Aufrid T.M. Lenferink

Nonresonant Confocal Raman Imaging of DNA and Protein Distribution in Apoptotic Cells

Nonresonant confocal Raman imaging has been used to map the DNA and the protein distributions in individual single human cells. The images are obtained on an improved homebuilt confocal Raman microscope. After statistical analysis, using singular value decomposition, the Raman images are reconstructed from the spectra covering the fingerprint region. The data are obtained at a step interval of approximately 250 nm and cover a field from 8- to 15- micro m square in size. Dwell times at each pixel are between 0.5 and 2 s, depending on the nature and the state of the cell under investigation. High quality nonresonant Raman images can only be obtained under these conditions using continuous wave high laser powers between 60 and 120 mW. We will present evidence that these laser powers can still safely be used to recover the chemical distributions in fixed cells. The developed Raman imaging method is used to image directly, i.e., without prior labeling, the nucleotide condensation and the protein distribution in the so-called nuclear fragments of apoptotic HeLa cells. In the control (nonapoptotic) HeLa cells, we show, for the first time by Raman microspectroscopy, the presence of the RNA in a cell nucleus.

Combined AFM and confocal fluorescence microscope for applications in bio‐nanotechnology

We present a custom‐designed atomic force fluorescence microscope (AFFM), which can perform simultaneous optical and topographic measurements with single molecule sensitivity throughout the whole visible to near‐infrared spectral region. Integration of atomic force microscopy (AFM) and confocal fluorescence microscopy combines the high‐resolution topographical imaging of AFM with the reliable (bio)‐chemical identification capability of optical methods. The AFFM is equipped with a spectrograph enabling combined topographic and fluorescence spectral imaging, which significantly enhances discrimination of spectroscopically distinct objects. The modular design allows easy switching between different modes of operation such as tip‐scanning, sample‐scanning or mechanical manipulation, all of which are combined with synchronous optical detection. We demonstrate that coupling the AFM with the fluorescence microscope does not compromise its ability to image with a high spatial resolution. Examples of several modes of operation of the AFFM are shown using two‐dimensional crystals and membranes containing light‐harvesting complexes from the photosynthetic bacterium Rhodobacter sphaeroides.

Feasibility of evanescent wave interferometer immunosensors for pesticide detection: chemical aspects

A waveguide Mach-Zehnder Interferometer (MZI) immunosensor has been developed which can detect, in a direct way, a minimum average layer growth thickness of the analyte of 2×10−3 nm (bound mass, I′=1×10−4 δ cm−2). The design and fabrication of the sensor and the experimental set-up are aimed at decreasing the detection limit. A flow cell has been designed and incorporated into the set-up to allow a high mass-transport rate of the analyte molecules to the sensor surface, in this way decreasing the net response time. Results for the immunosystem α-hSA/hSA are presented. All improvements have been done as a first step towards the use of the MZI immunosensor in the direct detection of low coverages of pesticides with an expected layer growth of 2×10−4 nm.

An improved optical method for surface plasmon resonance experiments

In this paper an inexpensive optical device is described, which is capable of measuring the optical reflectance at different angles, while keeping the laser spot stationary at one point of the surface. This is accomplished by applying cylindrical optics. Its use is demonstrated in a surface plasmon resonance (SPR) sensor. A coil-operated vibrating mirror is used to obtain an angle scan of about 4 degrees. The angle of minimum reflectance can be detected with an accuracy of approximately 2 × 10−3 degrees. Despite the use of simple optical components, disturbance of laser beam parallelism is no more than 0.02 degrees. Displacement of the laser spot at the surface during the angle scan is kept within 0.2 mm. The device eliminates disturbances due to surface irregularities in measurements.

Absence of amyloid-beta in lenses of Alzheimer patients: A confocal Raman microspectroscopic study

We have compared the protein profiles in plaques and tangles in the hippocampus of post-mortem Alzheimer brains and in opaque and clear regions in the deep cortex of eye lenses of the same donors. From the 7 Alzheimer donors studied, 1 had pronounced bilateral cortical lens opacities, 1 moderate and 5 only minor or no cortical opacities. We focused on beta-sheet levels, a hallmarking property of amyloid-beta, the major protein of plaques and tau protein, the major protein of tangles in Alzheimer brains. Confocal Raman microspectroscopy and imaging was used in combination with hierarchical cluster analysis. Plaques and tangles show high levels of beta-sheets with a beta-sheet to protein ratio of 1.67. This ratio is 1.12 in unaffected brain tissue surrounding the plaques and tangles. In the lenses this ratio is 1.17 independently of the presence or absence of opacities. This major difference in beta-sheet conformation between hippocampus and lens is supported by Congo red and immunostaining of amyloid-beta and tau which were positive for plaques and tangles in the hippocampus but fully negative for the lens irrespective of the presence or absence of opacities. In line with a previous study (Michael etxa0al., 2013) we conclude that cortical lens opacities are not typical for Alzheimer patients and are not hallmarked by accumulation of amyloid-beta, and can thus not be considered as predictors or indicators of Alzheimer disease as claimed by Goldstein etxa0al. (2003).

Raman and Fluorescence Spectral Imaging of Live Breast Cancer Cells Incubated with PEGylated Gold Nanorods

The optical properties of PEGylated gold nanorods (PEG-GNR) in interaction with cells have been investigated with Raman and fluorescence microspectroscopic imaging. The emission spectra were compared with those from dispersions of GNR, which can be characterized by a broad emission bandwidth of approximately 60 nm with a band maximum around 675 nm. These properties are in good agreement with observations from various other gold substrates and (nano)particles. The emission spectra from cells incubated with PEG-GNR were dominated by Raman scattering from locations where no GNR were present. Intense fluorescence spectral lines, with peak amplitude comparable with the Raman scattering from cells, were observed from locations containing GNR. The frequency range of the fluorescence emission spectra coincided mainly with the Raman fingerprint region from 500 cm−1 to 1800 cm−1, excited by the laser emission line at 647.1 nm. No surface-enhanced Raman spectra were observed. It was furthermore observed from cluster analysis of the Raman and fluorescence hyperspectral datasets that the GNR-related integrated fluorescence emission band from an individual cell could be sub-divided in multiple bands with slightly varying band maxima. Raman difference spectra of cells with GNR minus control cells showed that the amplitude of lipid signal in cells incubated with PEG-GNR was increased. An excellent correlation was found between the increased lipid signals and locations of the nanorods. This positive correlation between Raman signals from lipids and fluorescence signals from gold nanorods supports that gold nanorods are locally accumulating in lipid vesicles within the cells.

Improved detection method for evanescent wave interferometric chemical sensing

A phase modulation system for a waveguide Mach–Zehnder interferometer sensor system has been developed which is capable of direct detection of optical phase changes smaller than 2.2π milliradians. When implemented in a chemical sensor interferometer system this corresponds to a sensitivity for refractive index changes of the analyte of better than 10−6, provided temperature variations are kept below 0.1u2009°C during the measuring period. The sensitivity of the instrument is demonstrated by a stepwise addition of low concentrations of glucose in water. The possibility to reset the instrument by a whole number of fringes makes it feasible to combine the high sensitivity with a virtually unlimited range. The accuracy of the detection system is independent of interferometer visibility changes during the measurement. This makes it possible to perform accurate measurements in solutions with varying degrees of opaqueness, such as milk, blood, or wastewater. These features are demonstrated with adsorption experiment...

A microfluidic chip for high resolution Raman imaging of biological cells

A microfluidic chip was designed, prepared and tested for integration with a confocal Raman imaging spectrometer with the specific purpose of enabling studies of individual biological cells. The design of the chip effectively overcomes the limitations arising from the high numerical aperture (NA) and short working distance objectives, which are necessary for high resolution imaging. The high confocal spatial resolution was achieved by a careful design of the geometry of the chip together with a thin, optically and Raman silent sealing window as the embedding medium for the channels. A leak-free microfluidic chip was obtained by surface plasma modification of polydimethylsiloxane (PDMS) and optimization of the liquid loading parameters. Raman images of biological cells, which were transported by flow into the microfluidic chip, are presented as an example of a Raman-microfluidics application. The broad band Raman spectra from −50 cm−1 to 3650 cm−1 were recorded in 1600 frequency intervals without any signal enhancement or sample labeling. Raman images were recorded in ∼400 seconds and they typically consisted of 64 × 64 pixels with a step size of 250 nm, thus containing ∼4 × 106 data points altogether.

Quantitative fluorescence correlation spectroscopy reveals a 1000-fold increase in lifetime of protein functionality

We have investigated dilute protein solutions with fluorescence correlation spectroscopy (FCS) and have observed that a rapid loss of proteins occurs from solution. It is commonly assumed that such a loss is the result of protein adsorption to interfaces. A protocol was developed in which this mode of protein loss can be prevented. However, FCS on fluorescent protein (enhanced green fluorescent protein, mCherry, and mStrawberry) solutions enclosed by adsorption-protected interfaces still reveals a decrease of the fluorescent protein concentration, while the diffusion time is stable over long periods of time. We interpret this decay as a loss of protein functionality, probably caused by denaturation of the fluorescent proteins. We show that the typical lifetime of protein functionality in highly dilute, approximately single molecule per femtoliter solutions can be extended more than 1000-fold (typically from a few hours to >40 days) by adding compounds with surfactant behavior. No direct interactions between the surfactant and the fluorescent proteins were observed from the diffusion time measured by FCS. A critical surfactant concentration of more than 23 muM was required to achieve the desired protein stabilization for Triton X-100. The surfactant does not interfere with DNA-protein binding, because similar observations were made using DNA-cutting restriction enzymes. We associate the occurrence of denaturation of proteins with the activity of water at the water-protein interface, which was recently proposed in terms of the water attack model. Our observations suggest that soluble biomolecules can extend an influence over much larger distances than suggested by their actual volume.

Growth Rate and Morphology of a Single Calcium Carbonate Crystal on Polysulfone Film Measured with Time Lapse Raman Micro Spectroscopy

The growth of single, self- nucleated calcium carbonate crystals on a polysulfone (PSU) film was investigated with high resolution, time lapse Raman imaging. The Raman images were acquired on the interface of the polymer with the crystal. The growth of crystals could thus be followed in time. PSU is a polymer that is used as a membrane material in water cleaning technology. The intensity of the Raman band at the position of 1086 cm-1, which is due to the symmetric stretching of the C-O bonds in the carbonate group of calcite was used to translate the number of CO3 2- ions in a crystal to the growth in time. The growth rate of single crystals of calcium carbonate on a surface was obtained from successive Raman images. We are presenting for the first time time-lapse Raman images of single crystal growth as a direct method to determine a crystal growth rate on an industrially relevant membrane material, like polysulfone.