C.N.B. Udalagama

High-Resolution 3D Imaging and Quantification of Gold Nanoparticles in a Whole Cell Using Scanning Transmission Ion Microscopy

Increasing interest in the use of nanoparticles (NPs) to elucidate the function of nanometer-sized assemblies of macromolecules and organelles within cells, and to develop biomedical applications such as drug delivery, labeling, diagnostic sensing, and heat treatment of cancer cells has prompted investigations into novel techniques that can image NPs within whole cells and tissue at high resolution. Using fast ions focused to nanodimensions, we show that gold NPs (AuNPs) inside whole cells can be imaged at high resolution, and the precise location of the particles and the number of particles can be quantified. High-resolution density information of the cell can be generated using scanning transmission ion microscopy, enhanced contrast for AuNPs can be achieved using forward scattering transmission ion microscopy, and depth information can be generated from elastically backscattered ions (Rutherford backscattering spectrometry). These techniques and associated instrumentation are at an early stage of technical development, but we believe there are no physical constraints that will prevent whole-cell three-dimensional imaging at <10 nm resolution.

Ionoluminescence and ion beam induced secondary electron imaging of cubic boron nitride

Abstract In this work, we introduce the use of ionoluminescence (IL) with ion beam induced secondary electron (IBISE) imaging to correlate the surface topography and crystal faces with luminescence properties. Since both IL and IBISE require low beam currents of

Fabrication of integrated channel waveguides in polydimethylsiloxane (PDMS) using proton beam writing (PBW): applications for fluorescence detection in microfluidic channels

Proton beam writing (PBW) is a lithographic technique that utilizes MeV protons in a direct write mode to fabricate micro/nano features in suitable resist material (E.g PMMA, SU-8, silicon, Foturan). These micro/nano structures may be used in an electroplating step to yield robust metallic stamps/molds for the replication of the original and lends itself to the fabrication of micro/nano fluidic channels that are important components in devices such as biophotonic chips. Another feature of proton bombardment is its ability to induce an increase in refractive index along the ions path, in particular at the end of its range where there is substantial nuclear scattering. This allows PBW to directly write buried waveguides that can be accurately aligned with fluidic channels. Polydimethylsiloxane (PDMS) is an optically clear, biocompatible polymer that can be readily used with a mold (such as that created with PBW) and easily sealed so as to produce biophotonic chips containing micro/nano fluidic channels. This has lead us to favour PDMS as the base material for our work on the development of these biophotonic chips. The present work is concerned with the production of integrating channel waveguides in PDMS chips, so as to have a working device that may be used to detect fluorescently tagged biological samples. For this we have adopted two approaches, namely(1) directly embedding optical fibres in the polymer and (2) using PBW to directly write buried waveguides in the polymer.

Integrating photonic and microfluidic structures on a device fabricated using proton beam writing

Proton beam writing is a lithographic technique that can be used to fabricate microstructures in a variety of materials including PMMA, SU-8 and FoturanTM. The technique utilizes a highly focused mega-electron volt beam of protons to direct write latent images into a material which are subsequently developed to form structures. Furthermore, the energetic protons can also be used to modify the refractive index of the material at a precise depth by using the end of range damage. In this paper we apply the proton beam writing technique to the fabrication of a lab-on-a-chip device that integrates buried waveguides with microfluidic channels. We have chosen to use FoturanTM photostructurable glass for the device because both direct patterning and refractive index modification is possible with MeV protons.

FAST ION BEAM MICROSCOPY OF WHOLE CELLS

The way in which biological cells function is of prime importance, and the determination of such knowledge is highly dependent on probes that can extract information from within the cell. Probing deep inside the cell at high resolutions however is not easy: optical microscopy is limited by fundamental diffraction limits, electron microscopy is not able to maintain spatial resolutions inside a whole cell without slicing the cell into thin sections, and many other new and novel high resolution techniques such as atomic force microscopy (AFM) and near field scanning optical microscopy (NSOM) are essentially surface probes. In this paper we show that microscopy using fast ions has the potential to extract information from inside whole cells in a unique way. This novel fast ion probe utilises the unique characteristic of MeV ion beams, which is the ability to pass through a whole cell while maintaining high spatial resolutions. This paper first addresses the fundamental difference between several types of charged particle probes, more specifically focused beams of electrons and fast ions, as they penetrate organic material. Simulations show that whereas electrons scatter as they penetrate the sample, ions travel in a straight path and therefore maintain spatial resolutions. Also described is a preliminary experiment in which a whole cell is scanned using a low energy (45 keV) helium ion microscope, and the results compared to images obtained using a focused beam of fast (1.2 MeV) helium ions. The results demonstrate the complementarity between imaging using low energy ions, which essentially produce a high resolution image of the cell surface, and high energy ions, which produce an image of the cell interior. The characteristics of the fast ion probe appear to be ideally suited for imaging gold nanoparticles in whole cells. Using scanning transmission ion microscopy (STIM) to image the cell interior, forward scattering transmission ion microscopy (FSTIM) to improve the contrast of the gold nanoparticles, and Rutherford Backscattering Spectrometry (RBS) to determine the depth of the gold nanoparticles in the cell, a 3D visualization of the nanoparticles within the cell can be constructed. Finally a new technique, proton induced fluorescence (PIF), is tested on a cell stained with DAPI, a cell-nucleic acid stain that exhibits a 20-fold increase in fluorescence when binding to DNA. The results indicate that the technique of PIF, although still at an early stage of development, has high potential since there does not seem to be any physical barrier to develop simultaneous structural and fluorescence imaging at sub 10 nm resolutions.

Imaging using pulses: a simple and fast (>100 kHz) solution

Imaging is an important component of spectroscopy. A good imaging system is expected to work with a high‐pixel resolution using signals of high count‐rates with as little dead time as possible to deliver an image quickly and reliably. It is not uncommon for such a system to be highly specialized, expensive and to consist of many dedicated electronic components. In this work, we present a simple imaging algorithm that can be used with a pulse (TTL) data signal, such as that produced by some photomultipliers and electron detectors. This algorithm works with only a simple general purpose data acquisition computer card (NI PXI/PCI‐6259) from National Instruments residing in a computer. The system has been tested with signal rates in excess of 100 kHz to produce images at a pixel resolution of 512 × 512. The systems ability to handle such high count‐rates hinges on utilizing the buffered data collection feature on the said card, in a hitherto unreported configuration. This system now offers a simple and cost‐effective manner of incorporating high count‐rate imaging features, such as in a scanning electron microscope, into a purely spectroscopic system. Further, since the use of the NI DAQ cards are supported under other computer platforms, the current imaging formalism is readily transferrable to computer platforms such as Linux or Mac OS.

A new technology for revealing the flow profile in integrated lab‐on‐a‐chip

PURPOSE Recently, greater attention has been paid to interactions between biomolecules particularly at the single-molecular level. Due to their novel properties, integrated lab-on-a-chip (LOC) devices and fluorescence correlation spectroscopy (FCS) are in high demand. METHODS The LOC was manufactured using the technique of proton beam writing. The biomolecule fluorescein was used to probe flow profiles in the micro∕nanochannels on the LOC by FCS. The FCS optical system was based on a confocal microscopy setup. At different locations on the LOC, the numbers and traveling time of the fluorescein fluidic solution were investigated. RESULTS From calibrations, ω(0) and τ(D) were 217 nm and 2.2 × 10(-5) s, respectively. Particle number and duration in passing through the detect volume, τ(F), were investigated. Results indicated that particle number was proportional to the size of micro∕nanochannels in the LOC. The particle number distribution and speed of the flow were mirror-symmetric in the two parallel (inlet∕outlet) microchannels. The distribution of the fluidic particles remained stable and the speed of the flow was nearly symmetrical when being transferred in the nanochannels. CONCLUSIONS The results were realistic and in line with the hydromechanics, indicating that in multidisciplinary areas measurements of flow profiles by FCS are possible inside the LOC channels. This study paves the way to investigate biomolecular interactions at the single-molecular level.

MeV Helium Ion Imaging of Gold Nanoparticles in Whole Cells

Observation of the interior structure of cells and sub-cellular organelles at high spatial resolutions is important for determining the functioning mechanisms of biological cells. Conventional optical microscopies have resolutions limited to around 300 nm due to the diffraction limits of light, and the new super-resolution techniques such as Stimulated Emission Depletion microscopy (STED) have stringent requirements that limit their application areas. Electron microscopy has an important role to play, but is only useful when imaging very thin sections due to electron/electron large angle scattering within the sample. Our results indicate that microscopy using MeV ions has high potential for the imaging of thick samples, e.g. whole cells, at high spatial resolutions. The reason for this is that MeV ions (e.g. protons or alpha particles (helium ions)) maintain a straight trajectory when traversing material, therefore preserving spatial resolution. The interaction of MeV ions with matter is mainly through ion/electron collisions. Due to the high mass mismatch with electrons, ions suffer very low energy transfer for each collision and as a result thousands of collisions can occur before they stop. In addition, there is very little primary ion scattering, and therefore the MeV proton and alpha particle paths are characterised by a straight, deep penetration into the material [1]. As well as ion electron scattering, there is also a much smaller probability of the MeV ion undergoing elastic scattering from an atomic nucleus. In these nuclear collisions the ion can be backscattered (Rutherford backscattering spectrometry RBS), and by measuring the energy of the backscattered ion, the target atom can be identified. RBS is particularly efficient at identifying and measuring heavy metals in low mass matrices such as organic materials. The Centre for Ion Beam Applications, National University of Singapore has recently built up a high resolution single cell imaging facility with the ability of focusing for MeV proton and alpha particle beams down to 30nm spot sizes [2]. The facility incorporates a variety of techniques, including Scanning Transmission Ion Microscopy (STIM) and Rutherford Backscattering Spectroscopy (RBS), with the microanalytical technique of Particle Induced X-ray Emission (PIXE) being added at a later stage. STIM is a technique which relies on the measurement of the energy loss of transmitted ions. MeV helium ions or protons are energetic enough to pass through single whole cells, and by measuring the energy loss of the transmitted ions, a structural image can be assembled which has high density contrast. Gold nanoparticles have great potential uses for medical diagnostics and drug delivery, as tracers and for other biological applications. Although Transmission Electron Microscopy (TEM) has been very successful in identifying nanoparticles in thin cellular sections, it remains difficult to resolve the gold nanoparticles at nanoscale resolution in the whole cell. In this work, we have used STIM and RBS to image normal human lung fibroblast cells (MRC-5) cultured in an environment of 50 nm gold nanoparticles. Cells were grown on 100 nm silicon nitride windows in cell culture medium (RPMI supplemented with fetal bovine serum) containing 50 nm gold nanoparticles (0.2 nM). Following exposure to the nanoparticles for 72 hours, the cells were critically point dried. The STIM measurements were carried out using a silicon surface barrier ion detector positioned behind the cell, and the RBS measurements were carried out using an annular surface barrier detector in front of the cell. The STIM image in Fig. 1 shows the structural view of the whole cell cultured in a gold nanoparticle environment, and Fig. 2 shows a similar control cell grown in normal conditions. The 662 doi:10.1017/S1431927611004181 Microsc. Microanal. 17 (Suppl 2), 2011