Dark-field Imaging System Research Articles

Femtomolar oligonucleotide detection by a one-step gold nanoparticle-based assay

A sequence-specific oligonucleotide detection method based on the tail-to-tail aggregation of functionalized gold nanoparticles in the presence of target analytes is presented together with its optimization and capabilities for detection of single nucleotide polymorphisms (SNPs). In this single-step method, capture probes are freely accessible for hybridization, resulting in an improved assay performance compared to substrate-based assays. The analytes bring the nanoparticles close to each other via hybridization, causing a red shift of the nanoparticle plasmon peak detected by a spectrophotometer or CCD camera coupled to a darkfield imaging system. Optimal conditions for the assay were found to be (i) use of capture probes complementary to the target without any gap, (ii) maximum possible probe density on the gold nanoparticles, and (iii) 1M ionic strength buffer. The optimized assay has a 1fM limit of detection andfM to 10pM dynamic range, with detection of perfect match sequences being three orders of magnitude more sensitive than targets with single nucleotide mismatches.

Dark-field digital holographic microscopy by using vortex beam illumination

We propose a dark-field digital holographic microscopy (DHM) by using vortex beam illumination. In this paper, the annular illumination of vortex beam and the dark-field DHM imaging system are theoretically analyzed, and the quasi-nondiffracting property of the vortex beam is discussed. A corresponding DHM imaging system is established. The polystyrene spheres each with a size of 690 nm are utilized as objects in the experiment. By comparing the results of reconstructed images under bright-field illumination with those under dark-field illumination DHM, it is proved that the resolution of dark-field DHM under speckle-field illumination is improved and the contrast of its reconstructed image is enhanced accordingly.

Hyperspectral darkfield microscopy of PEGylated gold nanoparticles targeting CD44‐expressing cancer cells

We present a new hyperspectral darkfield imaging system with a scanned broadband supercontinuum light source. We observed the specific attachment of the functionalized gold plasmonic nanoparticles (AuNPs) targeting CD44(+) human breast cancer cells by conventional and by proposed hyperspectral darkfield microscopy. This wide-field and low phototoxic hyperspectral imaging system has been successful for performing spectral three-dimensional (3D) localization and spectroscopic identification of CD44-targeted PEGylated AuNPs in fixed cell preparations. Such spatial and spectral information is essential for the improvement of nanoplasmonic-based imaging, disease detection and treatment in complex biological environment. Presented system capability for 3D NP tracking will also enable investigation of specific sub-cellular activity with the use of NPs as spectral sensors.

Iterative reconstruction algorithm for analyzer-based phase-contrast computed tomography of hard and soft tissue

We propose a reconstruction algorithm for analyzer-based phase-contrast computed tomography (CT) applicable to biological samples including hard tissue that may generate conspicuous artifacts with the conventional reconstruction method. The algorithm is an iterative procedure that goes back and forth between a tomogram and its sinogram through the Radon transform and CT reconstruction, while imposing a priori information in individual regions. We demonstrate the efficacy of the algorithm using synthetic data generated by computer simulation reflecting actual experimental conditions and actual data acquired from a rat foot by a dark field imaging system.

Hyperspectral darkfield microscopy of single hollow gold nanoparticles for biomedical applications

Hyperspectral microscopy is a versatile method for simultaneous spatial and spectroscopic characterization of nonfluorescent samples. Here we present a hyperspectral darkfield imaging system for spectral imaging of single nanoparticles over an area of 150 × 150 μm(2) and at illumination intensities compatible with live cell imaging. The capabilities of the system are demonstrated using correlated transmission electron microscopy and single-particle optical studies of colloidal hollow gold nanoparticles. The potential of the system for characterizing the interactions between nanoparticles and cells has also been demonstrated. In this case, the spectral information proves a useful improvement to standard darkfield imaging as it enables differentiation between light scattered from nanoparticles and light scattered from other sources in the cellular environment. The combination of low illumination power and fast integration times makes the system highly suitable for nanoparticle tracking and spectroscopy in live-cell experiments.

1P168 バクテリアべん毛モーターの高時間分解能回転ステップ計測系の開発(11.分子モーター,ポスター,日本生物物理学会年会第51回(2013年度))

1P168 バクテリアべん毛モーターの高時間分解能回転ステップ計測系の開発(11.分子モーター,ポスター,日本生物物理学会年会第51回(2013年度))

Expanding the toolbox for nanoparticle trapping and spectroscopy with holographic optical tweezers

We have developed a workstation based on holographic tweezers to optically trap, move and characterize metal nanoparticles. Our advanced darkfield imaging system allows us to simultaneously image and take spectra of single trapped metal nanoparticles. We take advantage of the beamshaping abilities of the spatial light modulator and correct for aberrations of the trapping optics. We monitor the improvement of the optical trap with video-based nanoparticle tracking. Furthermore we theoretically assess the capabilities and limitations of video-based tracking for nanoparticle position detection, in particular with respect to acquisition frequencies below the corner frequency.

Simple Dark-Field Microscopy with Nanometer Spatial Precision and Microsecond Temporal Resolution

Molecular motors such as kinesin, myosin, and F1-ATPase are responsible for many important cellular processes. These motor proteins exhibit nanometer-scale, stepwise movements on micro- to millisecond timescales. So far, methods developed to measure these small and fast movements with high spatial and temporal resolution require relatively complicated experimental systems. Here, we describe a simple dark-field imaging system that employs objective-type evanescent illumination to selectively illuminate a thin layer on the coverslip and thus yield images with high signal/noise ratios. Only by substituting the dichroic mirror in conventional objective-type total internal reflection fluorescence microscope with a perforated mirror, were nanometer spatial precision and microsecond temporal resolution simultaneously achieved. This system was applied to the study of the rotary mechanism of F1-ATPase. The fluctuation of a gold nanoparticle attached to the γ-subunit during catalytic dwell and the stepping motion during torque generation were successfully visualized with 9.1-μs temporal resolution. Because of the simple optics, this system will be applicable to various biophysical studies requiring high spatial and temporal resolution in vitro and also in vivo.

Real-time three-dimensional optoacoustic imaging using an acoustic lens system

In medical optoacoustics (photoacoustics), absorbing structures, such as blood vessels, hidden inside scattering media are illuminated with short laser pulses resulting in the generation of thermoelastic pressure transients. This initial three-dimensional (3D) acoustic pressure distribution, which exactly resembles the absorption distribution, was imaged into a water container with a 4f acoustic lens system. An optical dark-field stereo imaging system using a 30ns flash illumination light was used to capture a snapshot of the pressure-induced refraction index changes in the water container at a predetermined time after the original laser pulse. The imaging system works at 20Hz frame rate and was designed toward a theoretical resolution of 50μm. The proposed method directly provides 3D images of absorbing structures without the need of computational reconstruction algorithms.

X-ray dark-field imaging and its application to medicine

A novel and simple X-ray dark-field imaging (DFI) system comprising Bragg asymmetric monochro-collimator and a Laue analyzer is under development. Very high transmissivity for refracted X-rays from the object is given by a 1.075 mm thick silicon single crystal blade with 4,4,0 diffraction at 35 keV ( λ=0.0354 nm). This condition completely suppresses the forward beam, preventing it from interacting with the object. Images of biomedicine samples, thus taken, show exceptionally high contrast under DFI with a spatial resolution of 5 μm or better.