This work was partially supported by the National Key R&#38;D Program of China (Grant No. 2017YFA0304401) and the National Natural Science Foundation of China (Grant Nos. 11774108, 91336213, and 61875065).

1. Set up the reference directions for polarizers A and B
<ol>
	<li>Put polarizer A and polarizer B into the laser beam (280 nm fourth harmonic laser) path.</li>
	<li>Ensure that the laser beam is perpendicular to the surfaces of the polarizers by carefully adjusting the polarizer holders to keep the back-reflection light coincident with the incident light. 
	NOTE: All the following alignment procedures for the optics components must follow the same rule. The placement of polarizer A and B in the laser path is not important. The spacing between them should be large enough for the future convenient adjustment.</li>
	<li>Put a power meter behind polarizer A and rotate the polarizer to maximize the output power. Define the azimuthal angle (see Results and Discussion) of the optical axis of polarizer A as 0&#176;. Define the clockwise direction as the positive direction and the counterclockwise direction as the negative direction when observing along the direction of light propagation.
	<ol>
		<li>Use a stepper motor rotation stage to hold polarizer A and put the power meter behind polarizer A to record the rotation angles and the output laser powers. Fit the angle vs power curve with a sinusoidal function; the maximum output power position of polarizer A is 0&#176; azimuthal angle position.</li>
	</ol>
	</li>
	<li>Put the power meter behind polarizer B and rotate polarizer B to maximize the output power. The azimuthal angle of the optical axis of polarizer B is then also 0&#176;.
	<ol>
		<li>Use another stepper motor rotation stage to hold polarizer B and put the power meter behind polarizer B to record the rotation angles and the output laser powers. Fit the angle vs power curve with a sinusoidal function; the maximum output power position of polarizer B is 0&#176; azimuthal angle position (see Figure 1).</li>
	</ol>
	</li>
</ol>
2. Set up the reference directions for the azimuthal angles of the waveplates
<ol>
	<li>Put an HWP into the beam path between polarizer A and polarizer B and rotate the HWP to maximize the output power. The azimuthal angle of the optical axis of the HWP is then 0&#176;.
	<ol>
		<li>Use a stepper motor rotation stage to hold the HWP and put the power meter behind polarizer B to record the rotation angles and the output laser powers. Fit the angle vs power curve with a sinusoidal function; the maximum output power position of the HWP is 0&#176; azimuthal angle.</li>
	</ol>
	</li>
	<li>Put a QWP into the beam path between the HWP and polarizer B, rotate the QWP to maximize the output power. The azimuthal angle of the optical axis of the QWP is then 0&#176;.
	<ol>
		<li>Use a stepper motor rotation stage to hold the QWP and put the power meter behind polarizer B to record the rotation angles and the output laser powers. Fit the angle vs power curve with a sinusoidal function; the maximum output power position of the QWP is 0&#176; azimuthal angle position.</li>
	</ol>
	</li>
	<li>Remove polarizer B and the power meter from the beam path. Use two mirrors to direct laser beam into the vacuum chamber that houses an ion trap to interact with 25Mg+ ions. 
	NOTE: The laser propagation direction should be along the magnetic field direction inside the vacuum chamber. A magnetic field is used to define the quantization axis of the ions.</li>
</ol>
3. Doppler cooling of single 25Mg+ ions
<ol>
	<li>Turn on the 532 nm ablation laser, which is a Q-switched Nd:YAG laser. Its repetition rate is 1 kHz, with pulse energy of 150 &#181;J. The ablation laser irradiates a magnesium wire target surface inside the vacuum chamber, and then magnesium (Mg) atoms are ejected from the target surface. 
	NOTE: The power supply for the ion trap should be turned on.</li>
	<li>At the same time, turn on the 285 nm ionization laser to ionized Mg atoms. The ionization laser is a fourth harmonic laser with an output power of 1 mW. The ionization laser will illuminate the center of the ion trap.</li>
	<li>Make sure only one ion is trapped in the ion trap by looking at the image of an electron multiplied charged coupled device (EMCCD). An example image showing trapped ions is shown in Figure 2. Each bright spot is one ion. If there is more than one ion in the trap, turn off the power supply of the ion trap to release the ions. Then repeat steps 3.1-3.2 until only one (i.e., single) ion is trapped. 
	NOTE: The homemade imaging system of the EMCCD consists of four lenses, and its magnification is 10x. The ion spacing is about 2-10 &#181;m and the pixel spacing of the EMCCD is 16 &#181;m. The EMCCD can, therefore, be used to identify the existence of one single ion.</li>
	<li>Set the magnetic field to be 6.5 Gauss by adjusting the current of Helmholtz coils. The magnetic field is measured by comparing the different frequencies between the two ground state transitions, <img align="center" alt="Equation" src="/files/ftp_upload/61175/61175eq1.jpg" /> and <img align="center" alt="Equation" src="/files/ftp_upload/61175/61175eq2.jpg" />. For details of the method please refer11.</li>
</ol>
4. Lock the 280 nm Doppler cooling laser frequency to a wavelength meter12
<ol>
	<li>Scan the frequency of the 280 nm laser and count the fluorescence photon numbers collected by a photon multiplier tube (PMT) by a frequency counter. At the same time, record the frequency of the laser using a wavelength meter. Find the resonant frequency &#957;0 where the fluorescence rate reaches a maximum. 
	NOTE: The fluorescence counts will increase when the laser frequency is moving close to the ion resonant frequency and will reach a maximum at the resonant frequency <img align="center" alt="Equation" src="/files/ftp_upload/61175/61175eq3.jpg" />.</li>
	<li>Lock the laser frequency to the wavelength meter using a digital servo control program that is running on an accompanying computer. Click on the Lock button on the program graphic interface when the wavelength meter shows a reading of <img align="center" alt="Equation" src="/files/ftp_upload/61175/61175eq3.jpg" />.</li>
</ol>
5. Set the intensity of the laser to equal the saturation intensity12
<ol>
	<li>Change the power of the laser by adjusting the driving power of an acousto-optic-modulator (AOM), which is used in the beam path to change the frequency and power of the laser. Record the power and the fluorescence counts.</li>
	<li>Fit the curve of the power and the fluorescence counts with Equation (6), and obtain the saturation power <img align="center" alt="Equation" src="/files/ftp_upload/61175/61175eq4a.jpg" />.</li>
	<li>Set the laser power to <img align="center" alt="Equation" src="/files/ftp_upload/61175/61175eq4a.jpg" /> by adjusting the driving power of the AOM.</li>
</ol>
6. Measure the birefringence of the vacuum window.
<ol>
	<li>Alternately, adjust the azimuthal angles of the HWP and the QWP to maximize the fluorescence counts. Record the azimuthal angles of the HWP and the QWP at maximum counts, which are &#945; and &#946;.
	<ol>
		<li>Use the stepper motor rotation stages to rotate the HWP and the QWP and record the rotation angles and the corresponding fluorescence counts.</li>
	</ol>
	</li>
	<li>Use Equation (4) and Equation (5) to calculate the birefringence of the vacuum window &#952; and <img alt="Equation" src="/files/ftp_upload/61175/61175eq5.jpg" />.</li>
</ol>

<ol>
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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+Measurement+of+the+Electron's+Electric+Dipole+Moment+Using+Trapped+Molecular+Ions.">Precision Measurement of the Electron's Electric Dipole Moment Using Trapped Molecular Ions.</a> Physical Review Letters. 119 (15), 153001 (2017).</li><li>Bougas, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+cavity-enhanced+polarimetry+for+parity-nonconserving+optical+rotation+measurements:+Application+to+Xe,+Hg,+and+I.">Fundamentals of cavity-enhanced polarimetry for parity-nonconserving optical rotation measurements: Application to Xe, Hg, and I.</a> Physical Review A. 89 (5), 52127 (2014).</li><li>Bragin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Energy+Vacuum+Birefringence+and+Dichroism+in+an+Ultra-strong+Laser+Field.">High-Energy Vacuum Birefringence and Dichroism in an Ultra-strong Laser Field.</a> Physical Review Letters. 119 (25), 250403 (2017).</li><li>Nicholson, T. 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The authors have nothing to disclose.

This manuscript describes a method to perform in situ measurement of the birefringence of the vacuum window and the polarization states of the laser light inside the vacuum chamber. By adjusting the azimuthal angles of the HWP and the QWP (&#945; and &#946;), the effect of the birefringence of the vacuum window (&#948; and &#952;) can be compensated so that the laser inside the vacuum chamber is a pure circularly polarized light. At this point, there exists a definite relationship between the birefringence of the vacuum ...

In many research fields such as cold atom experiments1, measurement of the electric dipole moment2, test of parity-nonconservation3, measurement of vacuum birefringence4, optical clocks5, quantum optics experiments6, and liquid crystal study7, it is important to precisely measure and accurately control the polarization states of laser light.
In experiments involving the use of a vacuum environment, the stress-induced birefringence effect of vacuum windows will affect the polarization states of laser light. It is not feasible to put a polarization analyzer inside the vacuum chamber to directly measure the polarization states of the laser light. One solution is to use atoms or ions directly as an in situ polarization analyzer to analyze the birefringence of vacuum windows. The vector light shifts of Cs atoms8 are sensitive to the degrees of linear polarization of the incidence laser light9. But this method is time consuming and can only be applied to the linearly polarized laser light detection.
Presented is a new, quick, precise, in situ method to determine the polarization states of laser light inside the vacuum chamber based on maximizing single 25Mg+ fluorescence in an ion trap. The method is based on the relationship of the ion fluorescence to the polarization states of the laser light, which is affected by the birefringence of the vacuum window. The proposed method is used for detecting the birefringence of vacuum windows and degrees of circular polarization of laser light inside a vacuum chamber10.
The method is applicable to any atoms or ions whose fluorescence rate is sensitive to the polarization states of laser light. In addition, while the demonstration is used to prepare a pure circularly polarized light, with the knowledge of the birefringence of the vacuum window, arbitrary polarization states of laser light can be prepared inside the vacuum chamber. Therefore, the method is quite useful for a wide range of experiments.

<table><tbody><tr><td>280 nm Doppler cooling laser</td><td>Toptica</td><td>SYST DL-FHG Pro 280</td><td>Doppler cooling laser</td></tr><tr><td>285 nm ionization laser</td><td>Toptica</td><td>SYST DL-FHG Pro 285</td><td>ionization laser</td></tr><tr><td>Ablation laser</td><td>Changchun New Industries Optoelectronics Technology</td><td>EL-532-1.5W</td><td>Q-switched Nd:YAG laser</td></tr><tr><td>AOM</td><td>Gooch &amp;amp; Housego</td><td>AOMO 3200-1220</td><td>wavelengh down to 257 nm</td></tr><tr><td>EMCCD camera</td><td>Andor</td><td>iXon3 897</td><td>imaging of &lt;sup&gt;25&lt;/sup&gt;Mg&lt;sup&gt;+&lt;/sup&gt; in ion trap</td></tr><tr><td>Glan-Taylor polarizer</td><td>Union Optic</td><td>Custom</td><td>distinction ratio 1e-6</td></tr><tr><td>Half waveplate</td><td>Union Optic</td><td>Custom</td><td>made of quartz</td></tr><tr><td>Photon multiplier tube</td><td>Hamamatsu</td><td>H8259-09</td><td>fluorescent counting</td></tr><tr><td>Power meter</td><td>Thorlabs</td><td>PM100D</td><td>laser power monitor</td></tr><tr><td>Quarter waveplate</td><td>Union Optic</td><td>Custom</td><td>made of quartz</td></tr><tr><td>Mirror</td><td>Union Optic</td><td>Custom</td><td>dielectric coated for 280 nm</td></tr><tr><td>Stepper motor roation stage</td><td>Thorlabs</td><td>K10CR1/M</td><td>rotating wave plates</td></tr><tr><td>Vacuum chamber</td><td>Kimball Physics</td><td>MCF800-SphSq-G2E4C4</td><td>made of Titanium</td></tr><tr><td>Vacuum window</td><td>Union Optic</td><td>Custom</td><td>made of fused silica</td></tr></tbody></table>

in situ measurement of vacuum window birefringence using 25mg+ fluorescence

Accurate control of the polarization states of laser light is important in precision measurement experiments. In experiments involving the use of a vacuum environment, the stress-induced birefringence effect of the vacuum windows will affect the polarization states of laser light inside the vacuum system, and it is very difficult to measure and optimize the polarization states of the laser light in situ. The purpose of this protocol is to demonstrate how to optimize the polarization states of the laser light based on the fluorescence of ions in the vacuum system, and how to calculate the birefringence of vacuum windows based on azimuthal angles of external wave plates with Mueller matrix. The fluorescence of 25Mg+ ions induced by laser light that is resonant with the transition of |32P3/2,F = 4, mF&#160;= 4<img align="center" alt="Equation 100" src="/files/ftp_upload/61175/61175img1.jpg" style="color: rgb(68, 0, 68); font-size: 18px; font-weight: 700;" />&#160;&#8594;&#160;|32S1/2,F = 3, mF&#160;= 3<img align="center" alt="Equation 100" src="/files/ftp_upload/61175/61175img1.jpg" style="color: rgb(68, 0, 68); font-size: 18px; font-weight: 700;" />&#160;is sensitive to the polarization state of the laser light, and maximum fluorescence will be observed with pure circularly polarized light. A combination of half-wave plate (HWP) and quarter-wave plate (QWP) can achieve arbitrary phase retardation and is used for compensating the birefringence of the vacuum window. In this experiment, the polarization state of the laser light is optimized based on the fluorescence of 25Mg+ ion with a pair of HWP and QWP outside the vacuum chamber. By adjusting the azimuthal angles of the HWP and QWP to obtain maximum ion fluorescence, one can obtain a pure circularly polarized light inside the vacuum chamber. With the information on the azimuthal angles of the external HWP and QWP, the birefringence of the vacuum window can be determined.

Figure 3 shows the beam path of the experiment. Polarizer B in Figure 3a is removed after angle initialization (Figure 3b). The laser passed through a polarizer, an HWP, a QWP, and the vacuum window, sequentially. The Stokes vector of laser is <img align="center" alt="Equation" src="/files/ftp_upload/61175/61175eq6.jpg" />, where <img align="center" alt="Equation" sr...

Watch this Scientific Journal Video about In Situ Measurement of Vacuum Window Birefringence using 25Mg+ Fluorescence at JoVE.com

In Situ Measurement of Vacuum Window Birefringence using 25Mg+ Fluorescence

Presented here is a method to measure the birefringence of vacuum windows by maximizing the fluorescence counts emitted by Doppler cooled 25Mg+ ions in an ion trap. The birefringence of vacuum windows will change the polarization states of the laser, which can be compensated by changing the azimuthal angles of external wave plates.

In Situ Measurement of Vacuum Window Birefringence using 25Mg+ Fluorescence

Accurate control of the polarization states of laser light is important in precision measurement experiments. In experiments involving the use of a vacuum ...

in-situ-measurement-vacuum-window-birefringence-using-25mg

Research

JoVE Journal

Engineering

3.8K Views.  Huazhong University of Science and Technology. De methode die hier wordt gepresenteerd, kan worden gebruikt om te compenseren in een birefringence van vacuüm ramen, om een zuiver gepolariseerd licht te genereren, in een vacuümkamer. Het kan vele toepassingen in de procedure experimenten waarbij koude atomen of ionen. Het belangrijkste voordeel van deze techniek is dat niet atomen of ionen niet alleen worden gebruikt als objecten die moeten worden gestart, maar ook als in-situ detectoren van polarisatietoestanden.We kunnen voorkom...

Video: In Situ Measurement of Vacuum Window Birefringence using 25Mg+ Fluorescence

Synthesis and Operation of Fluorescent-core Microcavities for Refractometric Sensing

This paper discusses fluorescent core microcavity-based sensors that can operate in a microfluidic analysis setup. These structures are based on the formation of a fluorescent quantum-dot (QD) coating on the channel surface of a conventional microcapillary. Silicon QDs are especially attractive for this application, owing in part to their negligible toxicity compared to the II-VI and II-VI compound QDs, which are legislatively controlled substances in many countries. While the ensemble emission spectrum is broad and featureless, an Si-QD film on the channel wall of a capillary features a set of sharp, narrow peaks in the fluorescence spectrum, corresponding to the electromagnetic resonances for light trapped within the film. The peak wavelength of these resonances is sensitive to the external medium, thus permitting the device to function as a refractometric sensor in which the QDs never come into physical contact with the analyte. The experimental methods associated with the fabrication of the fluorescent-core microcapillaries are discussed in detail, as well as the analysis methods. Finally, a comparison is made between these structures and the more widely investigated liquid-core optical ring resonators, in terms of microfluidic sensing capabilities.

Fluorescent-core microcavity sensors employ a high-index quantum-dot coating in the channel of silica microcapillaries. Changes in the refractive index of fluids pumped into the capillary channel cause shifts in the microcavity fluorescence spectrum that can be used to analyze the channel medium.

This paper discusses  fluorescent core microcavity-based sensors that can operate in a microfluidic  analysis setup. These structures are based on the ...

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)

Surface topology, e.g. of cells growing on a substrate, is determined with nanometer precision by Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM). Cells are cultivated on transparent slides and incubated with a fluorescent marker homogeneously distributed in their plasma membrane. Illumination occurs by a parallel laser beam under variable angles of total internal reflection (TIR) with different penetration depths of the evanescent electromagnetic field. Recording of fluorescence images upon irradiation at about 10 different angles permits to calculate cell-substrate distances with a precision of a few nanometers. Differences of adhesion between various cell lines, e.g. cancer cells and less malignant cells, are thus determined. In addition, possible changes of cell adhesion upon chemical or photodynamic treatment can be examined. In comparison with other methods of super-resolution microscopy light exposure is kept very small, and no damage of living cells is expected to occur.

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM

Topology of cell adhesion on a substrate is measured with nanometre precision by variable-angle total internal reflection fluorescence microscopy (VA-TIRFM).

Surface topology, e.g. of cells growing on a substrate, is determined with nanometer precision by Variable-Angle Total Internal Reflection Fluorescence ...

Bioengineering

Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies

Bicelles are tunable disk-like polymolecular assemblies formed from a large variety of lipid mixtures. Applications range from membrane protein structural studies by nuclear magnetic resonance (NMR) to nanotechnological developments including the formation of optically active and magnetically switchable gels. Such technologies require high control of the assembly size, magnetic response and thermal resistance. Mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and its lanthanide ion (Ln3+) chelating phospholipid conjugate, 1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate (DMPE-DTPA), assemble into highly magnetically responsive assemblies such as DMPC/DMPE-DTPA/Ln3+ (molar ratio 4:1:1) bicelles. Introduction of cholesterol (Chol-OH) and steroid derivatives in the bilayer results in another set of assemblies offering unique physico-chemical properties. For a given lipid composition, the magnetic alignability is proportional to the bicelle size. The complexation of Ln3+ results in unprecedented magnetic responses in terms of both magnitude and alignment direction. The thermo-reversible collapse of the disk-like structures into vesicles upon heating allows tailoring of the assemblies&#39; dimensions by extrusion through membrane filters with defined pore sizes. The magnetically alignable bicelles are regenerated by cooling to 5 &#176;C, resulting in assembly dimensions defined by the vesicle precursors. Herein, this fabrication procedure is explained and the magnetic alignability of the assemblies is quantified by birefringence measurements under a 5.5 T magnetic field. The birefringence signal, originating from the phospholipid bilayer, further enables monitoring of polymolecular changes occurring in the bilayer. This simple technique is complementary to NMR experiments that are commonly employed to characterize bicelles.

Fabrication procedures for highly magnetically responsive lanthanide ion chelating polymolecular assemblies are presented. The magnetic response is dictated by the assembly size, which is tailored by extrusion through nanopore membranes. The assemblies&#39; magnetic alignability and temperature-induced structural changes are monitored by birefringence measurements, a complimentary technique to nuclear magnetic resonance and small angle neutron scattering.

Bicelles are tunable disk-like polymolecular assemblies formed from a large variety of lipid mixtures. Applications range from membrane protein structural ...

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.

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

Longitudinal Intravital Imaging Through Clear Silicone Windows

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging windows is particularly powerful because it allows longitudinal observation of the same tissue over days to weeks. Typical imaging windows comprise a glass coverslip in a biocompatible metal frame sutured to the mouse&#8217;s skin. These windows can interfere with the free movement of the mice, elicit a strong inflammatory response, and fail due to broken glass or torn sutures, any of which may necessitate euthanasia. To address these issues, windows for long-term abdominal organ and mammary gland imaging&#160;were developed from a thin film of polydimethylsiloxane (PDMS), an optically clear silicone polymer previously used for cranial imaging windows. These windows can be glued directly to the tissues, reducing the time needed for insertion. PDMS is flexible, contributing to its durability in mice over time&#8212;up to 35 days have been tested. Longitudinal imaging is imaging of the same tissue region during separate sessions. A stainless-steel grid was embedded within the windows to localize the same region, allowing the visualization of dynamic processes (like mammary gland involution) at the same locations, days apart. This silicone window also allowed monitoring of single disseminated cancer cells developing into micro-metastases over time. The silicone windows used in this study are simpler to insert than metal-framed glass windows and cause limited inflammation of the imaged tissues. Moreover, embedded grids allow for straightforward tracking of the same tissue region in repeated imaging sessions.

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin.&#160;These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging ...

Cancer Research

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Biology

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. Here, we detail the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized. The protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, we provide a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment and demonstrate how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. In conclusion, this fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.

This article aims to present a protocol on how to build a spot variation Fluorescence Correlation Spectroscopy (svFCS) microscope to measure molecular diffusion at the plasma membrane of living cells.

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ...

Biochemistry

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

High Speed Sub-GHz Spectrometer for Brillouin Scattering Analysis

The goal of this protocol is to build a parallel high-extinction and high-resolution optical Brillouin spectrometer. Brillouin spectroscopy is a non-contact measurement method that can be used to obtain direct readouts of viscoelastic material properties. It has been a useful tool in material characterization, structural monitoring and environmental sensing. In the past, Brillouin spectroscopy has usually employed scanning Fabry-Perot etalons to perform spectral analysis. This process requires high illumination power and long acquisition times, making the technique unsuitable for biomedical applications. A recently introduced novel spectrometer overcomes this challenge by employing two VIPAs in a cross-axis configuration. This innovation enables sub-Gigahertz (GHz) resolution spectral analysis with sub-second acquisition time and illumination power within the safety limits of biological tissue. The multiple new applications facilitated by this improvement are currently being explored in biological research and clinical application.

Here we present a protocol to build a rapid Brillouin spectrometer. Cascading virtually imaged phase array (VIPA) etalons achieve a measurement speed more than 1,000 times faster than traditional scanning Fabry-Perot spectrometers. This improvement provides the means for Brillouin analysis of tissue and biomaterials at low power levels in vivo.

The goal of this protocol is to build a parallel high-extinction and high-resolution optical Brillouin spectrometer. Brillouin spectroscopy is a ...

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

Here, we describe protocols using fluorescent lipid sensors and liposomes to determine whether a protein extracts and transports phosphatidylserine or phosphatidylinositol 4-phosphate in vitro.

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found ...

In Situ Measurement of Vacuum Window Birefringence using ²⁵Mg⁺ Fluorescence

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