Juergen Popp

The application of a SERS fiber probe for the investigation of sensitive biological samples

The applicability of an etched and silver or gold coated SERS fiber probe in combination with a commercially available laboratory micro-Raman setup or a home built mobile micro-Raman setup to perform on-site field measurements was evaluated and successfully tested on different biological samples. The SERS fiber probe allows one to perform measurements with high spatial resolution. Simultaneously, the laser power used for Raman spectroscopy on biological samples as compared with conventional Raman experiments can be reduced by more than two orders of magnitude. This experimental arrangement was tested to investigate sensitive biological samples like mint plants (Bergamot mint, spear mint) and citrus fruits (kumquat). Furthermore, traces of fungicides on wine leaves were detected by means of such a SERS fiber probe setup.

Multimodal imaging to study the morphochemistry of basal cell carcinoma

Basal cell carcinoma is the most abundant malignant neoplasm in humans, the pathology of which is characterized by an abnormal proliferation of basal cells. Basal cell carcinoma can show a variety of different morphologies, which are based on different cellular biology. Furthermore, the carcinoma often grows invisibly to the eye imbedded in the surrounding skin. Therefore, in some cases its clinical detection is challenging. Thus, our work aims at establishing an unsupervised tissue classification method based on multimodal imaging and the application of chemometrics to discriminate basal cell carcinoma from non-diseased tissue. A case study applying multimodal imaging to ex-vivo sections of basal cell carcinoma is presented. In doing so, we apply a combination of various linear and non-linear imaging modalities, i.e. fluorescence, Raman and second-harmonic generation microscopy, to study the morphochemistry of basal cell carcinoma. The joint information content obtained by such multimodal approach in studying various aspects of the malignant tissue alterations associated with basal cell carcinoma is discussed.

Protochlorophyllide a: A Comprehensive Photophysical Picture

The photochemistry of protochlorophyllide a, a precursor in the biosynthesis of chlorophyll and substrate of the light regulated enzyme protochlorophyllide oxidoreductase, is investigated by pump-probe spectroscopy. Upon excitation into the lowest lying Q-band the light induced changes are recorded over a wide range of probe wavelengths in the visible and near-IR region between 500 and 1000 nm. Following excitation, an initial ultrafast 450 fs process is observed related to the motion out of the Franck-Condon region on the excited state surface; thus directly unraveling previous suggestions based on time-resolved fluorescence measurements (ChemPhysChem 2006, 7, 1727-1733). Furthermore, the data reveals a previously concealed photointermediate, whose formation on a nanosecond timescale matches the overall fluorescence decay and is assigned to a triplet state. The implications of this finding with respect to the photochemistry of NADPH:protochlorophyllide oxidoreductase (POR) are discussed.

A Raman spectroscopic study of the adsorption of fibronectin and fibrinogen on titanium dioxide nanoparticles

The adsorption of proteins that contain the amino acid sequence Arg-Gly-Asp (RGD) plays a crucial role for the biocompatibility of implant materials. Detailed knowledge of the adsorption process is of great interest because it is a dominant factor that decides on the integration or rejection of an implant by the organism. We have studied the adsorption process on titanium dioxide nanoparticles via two proteins, namely fibrinogen and fibronectin. Bulk protein spectra are compared to spectra of proteins that were adsorbed on TiO2 nanoparticles (as an enlarged model surface for TiO2 implants). In the Raman spectra of the adsorbed proteins a characteristic band occurs that can be assigned to an interaction between TiO2 nanoparticles and the carboxylate groups of the protein. A moderate shift of the amide I band towards higher wavenumbers observed in the adsorbed fibrinogen spectrum in comparison to the bulk protein spectrum is due to conformational changes during the adsorption process. In the spectra of adsorbed fibrinogen the peak area of the multiplet of CH3 and CH2 deformation modes in relation to the amide I Raman peak area is decreased as compared to the spectra of bulk fibrinogen. These spectral features indicate an increasing content of β-sheet and a decrease of α-helical structure content for fibrinogen while for fibronectin an increase of β-sheet structure and a decreasing content of random coil structure was found. The adsorption takes place via the protein side-chains.

LOC-SERS: A Promising Closed System for the Identification of Mycobacteria

A closed droplet based lab-on-a-chip (LOC) device has been developed for the differentiation of six species of mycobacteria, i.e., both Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM), using surface-enhanced Raman spectroscopy (SERS). The combination of a fast and simple bead-beating module for the disruption of the bacterial cell with the LOC-SERS device enables the application of an easy and reliable system for bacteria discrimination. Without extraction or further treatment of the sample, the obtained SERS spectra are dominated by the cell-wall component mycolic acid. For the differentiation, a robust data set was recorded using a droplet based LOC-SERS device. Thus, more than 2100 individual SERS spectra of the bacteria suspension were obtained in 1 h. The differentiation of bacteria using LOC-SERS provides helpful information for physicians to define the conditions for the treatment of individual patients.

Combining multiset resolution and segmentation for hyperspectral image analysis of biological tissues.

Hyperspectral images can provide useful biochemical information about tissue samples. Often, Fourier transform infrared (FTIR) images have been used to distinguish different tissue elements and changes caused by pathological causes. The spectral variation between tissue types and pathological states is very small and multivariate analysis methods are required to describe adequately these subtle changes. In this work, a strategy combining multivariate curve resolution-alternating least squares (MCR-ALS), a resolution (unmixing) method, which recovers distribution maps and pure spectra of image constituents, and K-means clustering, a segmentation method, which identifies groups of similar pixels in an image, is used to provide efficient information on tissue samples. First, multiset MCR-ALS analysis is performed on the set of images related to a particular pathology status to provide basic spectral signatures and distribution maps of the biological contributions needed to describe the tissues. Later on, multiset segmentation analysis is applied to the obtained MCR scores (concentration profiles), used as compressed initial information for segmentation purposes. The multiset idea is transferred to perform image segmentation of different tissue samples. Doing so, a difference can be made between clusters associated with relevant biological parts common to all images, linked to general trends of the type of samples analyzed, and sample-specific clusters, that reflect the natural biological sample-to-sample variability. The last step consists of performing separate multiset MCR-ALS analyses on the pixels of each of the relevant segmentation clusters for the pathology studied to obtain a finer description of the related tissue parts. The potential of the strategy combining multiset resolution on complete images, multiset segmentation and multiset local resolution analysis will be shown on a study focused on FTIR images of tissue sections recorded on inflamed and non-inflamed palatine tonsils.

Studies of silicon nanoparticles uptake and biodegradation in cancer cells by Raman spectroscopy

In-vitro Raman micro-spectroscopy was used for diagnostics of the processes of uptake and biodegradation of porous silicon nanoparticles (SiNPs) in breast cancer cells (MCF-7 cell line). Two types of nanoparticles, with and without photoluminescence in the visible spectral range, were investigated. The spatial distribution of photoluminescent SiNPs within the cells obtained by Raman imaging was verified by high-resolution structured-illumination optical microscopy. Nearly complete biodegradation of SiNPs inside the living cells was observed after 13days of the incubation. The results reveal new prospects of multi-modal visualization of SiNPs inside cancer cells for theranostic applications.

The invention of immersion ultramicroscopy in 1912-the birth of nanotechnology?

Dawn of nanotechnology: the immersion ultramicroscope was patented a century ago. When an analyte was examined with an antique instrument and with state-of-the-art technology, the historic assumptions were confirmed: the size and shape of the nanoparticles are in the same range as that described 100 years ago. The spectra of the Tyndall cones caused by the shape of the nanoparticles were also described correctly-long before electron microscopy was able to image single nanoparticles.

Differentiation of MCF-7 tumor cells from leukocytes and fibroblast cells using epithelial cell adhesion molecule targeted multicore surface-enhanced Raman spectroscopy labels

Abstract. Identification of tumor and normal cells is a promising application of Raman spectroscopy. The throughput of Raman-assisted cell sorting is limited by low sensitivity. Surface-enhanced Raman spectroscopy (SERS) is a well-recognized candidate to increase the intensity of Raman signals of cells. First, different strategies are summarized to detect tumor cells using targeted SERS probes. Then, a protocol is described to prepare multicore-SERS-labels (MSLs) by aggregating gold nanoparticles, coating with a reporter molecule and a thin silver shell to further boost enhancement, encapsulating with a stable silica layer, and functionalizing by epithelial cell adhesion molecule (EpCAM) antibodies. Raman, dark field and fluorescence microscopy proved the specific and nonspecific binding of functionalized and nonfunctionalized MSLs to MCF-7 tumor cells, leukocytes from blood, and nontransformed human foreskin fibroblasts. Raman imaging and dark field microscopy indicated no uptake of MSLs, yet binding to the cellular membrane. Viability tests were performed with living tumor cells to demonstrate the low toxicity of MSL-EpCAM. The SERS signatures were detected from cells with exposure times down to 25 ms at 785-nm laser excitation. The prospects of these MSLs in multiplex assays, for enumeration and sorting of circulating tumor cells in microfluidic chips, are discussed.

Proof of concept of fiber dispersed Raman spectroscopy using superconducting nanowire single-photon detectors.

Due to its high molecular specificity, Raman spectroscopy is a well-established analytical tool. Usually the inelastically scattered Raman light is spectrally dispersed by a spectrometer. Here, we present an alternative method, using an optical fiber as dispersive element. As the group velocity within the fiber is wavelength-dependent, different Raman bands arrive at different times at the detector. In combination with time-correlated single-photon counting, Raman spectra can be measured in the time domain. As detector we implemented a Superconducting Nanowire Single-Photon Detector (SNSPD), which possesses a timing accuracy of about 20 ps. Within this contribution we show first results of Raman spectra measured in the time domain using gradient index fibers of varying length.