 신호의 측정이 매우 제한적이었습니다. 또한, 전통적인 필터 기반 이미징 접근법은 스펙트럼 크로스토크, 제한된 신호 강도 및 자동 형광을 포함한 다양한 요인으로 인해 국소화 된 세포 이형 지역에서 cAMP 신호를 정확하게 측정하는 데 효과가 없습니다. 이러한 한계를 극복하고 FRET 기반 바이오 센서를 여러 형광과 함께 사용할 수 있도록 FRET 기반 의 바이오 센서를 여러 형광과 함께 사용할 수 있도록 FRET 효율을 계산하기 위한 스펙트럼 특이성 및 추가 형광 라벨에서 접건 성 자동 형광 및/또는 신호를 반사적으로 분리하는 기능을 제공하는 하이퍼 스펙트럼 FRET 이미징 및 분석 접근 방식을 개발했습니다. 여기서는 하이퍼스펙트럼 FRET 이미징을 구현하기 위한 방법론과 스펙트럼 미혼을 수행하기 위해 샘플링되거나 과샘플링되지 않은 적절한 스펙트럼 라이브러리를 구성할 필요성을 제시합니다. 우리는 폐 미세 혈관 내피 세포 (PMVEC)에서 3 차원 cAMP 분포의 측정을위한이 방법론을 제시하는 동안,이 방법론은 세포 유형의 범위에서 cAMP의 공간 분포를 연구하는 데 사용할 수 있습니다.

이 프로토콜은 살아있는 세포에 있는 3개의 공간 차원에서 cAMP 그라데이션을 측정하기 위하여 hyperspectral FRET 화상 진찰 및 분석 접근의 사용을 기술합니다. 이러한 결과를 생성하는 데 관련된 몇 가지 주요 단계가 있으며, 데이터를 분석하고 정량화하는 동안 주의 깊게 주의를 기울여야 합니다. 이러한 주요 단계에는 적절한 스펙트럼 라이브러리 구성, 백그라운드 스펙트럼 해제, 셀 테두리 식별을...

Watch this Scientific Journal Video about Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional x, y, z, and Î» Hyperspectral FRET Imaging and Analysis at JoVE.com

Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional x, y, z, and &lambda; Hyperspectral FRET Imaging and Analysis

Fónster 공명 에너지 전송 (FRET) 기반 센서의 내재 된 낮은 신호 대 잡음 비율 (SNR)으로 인해, 특히 세 공간 차원에서 cAMP 신호의 측정이 도전하고있다. 여기서는 3개의 공간 차원에서 cAMP 분포를 측정할 수 있는 하이퍼스펙트럼 FRET 이미징 및 분석 방법론을 설명합니다.

4차원(x, y, z 및 λ) 하이퍼스펙트럼 FRET 이미징 및 분석을 사용하여 살아있는 세포에서 3차원 cAMP 분포 측정

Cyclic AMP is a second messenger that is involved in a wide range of cellular and physiological activities. Several studies suggest that cAMP signals are ...

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Research

JoVE Journal

Biology

Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional (x, y, z, and &lambda;) Hyperspectral FRET Imaging and Analysis

3.8K 조회수.  University of South Alabama. Fónster 공명 에너지 전송 (FRET) 기반 센서의 내재 된 낮은 신호 대 잡음 비율 (SNR)으로 인해, 특히 세 공간 차원에서 cAMP 신호의 측정이 도전하고있다. 여기서는 3개의 공간 차원에서 cAMP 분포를 측정할 수 있는 하이퍼스펙트럼 FRET 이미징 및 분석 방법론을 설명합니다.

비디오: Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional x, y, z, and λ Hyperspectral FRET Imaging and Analysis

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

3 차원 매트릭스에서 찬란 태그가 추가된 단백질을 표현 마이 그 레이션 전지의 라이브 세포 이미징

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

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생체공학

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

구조화 조명 현미경 데이터의 시공간적 이미지 상관 분광학에 의해 정량화 T 세포에서 대뇌 피질 굴지 흐름

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

면역학 및 감염병학

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

살아있는 세포 형광 기술의 조합을 사용 하 여 힘에 민감한 단백질 역학의 측정

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

안달 Ratiometric하여 세포주기를 통해 키나제와 인산 가수 분해 효소 활동을 모니터링

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

생물학

Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification of lymphocyte subtypes is important for the study of immunology as well as diagnosis and treatment of various diseases. Currently, standard methods for classifying lymphocyte types rely on labeling specific membrane proteins via antigen-antibody reactions. However, these labeling techniques carry the potential risks of altering cellular functions. The protocol described here overcomes these challenges by exploiting intrinsic optical contrasts measured by 3D quantitative phase imaging and a machine learning algorithm. Measurement of 3D refractive index (RI) tomograms of lymphocytes provides quantitative information about 3D morphology and phenotypes of individual cells. The biophysical parameters extracted from the measured 3D RI tomograms are then quantitatively analyzed with a machine learning algorithm, enabling label-free identification of lymphocyte types at a single-cell level. We measure the 3D RI tomograms of B, CD4+ T, and CD8+ T lymphocytes and identified their cell types with over 80% accuracy. In this protocol, we describe the detailed steps for lymphocyte isolation, 3D quantitative phase imaging, and machine learning for identifying lymphocyte types.

We describe a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and a machine learning algorithm. Measurements of 3D refractive index tomograms of lymphocytes present 3D morphological and biochemical information for individual cells, which is then analyzed with a machine-learning algorithm for identification of cell types.

라벨-무료 3 차원 양적 위상 이미징 사용 하 여 림프 구의 식별 및 기계 학습

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

3 차원 형광 이미지에서 세포 형태학의 변경 사항 단정 지을 분석 도구

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. It has been used in a wide range of biophysical and molecular biological experiments, including the measurement of molecular forces, characterization of conformational dynamics of biomolecules, observation of intracellular colocalization of proteins, and determination of receptor-ligand interaction times. In a widefield microscopy configuration, experiments are typically performed using surface-immobilized probes. Here, a method combining single-molecule tracking with alternating excitation (ALEX) smFRET experiments is presented, permitting the acquisition of smFRET time traces of surface-bound, yet mobile probes in plasma membranes or glass-supported lipid bilayers. For the analysis of recorded data, an automated, open-source software collection was developed supporting (i) the localization of fluorescent signals, (ii) single-particle tracking, (iii) determination of FRET-related quantities including correction factors, (iv) stringent verification of smFRET traces, and (v) intuitive presentation of the results. The generated data can conveniently be used as input for further exploration via specialized software, e.g., for the assessment of the diffusional behavior of probes or the investigation of FRET transitions.

This article presents a method for spatiotemporal analysis of mobile, single-molecule F&#246;rster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.

모바일 단일 분자 FRET 프로브의 자동화된 2차원 스파티오 측량 분석

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

일렉트로을 통해 생활 미생물의 국제화 및 형광 생체 분자의 관측

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

3 차원 히드로 겔의 이미지를 FRET

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

자동 형광 수명 이미징 현미경을 활용하여 오픈 소스 높은 콘텐츠 분석

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...