Research related to this paper and the development of the fBFP technology in the Zhu lab were supported by NIH grants AI044902, AI077343, AI038282, HL093723, HL091020, GM096187, and TW008753. We thank Evan Evans for inventing this empowering experimental tool, and members of the Evans lab, Andrew Leung, Koji Kinoshita, Wesley Wong, and Ken Halvorsen, for helping us to build the BFP. We also thank other Zhu lab members, Fang Kong, Chenghao Ge and Kaitao Li, for their helps in the instrumentation development.

<ol>
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The authors have nothing to disclose.

A experiência bem sucedida fBFP implica algumas considerações críticas. Em primeiro lugar, para o cálculo de força para ser confiável, micropipeta, a RBC, eo talão sonda deve ser alinhado como perto de coaxial possível. A projecção do RBC dentro da pipeta deve ser de cerca de um diâmetro de sonda de pipeta de modo que o atrito entre a RBC e a pipeta é negligenciável. Para um RBC humano típico, o diâmetro ideal pipeta é 2,0-2,4 mm, que produz um melhor ajuste da Equação 1 17,30. Em segundo l...

Célula-a-célula e célula-matriz extracelular de (ECM) adesão é mediada pela ligação entre os receptores da superfície celular, proteínas de ECM, e / ou lípidos 1. A ligação permite que as células de modo a formar uma estrutura funcional, bem como reconhecer, comunicar, e reagir ao ambiente 1-3. Ao contrário de proteínas solúveis (por exemplo, citoquinas e factores de crescimento) que se ligam de uma imagem tridimensional (3D) de fase fluida para os receptores da superfície celular, os receptores de adesão de células formar ligações com os seus ligandos através de uma das junções de hiato estreita para unir duas superfícies opostas que limitam molecular difusão numa dimensional (2D) de interface dois 4-7. Em contraste com a cinética de 3D ​​que são vulgarmente medidos por ensaios de ligação tradicionais (por exemplo, de ressonância de plasmon de superfície ou SPR), a cinética 2D têm de ser quantificado com técnicas especializadas, tais como microscopia de força atómica (AFM), 8-10, câmara de fluxo 11,12, micropipeta 13,14, ópticopinças 15 e sonda vigor biomembrana (BFP) 16-21. Mais do que simplesmente fornecendo ligação física para a coesão celular, moléculas de adesão são um dos principais componentes da maquinaria de sinalização para a célula comunique com o espaço envolvente. Tem havido um interesse crescente na compreensão de como ligando acoplamento de moléculas de adesão inicia a sinalização intracelular e como o sinal inicial é transduzida no interior da célula. Intuitivamente, propriedades de receptor-ligando de ligação pode ter um impacto que induz os sinais. No entanto, é difícil para dissecar as relações mecanicistas entre a interação extracelular e eventos de sinalização intracelular utilizando conjunto tradicional de ensaios bioquímicos por causa de suas muitas limitações, por exemplo, uma resolução temporal pobres e completa falta de resolução espacial. Métodos que permitem tanto biofísico (cinética de ligação ao receptor do ligando 2D) e (sinalização) observações bioquímicas em directo existentecélulas incluem substratos de rigidez ajustável 22, elastômero pilar matrizes 23 e dispositivos de câmara de fluxo / microfluídicos incorporados com capacidade de fluorescência 24-26. No entanto, as leituras de sinalização e de ligação ao receptor do ligando tem que ser obtido separadamente (na maioria das vezes através de métodos diferentes), o que torna difícil para dissecar as relações temporais e espaciais das características de títulos com eventos de sinalização. BFP convencional é uma espectroscopia de força ultra-sensível de alta resolução espaço-temporal 17. Ele usa uma célula de sangue vermelho flexível (RBC) na forma de um sensor de força, permitindo a medição da cinética de 2D única molécula, propriedades mecânicas e alterações conformacionais 14,16,19-21,27-29. A BFP baseado imagens fluorescentes (fBFP) correlaciona-se a cinética de ligação ligando-receptor com a sinalização celular desencadeada obrigatório-a escala única molécula. Com esta configuração, em atividades de sinalização celular situ no contexto de mechani superfíciecal estimulação foi observada em células T-27. O fBFP é versátil e pode ser utilizado para estudos de adesão celular e de sinalização mediadas por outras moléculas em outras células.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="4">Table 1: Reagents/Equipment</td>
		</tr>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic Monohydrate (NaH2PO4&#8226;H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td>Phosphate buffer preparation</td>
		</tr>
		<tr>
			<td>Anhy. Sodium Phosphate Dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td>S7907</td>
			<td>Phosphate buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium Carbonate (Na2CO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S2127</td>
			<td>Carbonate/bicarbonate buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>Carbonate/bicarbonate buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>N2-5% buffer preparation</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td>N2-5% buffer preparation</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>N2-5% buffer preparation</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>N2-5% buffer preparation</td>
		</tr>
		<tr>
			<td>MAL-PEG3500-NHS</td>
			<td>JenKem</td>
			<td>A5002-1</td>
			<td>Bead functionalization</td>
		</tr>
		<tr>
			<td>Biotin-PEG3500-NHS</td>
			<td>JenKem</td>
			<td>A5026-1</td>
			<td>RBC biotinylation</td>
		</tr>
		<tr>
			<td>Nystatin</td>
			<td>Sigma-Aldrich</td>
			<td>N6261</td>
			<td>RBC osmolarity adjustment</td>
		</tr>
		<tr>
			<td>Ammonium Hydroxide (NH4OH)</td>
			<td>Sigma-Aldrich</td>
			<td>A-6899</td>
			<td>Glass bead silanization</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>BDH</td>
			<td>67-56-1</td>
			<td>Glass bead silanization</td>
		</tr>
		<tr>
			<td>30% Hydrogen Peroxide (H2O2)</td>
			<td>J. T. Barker</td>
			<td>Jan-86</td>
			<td>Glass bead silanization</td>
		</tr>
		<tr>
			<td>Acetic Acid (Glacial)</td>
			<td>Sigma-Aldrich</td>
			<td>ARK2183</td>
			<td>Glass bead silanization</td>
		</tr>
		<tr>
			<td>3-MERCAPTOPROPYLTRIMETHOXYSILANE(MPTMS)</td>
			<td>Uct Specialties, llc</td>
			<td>4420-74-0</td>
			<td>Glass bead functionalization</td>
		</tr>
		<tr>
			<td>Borosilicate Glass beads</td>
			<td>Distrilab Particle Technology</td>
			<td>9002</td>
			<td>Glass bead functionalization</td>
		</tr>
		<tr>
			<td>Streptavidin&#8722;Maleimide</td>
			<td>Sigma-Aldrich</td>
			<td>S9415</td>
			<td>Glass bead functionalization</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A0336</td>
			<td>Ligand functionalizing</td>
		</tr>
		<tr>
			<td>Fura2-AM</td>
			<td>Life Technologies</td>
			<td>F-1201</td>
			<td>Intracellular calcium fluorescence dye loading</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td>Intracellular calcium fluorescence dye loading</td>
		</tr>
		<tr>
			<td>Quantibrite PE Beads</td>
			<td>BD Biosciences</td>
			<td>340495</td>
			<td>Density quantification</td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>BD LSR II</td>
			<td>Density quantification</td>
		</tr>
		<tr>
			<td>Capillary Tube 0.7-1.0mm x 30&#34;</td>
			<td>Kimble Chase</td>
			<td>46485-1</td>
			<td>Micropipette making</td>
		</tr>
		<tr>
			<td>Flaming/Brown Micropipette Puller</td>
			<td>sutter instrument</td>
			<td>P-97</td>
			<td>Micropipette making</td>
		</tr>
		<tr>
			<td>Pipette microforce</td>
			<td>Narishige</td>
			<td>MF-900</td>
			<td>Micropipette making</td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Fisher Scientific</td>
			<td>BP2629-1</td>
			<td>Chamber assembly</td>
		</tr>
		<tr>
			<td>Microscope Cover Glass</td>
			<td>Fisher Scientific</td>
			<td>12-544-G</td>
			<td>Chamber assembly</td>
		</tr>
		<tr>
			<td>Micro-injector</td>
			<td>World Precision Instruments</td>
			<td>MF34G-5</td>
			<td>Chamber assembly</td>
		</tr>
		<tr>
			<td>1ml Syringe</td>
			<td>BD&#160;</td>
			<td>309602</td>
			<td>Chamber assembly</td>
		</tr>
		<tr>
			<td>Micropipette holder</td>
			<td>Narishige</td>
			<td>HI-7</td>
			<td>Chamber assembly</td>
		</tr>
		<tr>
			<td>Home-designed mechanical parts and adaptors fabrications using CNC machining.&#160;</td>
			<td>Biophysics Instrument</td>
			<td>All parts are customized according to the CAD designs.</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Microscope (TiE inverted)</td>
			<td>Nikon</td>
			<td>MEA53100</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Objective CFI Plan Fluor 40x (NA 0.75, WD 0.72mm, Spg)</td>
			<td>Nikon</td>
			<td>MRH00401</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Camera, GE680, 640x480, GigE, 1/3&#34; CCD, mono</td>
			<td>Graftek Imaging</td>
			<td>02-2020C</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Prosilica GC1290 - ICX445, 1/3&#34;, C-Mount, 1280x960, Mono., CCD, 12 Bit ADC</td>
			<td>Graftek Imaging</td>
			<td>02-2185A</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Manual submicron probehead with high resolution remote control</td>
			<td>Karl Suss</td>
			<td>PH400</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Anti-vibration table (5&#8217; x 3&#8217;)</td>
			<td>TMC</td>
			<td>77049089</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>3D manual translational stage</td>
			<td>Newport</td>
			<td>462-XYZ-M</td>
			<td></td>
		</tr>
		<tr>
			<td>SolidWorks 3D CAD software</td>
			<td>SOLIDWORKS Corp.</td>
			<td>Version 2012 SP5</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>LabVIEW software</td>
			<td>National Instruments</td>
			<td>Version 2009</td>
			<td>BFP system, BFP program</td>
		</tr>
		<tr>
			<td>3D piezo translational stage</td>
			<td>Physik Instrumente</td>
			<td>M-105.3P</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Linear piezo accuator</td>
			<td>Physik Instrumente</td>
			<td>P-753.1CD</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Micromanager software</td>
			<td></td>
			<td>Version 1.4</td>
			<td>fBFP system, fluorescence imaging program</td>
		</tr>
		<tr>
			<td>Dual Cam (DC-2)</td>
			<td>Photometrics</td>
			<td>77054724</td>
			<td>fBFP system</td>
		</tr>
		<tr>
			<td>Dual Cam emission filter (T565LPXR)</td>
			<td>Photometrics</td>
			<td>77054725</td>
			<td>fBFP system</td>
		</tr>
		<tr>
			<td>Fluorescence Camera</td>
			<td>Hamamatsu</td>
			<td>ORCA-R2 C10600-10B</td>
			<td>fBFP system</td>
		</tr>
		<tr>
			<td>Plastic paraffin film (Parafilm)</td>
			<td>Bemis Company, Inc</td>
			<td>PM996</td>
			<td>bottle sealing</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Table 2: Buffer solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonate/bicarbonate buffer (pH 8.5)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Carbonate (Na2CO3)</td>
			<td>8.4g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>10.6g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer (pH 6.5-6.8)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaPhosphate monobasic&#160;&#160;&#160;NaH2PO4&#8226;H2O</td>
			<td>27.6g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anhy. NaPhosphate dibasic&#160;&#160; Na2HPO4</td>
			<td>28.4g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N2-5% buffer (pH 7.2)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>20.77g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>2.38g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>0.13g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anhy. Sodium Phosphate Dibasic (Na2HPO4)</td>
			<td>0.71g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>9.70g/L</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescence biomembrane force probe: concurrent quantitation of receptor-ligand kinetics and binding-induced intracellular signaling on a single cell

Interacções ligando-receptor de membrana medeiam muitas funções celulares. Encadernação cinética e sinalização a jusante desencadeada por essas interações moleculares são provavelmente afectado pelo ambiente em que a ligação mecânica e sinalização ocorrem. Um estudo recente demonstrou que a força mecânica pode regular o reconhecimento do antigénio pelo desencadeamento e do receptor de células T (TCR). Isso foi possível graças a uma nova tecnologia que nós desenvolvemos e fluorescência denominadas sonda vigor biomembrana (fBFP), que combina força espectroscopia única molécula com microscopia de fluorescência. Usando um glóbulo vermelho humano ultra-suave como o sensor de força sensível, uma câmera de alta velocidade e em tempo real técnicas de rastreamento de imagens, o fBFP é de ~ 1 pN (10 -12 N), a 3 nm e ~ 0,5 ms em vigor, a resolução espacial e temporal. Com o fBFP, pode-se medir com precisão cinética de ligação ligando-receptor individuais no âmbito da regulamentação vigente e cal intracelular simultaneamente imagem ligação desencadeadaCium sinalização sobre uma única célula viva. Esta nova tecnologia pode ser utilizada para estudar a interacção receptor-ligando outra membrana e sinalização em outras células sob regulação mecânica.

A técnica BFP foi iniciada pelo laboratório Evans em 1995 17. Esta ferramenta picoforce tem sido extensivamente utilizado para medir as interacções de proteínas imobilizadas em superfícies, de modo a analisar a cinética bidimensionais de moléculas de adesão que interage com os seus ligandos 16,19,20, 30, para medir a elasticidade molecular 21,29, e para determinar conformacional proteína muda 21. Para uma fBFP, um conjunto adicional de dispositivos relacionados-epi-f...

Watch this Scientific Journal Video about Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell at JoVE

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Fluorescência Biomembrane Força Probe: quantificação simultânea de Kinetics receptor de ligando e induzida Encadernação-Sinalização Intracelular em uma única célula

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

fluorescence-biomembrane-force-probe-concurrent-quantitation-receptor

Research

JoVE Journal

Bioengineering

12.3K visualizações.  Georgia Institute of Technology. We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Vídeo: Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

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.

Time-lapse imagens de fluorescência de crescimento de raiz Arabidopsis com Manipulação acelerada do meio root usando o 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, ...

Bioengenharia

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Investigando Sistemas receptor-ligante do Celulossoma com base-AFM única molécula Força Espectroscopia

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Espremendo celular como uma plataforma de entrega intracelular Robust Microfluidic

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

A técnica de microfluídica para sondar Deformabilidade celular

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Formação de biomembrana Microarrays com um método de montagem baseada em Rodo

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

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

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

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

3D Orbital Rastreamento em um microscópio de dois fotões Modificação: Uma Aplicação para o rastreamento de vesículas intracelulares

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

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

De fast imagens de fluorescência com a Lei difusão molecular em membranas de células vivas em um microscópio comercial

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.

Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces.

Superfície de Medição de Potencial de Bactérias Usando Kelvin Probe Microscopia de Força

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

Biomembrane Fabrication pelo Solvent-assistida Lipid bicamada (SALB) Método

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

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

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

Bicamadas única molécula de microscopia de fluorescência em Planar Apoiado

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