A versatile multimode microscope to probe and manipulate nanoparticles and biomolecules

We describe a flexible, multifaceted optical setup that allows quantitative measurement and manipulation of biomolecules and nanoparticles in biomimetic and cellular systems. We have implemented integrated biophotonics techniques (i.e. differential interference contrast, wide‐field fluorescence, prism‐ and objective‐based total internal reflection excitation, single particle tracking, fluorescence correlation spectroscopy and dynamic holographic optical trapping) on a single platform. The adaptability of this versatile, custom‐designed system allows us to simultaneously monitor cell morphology, while measuring lateral diffusion of biomolecules or controlling their cellular location or interaction partners.     

Microscopy of biological sample through advanced diffractive optics from visible to X-ray wavelength regime

The aim of this report is to demonstrate a unified version of microscopy through the use of advanced diffractive optics. The unified scheme derives from the technical possibility of realizing front wave engineering in a wide range of electromagnetic spectrum. The unified treatment is realized through the design and nanofabrication of phase diffractive elements (PDE) through which wave front beam shaping is obtained. In particular, we will show applications, by using biological samples, ranging from micromanipulation using optical tweezers to X-ray differential interference contrast (DIC) microscopy combined with X-ray fluorescence. We report some details on the design and physical implementation of diffractive elements that besides focusing also perform other optical functions: beam splitting, beam intensity, and phase redistribution or mode conversion. Laser beam splitting is used for multiple trapping and independent manipulation of micro-beads surrounding a cell as an array of tweezers and for arraying and sorting microscopic size biological samples. Another application is the Gauss to Laguerre-Gauss mode conversion, which allows for trapping and transfering orbital angular momentum of light to micro-particles immersed in a fluid. These experiments are performed in an inverted optical microscope coupled with an infrared laser beam and a spatial light modulator for diffractive optics implementation. High-resolution optics, fabricated by means of e-beam lithography, are demonstrated to control the intensity and the phase of the sheared beams in x-ray DIC microscopy. DIC experiments with phase objects reveal a dramatic increase in image contrast compared to bright-field x-ray microscopy. Besides the topographic information, fluorescence allows detection of certain chemical elements (Cl, P, Sc, K) in the same setup, by changing the photon energy of the x-ray beam.     

Development of a simultaneous Hugoniot and temperature measurement for preheated-metal shock experiments: melting temperatures of Ta at pressures of 100 GPa

Equations of state of metals are important issues in earth science and planetary science. A major limitation of them is the lack of experimental data for determining pressure-volume and temperature of shocked metal simultaneously. By measuring them in a single experiment, a major source of systematic error is eliminated in determining from which shock pressure release pressure originates. Hence, a non-contact fast optical method was developed and demonstrated to simultaneously measure a Hugoniot pressure-volume (P(H)-V(H)) point and interfacial temperature T(R) on the release of Hugoniot pressure (P(R)) for preheated metals up to 1000 K. Experimental details in our investigation are (i) a Ni-Cr resistance coil field placed around the metal specimen to generate a controllable and stable heating source, (ii) a fiber-optic probe with an optical lens coupling system and optical pyrometer with ns time resolution to carry out non-contact fast optical measurements for determining P(H)-V(H) and T(R). The shock response of preheated tantalum (Ta) at 773 K was investigated in our work. Measured data for shock velocity versus particle velocity at an initial state of room temperature was in agreement with previous shock compression results, while the measured shock data between 248 and 307 GPa initially heated to 773 K were below the Hugoniot evaluation from its off-Hugoniot states. Obtained interfacial temperatures on release of Hugoniot pressures (100-170 GPa) were in agreement with shock-melting points at initial ambient condition and ab initio calculations of melting curve. It indicates a good consistency for shock melting data of Ta at different initial temperatures. Our combined diagnostics for Hugoniot and temperature provides an important approach for studying EOS and the temperature effect of shocked metals. In particular, our measured melting temperatures of Ta address the current controversy about the difference by more than a factor of 2 between the melting temperatures measured under shock and those measured in a laser-heated diamond anvil cell at ～100 GPa.     

Internet-based robotic laser scissors and tweezers microscopy

We have engineered a robotic laser ablation and tweezers microscope that can be operated via the internet using most internet accessible devices, including laptops, desktop computers, and personal data assistants (PDAs). The system affords individual investigators the ability to conduct micromanipulation experiments (cell surgery or trapping) from remote locations (i.e., between the US and Australia). This system greatly expands the availability of complex and expensive research technologies via investigator-networking over the internet. It serves as a model for other "internet-friendly" technologies leading to large scale networking and data-sharing between investigators, groups, and institutions on a global scale. The system offers three unique features: (1) the freedom to operate the system from any internet-capable computer, (2) the ability to image, ablate, and/or trap cells and their organelles by "remote-control," and (3) the security and convenience of controlling the system in the laboratory on the user's own personal computer and not on the host machine. Four "proof of principle" experiments were conducted: (1) precise control of microscope movement and live cell visualization, (2) subcellular microsurgery on the microtubule organizing center of live cells viewed under phase contrast and fluorescence microscopy, (3) precise targeting of multiple sites within single red blood cells, and (4) optical trapping of 10 microm diameter polystyrene microspheres.     

Temperature dependence dynamical permeability characterization of magnetic thin film using near-field microwave microscopy

A temperature dependence characterization system of microwave permeability of magnetic thin film up to 5 GHz in the temperature range from room temperature up to 423 K is designed and fabricated as a prototype measurement fixture. It is based on the near field microwave microscopy technique (NFMM). The scaling coefficient of the fixture can be determined by (i) calibrating the NFMM with a standard sample whose permeability is known; (ii) by calibrating the NFMM with an established dynamic permeability measurement technique such as shorted microstrip transmission line perturbation method; (iii) adjusting the real part of the complex permeability at low frequency to fit the value of initial permeability. The algorithms for calculating the complex permeability of magnetic thin films are analyzed. A 100 nm thick FeTaN thin film deposited on Si substrate by sputtering method is characterized using the fixture. The room temperature permeability results of the FeTaN film agree well with results obtained from the established short-circuited microstrip perturbation method. Temperature dependence permeability results fit well with the Landau-Lifshitz-Gilbert equation. The temperature dependence of the static magnetic anisotropy H(K)(sta), the dynamic magnetic anisotropy H(K)(dyn), the rotational anisotropy H(rot), together with the effective damping coefficient α(eff), ferromagnetic resonance f(FMR), and frequency linewidth Δf of the thin film are investigated. These temperature dependent magnetic properties of the magnetic thin film are important to the high frequency applications of magnetic devices at high temperatures.     

Three-dimensional reconstruction of the guinea pig inner ear, comparison of OPFOS and light microscopy, applications of 3D reconstruction

Three-dimensional (3D) reconstruction of anatomical structures can give additional insight into the morphology and function of these structures. We compare 3D reconstructions of the guinea pig inner ear, using light microscopy and orthogonal plane fluorescence optical sectioning microscopy. Applications of 3D reconstruction of the inner ear are further explored. For each method two bullas were prepared for 3D reconstruction. Both methods are explained. In general, the 3D reconstructions using orthogonal plane fluorescence optical sectioning microscopy are superior to light microscopy. The exact spiral shape of the cochlea could be reconstructed using orthogonal plane fluorescence optical sectioning microscopy and the length of the basilar membrane measured. When a resolution of 20 μm is sufficient, orthogonal plane fluorescence optical sectioning microscopy is a superior technique for 3D reconstruction of inner ear structures in animals.     

Analytical Behavior of Fura-2 and Its Determination of [Ca2+]i in Lymphocytes Treated with Cefotaxime

The interaction of Fura-2 with Ca2+ is studied using steady fluorescence technique.The effect of pH on the spectra behavior of Fura-2 in the presence of Ca2+ is investigated,the excitation maxima(340 nm)and the isobestic point(360 nm)for the fluorescence spectra of Fura-2 depend on pH.At different temperatures the apparent dissociation constants(Kd)of Fura-2-Ca2+ complex are examined,Kd is found to decrease with increasing temperatures(20 ℃,37 ℃,50 ℃)and ΔH is calculated to be 21.16 kJ/mol by using the Van't Hoff equation at pH 7.4 for all the temperatures tested.The determination of intracellular Ca2+ concentration([Ca2+]i)in lymphocyte is developed by using Fura-2 as a fluorescence probe in the presence of Cefotaxime at 37 ℃ and pH 7.4.     

Effect of a confocal pinhole in two-photon microscopy.

We investigated the effect of a finite-sized confocal pinhole on the performance of nonlinear optical microscopes based on two-photon excited fluorescence and second-harmonic generation. These techniques were implemented using a modified inverted commercial confocal microscope coupled to a femtosecond Ti:sapphire laser. Both the transverse and axial resolutions are improved when the confocal pinhole is used, albeit at the expense of the signal level. Therefore, the routine use of a confocal pinhole of optimized size is recommended for two-photon microscopy wherever the fluorescence or harmonic signals are large.     

A combined optical and atomic force microscope for live cell investigations

We present an easy-to-use combination of an atomic force microscope (AFM) and an epi-fluorescence microscope, which allows live cell imaging under physiological conditions. High-resolution AFM images were acquired while simultaneously monitoring either the fluorescence image of labeled membrane components, or a high-contrast optical image (DIC, differential interference contrast). By applying two complementary techniques at the same time, additional information and correlations between structure and function of living organisms were obtained. The synergy effects between fluorescence imaging and AFM were further demonstrated by probing fluorescence-labeled receptor clusters in the cell membrane via force spectroscopy using antibody-functionalized tips. The binding probability on receptor-containing areas identified with fluorescence microscopy ("receptor-positive sites") was significantly higher than that on sites lacking receptors.     

Combining ocFLIM and FIDSAM reveals fast and dynamic physiological responses at subcellular resolution in living plant cells

For a deeper understanding of molecular mechanisms within cells and for the realization of predictive biology for intracellular processes at subcellular level, quantitative biology is required. Therefore, novel optical and spectroscopic technologies with quantitative and dynamic output are needed in cell biology. Here, we present a combined approach of novel one-chromophore fluorescence lifetime imaging microscopy to probe the local environment of fluorescent fusion proteins and fluorescence intensity decay shape analysis microscopy to suppress interfering autofluorescence. By applying these techniques, we are able to analyse the subcellular localization and partitioning of a green fluorescence protein fusion of the salt stress-induced protein low temperature induced (LTI)6b in great detail with high spatial and temporal resolution in living cells of Arabidopsis plants.     

Cryogenic gas loading in a Mao-Bell-type diamond anvil cell for high pressure-high temperature investigations

A simple system for loading argon fluid at cryogenic temperatures in a Mao-Bell-type diamond anvil cell (DAC) has been developed. It is done in a two step process in which the piston-cylinder assembly alone is submerged in the cryogenic chamber for trapping the liquefied inert gas. Liquid nitrogen is used for condensing the argon gas. This system is now being efficiently used for loading liquid argon in the DAC for high pressure-high temperature experiments. The success rate of trapping liquefied argon in the sample chamber is about 75%. The performance of the gas loading system is successfully tested by carrying out direct conversion of pyrolitic graphite to diamond under high pressure-high temperature using laser heated DAC facility.     

Combining optical tweezers and patch clamp for studies of cell membrane electromechanics

We have designed and implemented a novel experimental setup which combines optical tweezers with patch-clamp apparatus to investigate the electromechanical properties of cellular plasma membranes. In this system, optical tweezers provide measurement of forces at piconewton scale, and the patch-clamp technique allows control of the cell transmembrane potential. A micron-size bead trapped by the optical tweezers is brought in contact with the membrane of a voltage-clamped cell, and subsequently moved away to form a plasma membrane tether. Bead displacement from the trapping center is monitored by a quadrant photodetector for dynamic measurements of tether force. Fluorescent beads and the corresponding fluorescence imaging optics are used to eliminate the shadow of the cell projected on the quadrant photodetector. Salient information associated with the mechanical properties of the membrane tether can thus be obtained. A unique feature of this setup is that the patch-clamp headstage and the manipulator for the recording pipette are mounted on a piezoelectric stage, preventing relative movements between the cell and the patch pipette during the process of tether pulling. Tethers can be pulled from the cell membrane at different holding potentials, and the tether force response can be measured while changing transmembrane potential. Experimental results from mammalian cochlear outer hair cells and human embryonic kidney cells are presented.     

Near-field fluorescence imaging and simultaneous observation of surface potentials

We present a new detection method to measure simultaneously surface potential and fluorescence intensity distributions using a combined scanning near-field optical microscope-atomic force microscope (SNOM-AFM). A surface potential image of phospholipid monolayers was obtained in non-contact mode using the SNOM-AFM with a thin-step etched optical fibre probe. For applying this technique, a phospholipid of dipalmitoylphosphatidylethanolamine labelled at the head with a nitrobenzoxadiazole group was used as a fluorescent and single component Langmuir–Blodgett film. It is well known that aggregation of the lipid molecules and their fluorescence intensities are very sensitive to its environmental conditions such as humidity and temperature. We demonstrated for the first time the near-field optical imaging and simultaneous observation of surface potentials with Maxwell stress microscopy.     

Development and testing of hyperbaric atomic force microscopy (AFM) and fluorescence microscopy for biological applications

A commercially available atomic force microscopy and fluorescence microscope were installed and tested inside a custom-designed hyperbaric chamber to provide the capability to study the effects of hyperbaric gases on biological preparations, including cellular mechanism of oxidative stress. In this report, we list details of installing and testing atomic force microscopy and fluorescence microscopy inside a hyperbaric chamber. The pressure vessel was designed to accommodate a variety of imaging equipment and ensures full functionality at ambient and hyperbaric conditions (≤85 psi). Electrical, gas and fluid lines were installed to enable remote operation of instrumentation under hyperbaric conditions, and to maintain viable biological samples with gas-equilibrated superfusate and/or drugs. Systems were installed for vibration isolation and temperature regulation to maintain atomic force microscopy performance during compression and decompression. Results of atomic force microscopy testing demonstrate sub-nanometre resolution at hyperbaric pressure in dry scans and fluid scans, in both contact mode and tapping mode. Noise levels were less when measurements were taken under hyperbaric pressure with air, helium (He) and nitrogen (N(2) ). Atomic force microscopy and fluorescence microscopy measurements were made on a variety of living cell cultures exposed to hyperbaric gases (He, N(2) , O(2) , air). In summary, atomic force microscopy and fluorescence microscopy were installed and tested for use at hyperbaric pressures and enables the study of cellular and molecular effects of hyperbaric gases and pressure per se in biological preparations.     

The three-dimensional architecture of the mitotic spindle,analyzed by confocal fluorescence and electron microscopy

Fluorescence microscopy techniques have become important tools in mitosis research. The well-known disadvantages of fluorescence microscopy, rapid bleaching, phototoxicity and out-of-focus contributions blurring the in-focus image are obstacles which still need to be overcome. Confocal fluorescence microscopy has the potential to improve our capabilities of analyzing cells, because of its excellent depth-discrimination and image processing power. We have been using a confocal fluorescence microscope for the study of the mechanism of poleward chromosome movement, and report here (1) a cell preparation technique, which allows labeling of fixation sensitive spindle antigens with acceptable microtubule preservation; (2) the use of image processing methods to represent the spatial distribution of various labeled elements in pseudocolour; (3) a novel immunoelectron microscopic labeling method for microtubules, which allows the visualization of their distribution in semithin sections at low magnification; and (4) a first attempt to study microtubule dynamics with a confocal fluorescence microscope in living cells, microinjected with rhodamine labeled tubulin. Our experience indicates that confocal fluorescence microscopy provides real advantages for the study of spatial colocalization of antigens in the mitotic spindle. It does not, however, overcome the basic limits of resolution of the light microscope. Therefore, it has been necessary to use an electron microscopic method. Our preliminary results with living cells show that it is possible to visualize the entire microtubule network in stereo, but that the sensitivity of the instrument is still too low to perform dynamic time studies. It will be worthwhile to further develop this new type of optical instrumentation and explore its usefulness on both fixed and living cells.     

Optimizing multiphoton fluorescence microscopy light collection from living tissue by noncontact total emission detection (epiTED)

A benefit of multiphoton fluorescence microscopy is the inherent optical sectioning that occurs during excitation at the diffraction-limited spot. The scanned collection of fluorescence emission is incoherent; that is, no real image needs to be formed on the detector plane. The nearly isotropic emission of fluorescence excited at the focal spot allows for new detection schemes that efficiently funnel all attainable photons to detector(s). We previously showed [Combs, C.A., et al. (2007) Optimization of multiphoton excitation microscopy by total emission detection using a parabolic light reflector. J. Microsc. 228, 330-337] that parabolic mirrors and condensers could be combined to collect the totality of solid angle around the excitation spot for tissue blocks, leading to ～8-fold signal gain. Using a similar approach, we have developed an in vivo total emission detection (epiTED) instrument modified to make noncontact images from outside of living tissue. Simulations suggest that a ～4-fold enhancement may be possible (much larger with lower NA objectives than the 0.95 NA used here) with this approach, depending on objective characteristics, imaging depth and the characteristics of the sample being imaged. In our initial prototype, 2-fold improvements were demonstrated in the mouse brain and skeletal muscle as well as the rat kidney, using a variety of fluorophores and no compromise of spatial resolution. These results show this epiTED prototype effectively doubles emission signal in vivo; thus, it will maintain the image signal-to-noise ratio at two times the scan rate or enable full scan rate at approximately 30% reduced laser power (to minimize photo-damage).     

Atomic force microscopy and cells: Indentation profiles around the AFM tip,cell shape changes,and other examples of experimental factors affecting modeling

We use atomic force microscopy in conjunction with a fluorescence microscope capable of optical sectioning to acquire images of white blood cells while force is applied with the AFM tip. The indentation profile within the cell is compared to the profile of the AFM tip: examples are shown for indentations at the center of the cell which are reasonable matches to the tip profile, and an additional example is shown for an indentation that is on the tilted side of a highly rounded cell and that differs from the tip shape. We also demonstrate that the AFM tip can interact with internal cell structures, we show that the contact area between the cell and the substrate can increase under applied pressure, that the main body of the cell can fuse with the extended lamellipodium, and that the cell can be displaced laterally by the AFM tip. The features illustrated here are relevant to the interpretation of indentation experiments that measure cell elasticity properties, as is discussed briefly. Microsc. Res. Tech. 78:626–632, 2015. © 2015 Wiley Periodicals, Inc.     

Characterization of surface grooves and scratches induced by friction of C/C composites at low and high temperatures

Friction experiments were conducted on C/C composites at low and high temperatures during braking with the use of a pin-on-disc tribometer. The surface grooves formed were investigated by an optical camera and a laser profilometer, while scratches were characterized by optical microscopy. Damages were correlated with tribological performances (friction and wear). It is shown that friction at low temperature leads to high friction coefficient and wear rate, and to surfaces strongly grooved and abraded. For friction experiments performed at high temperature, they lead to lower friction coefficient and wear, and the resulting surfaces are rather smooth and slightly grooved.     

Contrast,resolution, pixelation,dynamic range and signal-to-noise ratio: fundamental limits to resolution in fluorescence light microscopy

In a perfect optical system numerical aperture and wavelength determine resolution. In a real optical system, however, the number of photons collected from a specimen determines the contrast and this limits the resolution. Contrast is affected by the number of picture elements per unit area, the number of photons and the aberrations present in every optical system. The concept of contrast vs. distance functions is used to compare the resolution achievable in confocal and wide-field fluorescence microscopes and the effect of a further reduction of the observable volume. In conclusio: (a) real optical systems will never be able to achieve the theoretical resolution, (b) wide-field fluorescence microscopy will often provide a better resolution than confocal fluorescence microscopy, (c) decreasing the observed volume does not necessarily increase the resolution and (d) using multiple fluorophores can improve the accuracy with which distances are measured. Some numbers for typical situations are provided.     

Two-photon fluorescence surface wave microscopy

This paper demonstrates the principle of two-photon surface wave microscopy with a view to applications on biological samples. We describe a modified scanning optical microscope, which uses specially prepared coverslips. These coverslips are designed to support the propagation of surface waves capable of large field enhancements. We also discuss the beam conditioning necessary to ensure efficient use of the available illumination. Two-photon surface wave fluorescent excitation is demonstrated on fluorescent nanospheres, demonstrating a point spread function width of ≈220 nm at an illumination wavelength of 925 nm. The potential of non-linear surface wave excitation for both fluorescence and harmonic imaging microscopy is discussed.