Andrew Downes

Raman Spectroscopy and Related Techniques in Biomedicine

In this review we describe label-free optical spectroscopy techniques which are able to non-invasively measure the (bio)chemistry in biological systems. Raman spectroscopy uses visible or near-infrared light to measure a spectrum of vibrational bonds in seconds. Coherent anti-Stokes Raman (CARS) microscopy and stimulated Raman loss (SRL) microscopy are orders of magnitude more efficient than Raman spectroscopy, and are able to acquire high quality chemically-specific images in seconds. We discuss the benefits and limitations of all techniques, with particular emphasis on applications in biomedicine—both in vivo (using fiber endoscopes) and in vitro (in optical microscopes).

Optical Spectroscopy for Noninvasive Monitoring of Stem Cell Differentiation

There is a requirement for a noninvasive technique to monitor stem cell differentiation. Several candidates based on optical spectroscopy are discussed in this review: Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, and coherent anti-Stokes Raman scattering (CARS) microscopy. These techniques are briefly described, and the ability of each to distinguish undifferentiated from differentiated cells is discussed. FTIR spectroscopy has demonstrated its ability to distinguish between stem cells and their derivatives. Raman spectroscopy shows a clear reduction in DNA and RNA concentrations during embryonic stem cell differentiation (agreeing with the well-known reduction in the nucleus to cytoplasm ratio) and also shows clear increases in mineral content during differentiation of mesenchymal stem cells. CARS microscopy can map these DNA, RNA, and mineral concentrations at high speed, and Mutliplex CARS spectroscopy/microscopy is highlighted as the technique with most promise for future applications.

Heating effects in tip-enhanced optical microscopy

Finite element simulations of laser-induced heating in scanning probe microscopy are presented. The electromagnetic field is first simulated for a variety of tip and substrate materials, and for air and aqueous environments. This electromagnetic field, in the end of the tip and substrate under the tip, produces Joule heating. Using this Joule heat source, steady state thermal simulations are performed. As a result of the large enhancement of optical power by the tip-substrate cavity, predicted temperature rises can be over 3 orders of magnitude higher than the values predicted without a tip present, but the optical signal can be enhanced by over 10 orders. Gold tips and substrates are predicted to give the highest optical signal for a given temperature increase.

CENP-A confers a reduction in height on octameric nucleosomes

Nucleosomes with histone H3 replaced by CENP-A direct kinetochore assembly. CENP-A nucleosomes from human and Drosophila have been reported to have reduced heights as compared to canonical octameric H3 nucleosomes, thus suggesting a unique tetrameric hemisomal composition. We demonstrate that octameric CENP-A nucleosomes assembled in vitro exhibit reduced heights, indicating that they are physically distinct from H3 nucleosomes and negating the need to invoke the presence of hemisomes.

Development of tip-enhanced optical spectroscopy for biological applications: a review

Tip-enhanced optical spectroscopy is an approach that holds a good deal of promise for the nanoscale characterisation of matter. Tip-enhanced Raman spectroscopy (TERS) has been demonstrated on a variety of samples: inorganic, organic and biological. Imaging using TERS has been shown for carbon nanotubes due to their high scattering efficiency. There are a number of compelling motivations to consider alternative approaches for biological samples; most importantly, the potential for heat damage of biomolecules and long acquisition times. These issues may be addressed through the development of tip-enhanced coherent anti-Stokes Raman scattering microscopy.

Label-free assessment of adipose-derived stem cell differentiation using coherent anti-Stokes Raman scattering and multiphoton microscopy

Abstract. Adult stem cells (SCs) hold great potential as likely candidates for disease therapy but also as sources of differentiated human cells in vitro models of disease. In both cases, the label-free assessment of SC differentiation state is highly desirable, either as a quality-control technology ensuring cells to be used clinically are of the desired lineage or to facilitate in vitro time-course studies of cell differentiation. We investigate the potential of nonlinear optical microscopy as a minimally invasive technology to monitor the differentiation of adipose-derived stem cells (ADSCs) into adipocytes and osteoblasts. The induction of ADSCs toward these two different cell lineages was monitored simultaneously using coherent anti-Stokes Raman scattering, two photon excitation fluorescence (TPEF), and second harmonic generation at different time points. Changes in the cell’s morphology, together with the appearance of biochemical markers of cell maturity were observed, such as lipid droplet accumulation for adipo-induced cells and the formation of extra-cellular matrix for osteo-induced cells. In addition, TPEF of flavoproteins was identified as a proxy for changes in cell metabolism that occurred throughout ADSC differentiation toward both osteoblasts and adipocytes. These results indicate that multimodal microscopy has significant potential as an enabling technology for the label-free investigation of SC differentiation.

Optical probing of sample heating in scanning near-field experiments with apertured probes

We have used the inherent thermochromism of conjugated polymers to investigate substrate heating effects in scanning near-field experiments with metal-coated “apertured” probes. Chemically etched and pulled fibers were used to provide near-field excitation of fully converted films of poly(p-phenylene vinylene), PPV, and of poly(4,4′-diphenylene diphenylvinylene). We detect no significant blueshift of the photoluminescence spectra generated with near-field excitation, in comparison to those collected with far-field excitation. We conclude that polymer heating in the region contributing to the luminescence is less than 40K. We also demonstrate that thermolithography of the PPV precursor is not significant by comparing UV (325nm) and red (670nm) illumination.

Photon emission from Ag and Au clusters in the scanning tunneling microscope

In principle, chemical information is obtainable from metal surfaces by means of photon emission from the scanning tunneling microscope (STM). However, the photon emission varies significantly with topography and choice of tip. We address the important issue of geometry by studying the emission characteristics of Ag and Au spheres. First, photon maps of Ag clusters, consisting in some cases of just a few atoms, demonstrate that they can be uniquely identified from other nonmetallic particles. Then, the bias at which there is an onset of photon emission for 1 nm Ag and Au clusters is measured and found to be ≈3.3 and ≈2.1 V, respectively. This allows for the demonstration of the ability of the STM to distinguish different metal particles by their photon emission. The value of the onset bias for each metal can be made almost invariant to sample topography by an appropriate choice of tip; only then is the photon emission related purely to the optical properties of the surface. We envisage a form of chemical ...

Simulations of tip-enhanced optical microscopy reveal atomic resolution

We have performed finite element electromagnetic analysis of ‘apertureless’ scanning near‐field optical microscope tips. A variety of radii was considered, from 40 nm down to 1 nm, and the enhancement of optical scattering from the region beneath the probe apex was modelled in air and water. We observed sizeable spectral shifts when the radius decreases, together with a surprising increase in the amount of scattering from small tips. The lateral resolution is considered, which is shown to be always smaller than the tip radius, and for 1‐nm‐radius tips can allow atomic resolution imaging with sufficient optical enhancement. Gold, silver, copper and aluminium were modelled as tip materials.

In vivo imaging of the tumor and its associated microenvironment using combined CARS / 2-photon microscopy

The use of confocal and multi-photon microscopy for intra-vital cancer imaging has impacted on our understanding of cancer cell behavior and interaction with the surrounding tumor microenvironment in vivo. However, many studies to-date rely on the use fluorescent dyes or genetically encoded probes that enable visualization of a structure or cell population of interest, but do not illuminate the complexity of the surrounding tumor microenvironment. Here, we show that multi-modal microscopy combining 2-photon fluorescence with CARS can begin to address this deficit, enabling detailed imaging of the tumor niche without the need for additional labeling. This can be performed on live tumor-bearing animals through optical observation windows, permitting real-time and longitudinal imaging of dynamic processes within the tumor niche.