Liquid Biological Samples Research Articles

Nanoscale Dynamics in Ultrathin Liquids Visualized with TEM

Nanoscale imaging of frozen aqueous specimens and solid materials with transmission electron microscopes (TEM) has revolutionized our understanding in biological and material sciences. However, there is an ample number of important problems in life and physical sciences that occur only in liquid environments. Therefore, there is an incredible advantage of being able to image nanoscale processes directly in liquids [1-3]. I will describe our recent work on development of platform for imaging soft materials and biological samples in liquids using TEM [4-6]. We use this platform to study liquid properties at nanoscale. Here we show that the properties of fluid at nanoscale dominated by its interfacial interaction with the solid substrate surface and drastically differ from the expected bulk behavior. For example, the diffusive movement and rotation of nanocrystals within liquid nanodroplets are severely dampened when compared with macroscopic fluids. We will describe dynamic processes in nanoscale fluids such as condensation of nanodroplets and flow of nanodroplets. Imaging nanoscale fluids also enabled us to observe nanocrystal nucleation through nanocluster aggregation that differs from predictions of classical nucleation theory. We observe that crystals form amorphous aggregates.

Visualizing Biological Samples in Liquid Solution by Electron Microscopy

Liquid water is essential to life on earth. The cell, the basic component of all living organisms, functions through the action of proteins in a liquid environment. Consequently, the ability to directly visualize protein structure at nanometer-resolution in a liquid environment would dramatically advance mechanistic understanding of protein function in cellular activity. Current techniques in imaging biological samples in liquid solution are limited by micrometer scale resolution, Fluorescence-labeled, dehydrated, fixed, stained or frozen in ice.Here, we report an electron microscopic approach for imaging label-free, near-native biological samples in liquid environment. By passing electrons through a thin layer of liquid-buffer sealed within a micro-chamber, we directly visualized the structure of proteins, viruses, bacteria and human cells at nanometer resolution, even achieved their 3D structures. For example, the electron tomographic 3D reconstruction (Figure) of a label-free living bacteria, Magnetospirillum magneticum in growth medium showed a smooth outer shell and internal components, such as poly(3-hydroxybutyrate) (PHB), chromosomal mass (CR), magnetosomes (MG) and internal septal layer. These results suggest the method offers both a general-purpose and high-throughput tool for imaging the structure of biological samples in near-native physiological liquid conditions.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Electrical current profile of a confined isotropic liquid sample: Biological systems and liquid crystals applications

The electrical current profile in a liquid sample containing ionic impurities, submitted to a time-dependent external voltage, is determined in the framework of the Poisson–Nernst–Planck (PNP) diffusional model. The boundary conditions at the limiting electrodes include the situations of blocking electrodes, generalized Chang–Jaffé (specific adsorption), and the adsorption–desorption of positive and negative ions at the interface, governed by a kinetic equation in the Langmuir’s approximation. The current–voltage characteristics of the confined system are obtained in the presence of an arbitrary time-dependent applied potential of small amplitude (linear approximation). The calculations are potentially useful for modeling and interpreting experimental situations found in biological cells and liquid crystals samples.

Light element quantification by lithium elastic scattering

Accurate differential cross sections have been measured at specific beam energies and angles to be used in a method proposed previously for the simultaneous quantification of light elements (Z<11) present in evaporated liquid biological samples. Targets containing 1H, 7Li, 12C, 16O, 19F, 28Si and 197Au have been bombarded with 13MeV 6Li3+ and 20MeV 16O5+ beams. The 16O+1H, 16O+12C, 16O+16O, 16O+19F, 16O+28Si and 16O+197Au cross sections, shown to be consistent with the Rutherford formula predictions at 15° and 20°, have been used to determine cross sections for the 6Li+1H, 6Li+12C, 6Li+16O, 6Li+19F, 6Li+28Si and 6Li+197Au scatterings respectively at 17.5°, 24°, 25°, 26°, 28° and 30°. Although 6Li+7Li cross sections have not been obtained from 16O+7 Li cross sections, they have been determined from measured 6Li+19F cross sections and, in addition, used to obtain 16O+7Li cross sections at 15° and 20°. The reliability of the new cross sections determined in this investigation for the 6Li+1H, 6Li+7Li and 6Li+19F scatterings is based on the Rutherford behavior of the measured 6Li+197Au scattering data as expected and the consistency observed between the 6Li+12C, 6Li+16O and 6Li+28Si cross sections obtained in this work and previously reported values. This research has important implications in applied physics.

Temperature-modulated differential scanning calorimetry in a MEMS device

We present a MEMS-based approach to temperature-modulated differential scanning calorimetry (AC-DSC) for biomolecular characterization. Based on a MEMS device integrating microfluidic handling with highly sensitive thermoelectric detection as well as on-chip AC heating and temperature sensing, we perform, for the first time, MEMS-based AC-DSC detection of liquid biological samples, demonstrated by the measurements of protein unfolding. The specific heat capacity and the melting temperature of the protein during the unfolding process are obtained and found to be consistent with published data. This MEMS AC-DSC approach has potential applications to label-free characterization of biomolecules.

Atomic force microscopy: A nanoscopic view of microbial cell surfaces

The atomic force microscope (AFM) is a powerful instrument for microbiological investigation. It has evolved from an imaging tool used to investigate microbial surfaces at high resolution in their physiological environment into a lab-on-a-tip device, which allows more quantitative analysis of biological samples (from molecules to cells) in aqueous liquids. Atomic force microscopy provides information about the nanoscale architecture of microbes and about the localization and interactions of their individual constituents. Microbial interactions play essential roles in biology, medicine, ecology, biotechnology, food science and contribute to phenomena as varied as bacterial infections, biofilm formation, and bacterial adhesion to surfaces. In this review, we focus on recent developments offered by the rapid advances in AFM imaging and force spectroscopy with emphasizes on microbial research.

Scanning ion conductance microscopy for imaging biological samples in liquid: A comparative study with atomic force microscopy and scanning electron microscopy

The present study was designed to show the applicability of scanning ion conductance microscopy (SICM) for imaging different types of biological samples. For this purpose, we first applied SICM to image collagen fibrils and showed the usefulness of the approach-retract scanning (ARS)/hopping mode for such samples with steep slopes. Comparison of SICM images with those obtained by AFM revealed that the ARS/hopping SICM mode can probe the surface topography of collagen fibrils and chromosomes at nanoscale resolution under liquid conditions. In addition, we successfully imaged cultured HeLa cells, with 15μm in height by ARS/hopping SICM mode. Because SICM can obtain non-contact (or force-free) images, delicate cellular projections were visualized on the surface of the fixed cell. SICM imaging of live HeLa cells further demonstrated its applicability to study the morphological dynamics associated with biological processes on the time scale of minutes under liquid conditions. We further applied SICM for imaging the luminal surface of the trachea and succeeded in visualizing the surface of both ciliated and non-ciliated cells. These SICM images were comparable with those obtained by scanning electron microscopy. Although the dynamic mode of AFM provides better resolution than the ARS/hopping mode of SICM in some samples, only the latter can obtain contact-free images of samples with steep slopes, rendering it an important tool for observing live cells as well as unfixed or fixed soft samples with complicated shapes. Taken together, we demonstrate that SICM imaging, especially using an ARS/hopping mode, is a useful technique with unique capabilities for imaging the three-dimensional topography of a range of biological samples under physiologically relevant aqueous conditions.

Topography and near-field image measurement of soft biological samples in liquid by using a tuning fork based bent optical-fiber sensor

The fabrication of a tuning fork based bent optical-fiber sensor and its application for topography and near-field image measurement of soft biological samples in physiological solution are reported. By adopting the bent optical fiber and tuning fork feedback scheme, the possibility of signal interference with stray light is minimized, which is especially important for near-field applications. From the measured tuning fork amplitude and its calibration with the preamplifier output voltage, it was determined that the interaction force between the fiber tip and a soft sample in liquid needs to be controlled within approximately 10 nN level and that the image quality depends sensitively to the interaction force. The results of topography measurements of fixed COS-7 and MCF-7 cells in phosphate buffered saline and of the near-field imaging of red blood cell also in phosphate buffered saline with a resolution of about 100 nm are presented.

Determination of Cu, Zn, and Se in microvolumes of liquid biological samples

Cu, Zn, and Se were successfully determined in a few microliters (<100 μl) of biological samples using discrete injection atomic absorption spectrometry. Different factors were investigated in order to obtain a biological sample volume which is valid for analysis. Among them are the effect of microsampling volume variations (starting from 40 to 200 μl), nebulization efficiency, detection limits, precision, and finally the calibration and sensitivity of the proposed method. It was found that 60 μl of the biological sample was adequate for the quantitative analysis with reasonable precision. The advantages of the proposed method are not only rapidity, simplicity, sensitivity, and good precision, but also, contrary to conventional flame atomic absorption spectrometry, the capability of analyzing microvolumes of samples.

Compositional Contrast of Biological Materials in Liquids Using the Momentary Excitation of Higher Eigenmodes in Dynamic Atomic Force Microscopy

Atomic Force microscope (AFM) cantilevers commonly used for imaging soft biological samples in liquids experience a momentary excitation of the higher eigenmodes at each tap. This transient response is very sensitive to the local sample elasticity under gentle imaging conditions because the higher eigenmode time period is comparable to the tip-sample contact time. By mapping the momentary excitation response, we demonstrate a new scanning probe spectroscopy capable of resolving with high sensitivity the variations in the elasticity of soft biological materials in liquids.

High resolution laser-based detection of ammonia

In the present paper we compare the response of two types of photoacoustic cells (resonant and nonresonant) to determine the amount of ammonia volatilized from biological liquid samples at constant temperature, pressure and pH. The home made detector was a photoacoustic spectroscopy apparatus developed by Molecular Spectroscopy Laboratory staff at ENEA Frascati Research Centre in Italy. The sensor makes use of photo-acoustic cells equipped with commercially available high sensitivity miniaturized microphones. The radiation source was a line-tunable stabilized 10 W CW CO2 laser. Ammonia measurements were performed by tuning the laser source on the 9R30 laser line (9.2197 µm radiation wavelength). Ammonium chloride standard solutions were prepared by us in laboratory, to serve as reproducible ideal samples. The photoacoustic response of the two type of photoacoustic cells was determined and compared. The feasibility study was reported.

Video-rate High-speed Atomic Force Microscopy for Biological Sciences

The atomic force microscope (AFM) is unique in its capability to capture high-resolution images of biological samples in liquids. This capability becomes more valuable to biological sciences if AFM additionally acquires an ability of high-speed imaging. “Direct and real-time visualization” is a straightforward and powerful means of understanding biomolecular processes. With conventional AFM, it takes more than a minute to capture an image, while biomolecular processes generally occur on a millisecond timescale. In order to fill this large gap,various efforts have been carried out in the past decade. Here, we review these past efforts, describe the current state of the capability and limitations of our high-speed AFM, and discuss possibilities that may break the limitations, leading to an innovative high-speed bioAFM.

High‐speed atomic force microscopy for observing dynamic biomolecular processes

The atomic force microscope (AFM) is unique in its capability to capture high-resolution images of biological samples in liquids. This capability will become more valuable to biological sciences if AFM additionally acquires an ability of high-speed imaging, because 'direct and real-time visualization' is a straightforward and powerful means to understand biomolecular processes. With conventional AFMs, it takes more than a minute to capture an image, while biomolecular processes generally occur on a millisecond timescale or less. In order to fill this large gap, various efforts have been carried out in the past decade. Here, we review these past efforts, describe the current state of the capability and limitations of high-speed AFM, and discuss possibilities that may break the limitations and lead to the development of a truly useful high-speed AFM for biological sciences.

Method of imaging low density lipoproteins by atomic force microscopy

This short paper reports a simple method to image low density lipoproteins (LDL) using atomic force microscopy (AFM). This instrument allows imaging of biological samples in liquid and presents the advantage of needing no sample preparation such as staining or fixation that may affect their general structure. Dimensions (diameter and height) of individual LDL particles were successfully measured. AFM imaging revealed that LDL have a quasi-spherical structure on the x and y axis with an oblate spheroid structure in the z axis (i.e., height). LDLs were found to have an average diameter of 23 +/- 3 nm. The obtained mean height was 10 +/- 2 nm.

Active bimorph-based tapping-mode distance control for scanning near-field optical microscopy of biological samples in liquid

Commonly used shear force scanning near-field optical microscopy (SNOM) of soft biological samples is more critical to implement in aqueous environment than in air. A tapping-mode distance control based on a rectangular piezoelectric bimorph cantilever attached vertically by a straight fiber tip as force sensor for SNOM is introduced. The bimorph lever serving as both the probe dither and the force responder operates in flexural mode with a spring constant k=3.7×103N∕m. The sensitivity of the sensor is enhanced through the increase in lever’s quality factor (Q) and the usage of a higher eigenmode. Experimental results reveal that the describe sensor can operate in liquid with an effective Q up to 103 at its second eigenfrequecy f2=18.8kHz. High sensitivity of the sensor is demonstrated by imaging soft biological samples. Near-field optical resolution of better than 100nm on red blood cells in water is obtained. Compared to the existing tapping mode SNOM setups, our approach is compact, sensitive, lacking in parasitic optical background, and easy to practice in liquid.

Scanning near-field optical microscopy of live cells in liquid

A scanning near-field optical microscope (SNOM) is applied to fluorescence imaging of biological samples in liquid, including live cells. The SNOM is mounted on a Zeiss Axiovert 135 TV fluorescence microscope. For feedback we use tuning fork shear force method. The scanning tip is produced from a 125 μm optical fibre (8.3 μm core diameter) in a commercial Sutter P-2000 pipette puller and is coated with aluminium. Other commercial tips have also been used. Coarse z-axis adjustment is hydraulic, and fine positioning is accomplished with piezoelectric tube units. We have constructed the original liquid chamber, which allows long term stability of scanning and highest values of the Q factor (300 or more). The depth of liquid layer was less than 40 μm. Near-field images – the topography and distribution of membrane fluorescence of live human epithelial A431 cells, stably transfected with an EGFP fusion protein of the epidermal growth factor transmembrane receptor protein (EGFR, erbB1), were obtained in liquid.

Phase Separation in Supported Phospholipid Bilayers Visualized by Near-Field Scanning Optical Microscopy in Aqueous Solution

An experimental approach for near-field scanning optical microscopy (NSOM) of biological samples in an aqueous environment with a resolution of <100 nm has been developed. This was accomplished by using high-throughput bent optical fiber probes prepared by a two-step chemical etching method followed by focused ion beam milling to fabricate a reproducible aperture. The utility of the method for high-resolution fluorescence imaging of biological samples in liquid was demonstrated using phase separated, supported phospholipid bilayers as models for natural membranes. Bilayers with a phase separated dipalmitoyl−phosphatidylcholine/dilauroyl−phosphatidylcholine (DPPC/DLPC) mixture in one or both leaflets were imaged by both atomic force microscopy and NSOM. The addition of dihexadecanoyl-sn-glycero-3-phosphoethanolamine−Texas Red (DHPE−TR) was used to visualize fluid and gel phases for the NSOM fluorescence measurements. Hybrid bilayers with 7:3 DLPC/DPPC in the bottom leaflet and DPPC on top showed phase sepa...

A non-contact mode scanning force microscope optimised to image biological samples in liquid

Given that biological samples are particularly soft in their natural state and that a liquid is the most difficult operating environment for non-contact mode scanning force microscopy, the development of a scanning force microscope that is optimised to image biological samples in vitro presents significant challenges. The performance of the instrument described here has been optimised for such an application by incorporating magnetic excitation and active-Q control of the cantilever. The application of this instrument has been validated by imaging monolayers of immobilised antibodies in liquid and achieving image qualities comparable to those achieved in air for such samples.

Polymer Track Membranes for Extraction of Ions from Aqueous Solutions at Atmospheric Pressure

The possibility of the application of the electromembrane technique for production of ions of biological molecules at atmospheric pressure is demonstrated. This technique has previously only been used for extraction of ions from liquids directly into vacuum. The membrane technique for ion extraction at atmospheric pressure was tested with both time-of-flight and Fourier transform ion cyclotron resonance mass spectrometers. The mass spectra of intact molecular ions obtained from aqueous solutions of peptides and proteins are presented. The possible mechanisms of non-destructive ion extraction are discussed. The new technique is promising for achieving absolute sensitivity (charging every analyte molecule) and for performing spatially-resolved analysis of liquid biological samples.

Use of nitric acid in sample pretreatment for determination of trace elements in various biological samples by ETAAS

Trace elements in liquid biological samples may be determined by direct electrothermal atomic absorption spectrometry (ETAAS). In our previous work it was found that samples containing proteins or DNA may leak out of the graphite tube before the drying step, despite the addition of various modifiers. In order to keep the sample to the graphite tube, samples were diluted before analysis 1+1 with 32% v/v nitric acid, or 5 μl of 32% v/v nitric acid was added to the graphite tube before ETAAS determination. Applying the proposed procedure, the concentrations of lead in eluted fractions after gel chromatographic separation of human cerebellar nucleus dentatus supernatant and platinum in isolated DNA samples were determined. The use of nitric acid in sample pretreatment prevent sample leakage out of the graphite tube, provided for even drying and considerably reduced nonspecific absorption in lead determination. The repeatability of measurements was better than ±6%. The accuracy of the procedure was checked by spiking samples. The recoveries for both elements lay between 93–104%. Nitric acid was found to be a better modifier than TRITON X-100.