It has been well documented that ideal SERS conditions occur in the nano sized gaps between metal nanoparticles 1,2 . Aggregation of the nanoparticles is a very simple and effective way to form these gaps3,4. We have investigated the 2 salts most commonly used throughout the literature as aggregating agents. Their absorption spectra were measured over time as the nanoparticles gradually aggregate and fall out of suspension, as well as their effects on SERS spectra. It was found that adding these salts to colloidal silver nanoparticles causes them to form large clusters which provide huge enhancement to the SERS spectra. This is due to the analyte molecules being situated in newly formed “hot spots” between the nanoparticles. This method is shown to be a cheap and simple way to achieve very large SERS enhancement.
Pterins are a group of biological compounds which have potential for use as a possible cancer diagnostic. This
paper considers reproducibility issues using Surface Enhanced Raman Scattering (SERS) for application in pterin
detection.
Raman spectroscopy is a very useful tool for analysing compounds, however its ability to detect low concentrations of a substance are very limited. Surface Enhanced Raman Spectroscopy (SERS) overcomes that issue and is reported to have achieved single molecule detection. Its main shortcoming is the reproducibility of SERS spectra. The variation in signal strength prevents SERS from being usable as a quantitative analytical technique. This variability have been investigated in this work and key factors in improving reproducibility have been considered.
Pterins, such as xanthopterin are studied in this paper. Pterins are a group of biological compounds that are found in
nature in colour pigmentation and in mammal’s metabolic pathways. Moreover, they have been identified in abnormal
concentrations in the urine of people suffering from certain kinds of cancer. The potential for pterin’s use as a cancer
diagnostic points to the importance of SERS detection for pterins.
Raman spectroscopy is a useful technique in the identification and characterisation of compounds, but in terms of
sensitivity its application is limited. With respect to this the discovery of the surface-enhanced Raman scattering (SERS)
phenomenon has proved monumental, and much research has been carried out over the past 30 years developing the
technique.
Pterins are biological compounds that are found in nature in colour pigmentation and in mammalian metabolic pathways.
Moreover, they have been identified in abnormal concentrations in cancer patients, suggesting potential applications in
cancer diagnostics.
SERS is an ideal technique to identify these compounds, and both nanoparticle suspensions and pulsed laser deposited
nanoparticle substrates have been used to examine the spectra of xanthopterin, both in aqueous solution and in different
pH environments.
Optical techniques toward the realization of sensitive and selective biosensing platforms have received considerable attention in recent times. Techniques based on interferometry, surface plasmon resonance, and waveguides have all proved popular, while spectroscopy in particular offers much potential. Raman spectroscopy is an information-rich technique in which the vibrational frequencies reveal much about the structure of a compound, but it is a weak process and offers poor sensitivity. In response to this problem, surface-enhanced Raman scattering (SERS) has received much attention, due to significant increases in sensitivity instigated by bringing the sample into contact with an enhancing substrate. Here we discuss a facile and rapid technique for the detection of pterins using SERS-active colloidal silver suspensions. Pterins are a family of biological compounds that are employed in nature in color pigmentation and as facilitators in metabolic pathways. In this work, small volumes of xanthopterin, isoxanthopterin, and 7,8-dihydrobiopterin have been examined while adsorbed to silver colloids. Limits of detection have been examined for both xanthopterin and isoxanthopterin using a 10-s exposure to a 12 mW 532 nm laser, which, while showing a trade-off between scan time and signal intensity, still provides the opportunity for the investigation of simultaneous detection of both pterins in solution.
Optical techniques towards the realisation of sensitive and selective biosensing platforms have received a
considerable amount of attention in recent times. Techniques based on interferometry, surface plasmon resonance,
field-effect transistors and waveguides have all proved popular, and in particular, spectroscopy offers a large range
of options. Raman spectroscopy has always been viewed as an information rich technique in which the vibrational
frequencies reveal a lot about the structure of a compound. The issue with Raman spectroscopy has traditionally
been that its rather low cross section leads to poor limits-of-detection. In response to this problem, Surface-enhanced
Raman Scattering (SERS), which increases sensitivity by bringing the sample in contact with many types of
enhanceing substrates, has been developed. Here we discuss a facile and rapid technique for the detection of pterins
using colloidal silver suspensions. Pteridine compounds are a family of biochemicals, heterocyclic in structure, and
employed in nature as components of colour pigmentation and also as facilitators for many metabolic pathways,
particularly those relating to the amino acid hydroxylases. In this work, xanthopterin, isoxanthopterin and 7,8-
dihydrobiopterin have been examined whilst absorbed to SERS-active silver colloids. SERS, while far more
sensitive than regular Raman spectroscopy, has its own issues relating to the reproducibility of substrates. In order to
obtain quantitative data for the pteridine compounds mentioned above, exploratory studies of methods for
introducing an internal standard for normalisation of the signals have been carried out.e
Microlenses have been implemented in confocal systems successfully as components of aperture arrays and as arrays of objective lenses. The use of the novel technology of variable focal length (VFL) microlenses in the confocal system has also shown potential. The properties of the VFL microlenses are controlled by the physical and chemical parameters of the microlenses. Arrays of microlenses with varying parameters are fabricated and their characteristics tuned to meet the demands of confocal microscopy.
In this paper, we experimentally studied both the bright-field and fluorescence images of microspheres by conventional and confocal scanning polarization microscopes. A qualitative analysis have been given to show a physical picture on the imaging of the microspheres. Emission spectra from melamine formaldehyde microspheres stained with Ethidium Bromide or covered by thin shell of CdTe nanocrystals have been experimentally studied. We adopted analytical expressions describing the resonance spacing in order to determine the size of the microspheres.
We present the investigation of integration of Variable Focal Length (VFL) microlenses into the confocal system. VFL microlenses acting as an array of objective lenses is examined with a novel method for scanning in the axial direction presented. By variation of the focal length of the lenses the focal plane can be scanned through the sample without the mechanical movement of the sample or the objective lens, we have named this 'focal scanning'. Some of the issues related to this experiment are noted and discussed, in particular with reference to the low Numerical Aperture (NA) of the VFL microlenses available. Proposed solutions to these issues deal with the design of higher NA microlenses.
Microlenses have been implemented in confocal systems successfully as components of aperture arrays and as arrays of objective lenses. The use of the novel technology of variable focal length (VFL) microlenses in the confocal system is investigated. The use of VFL microlenses as an aperture array in conjunction with an optical fiber as a pinhole array is examined. Axial responses of the system where measured and the Full-Width Half Maximum (FWHM) found to be ~16μm.
VFL microlenses as an array of objective lenses is investigated with a novel method for scanning in the axial direction presented. By variation of the focal length of the lenses the focal plane can be scanned through the sample without the mechanical movement of the sample or the objective lens, we have named this 'focal scanning'. It is shown that the limiting factor with this type of scanning is the low numerical aperture (NA) of the microlenses available. Both focal scanning and conventional scanning are examined for this experimental set-up.
Confocal microscopy has a unique optical sectioning property which allows three-dimensional images at different depths. Use of a microlens array is a potential alternative to the Nipkow disk for parallel imaging with high throughput in real-time confocal microscopy. The use of variable-focal-length microlenses can provide a way to axially scan the foci electronically avoiding the inflexible mechanical movement of the lens or the sample. Here we demonstrate a combination of a variable-focal-length microlens array and a fiber optic bundle as a way to create a high throughput aperture array that would be potentially applied as confocal imaging in vivo biological specimens. Variable focal length microlenses that we use consist of a liquid crystal film sandwiched between a pair of conductive substrates with patterned electrodes. The incident side of the microlens array was determined by examining the focus distribution in the axial direction. The variation of the focal length obtained by changing the voltage and corresponding focus intensity were measured through a conventional microscope. Meanwhile, the fiber bundle was characterized by coupling with either coherent or incoherent light source. We use the fiber bundle as both a multiple aperture and an image-carrying element and combine it with a microlens array to built up a confocal system. Axial responses are measured in two optical arrangements as a route to investigate endoscope potential.
We present a description of our work in recent years on imaging in
confocal microscopy in the context of biological applications. The
first system presented considers a Nipkow disc type arrangement where a detailed investigation of optimal aperture arrangements and spacings is performed. The effect of varying these parameters on the optical sectioning characteristics and on the light throughput is evaluated. Novel routes to achieving alternative multi-aperture configurations are presented. A programmable array microscope demonstrator is described using a ferroelectric liquid crystal SLM. A novel system is also proposed which uses variable focus microlenses in a confocal imaging system. We also discuss current trends in confocal microscopy in biology.
Images of the microspheres are studied in three-dimensions by using the confocal and conventional scanning polarization microscopes. It is found that the polarization of the detected signals is mainly parallel to the initial polarization which is due to the high extinction coefficient of the confocal system. Arc pairs are observed at the edge of the microspheres with the conventional polarization microscope with a crossed analyzer. Theoretical analysis are given by using the vector field theory and the image formations of the two systems.
The direct-view microscope is a confocal microscope which allows faster image acquisition rates than typical confocal scanning optical microscopes through the use of a pinhole array rather than the usual single pinhole. We present a theoretical investigation of the effects of source coherence on optical sectioning in direct-view microscopy. As a first step we present an equation which describes the optical sectioning strength of a coherent source brightfield DVM employing an infinite pinhole array. By simulation of both this and the finite array equation which show the existence of certain `principal' sidelobes which are likely to represent the most problematic artifact of coherent source imaging. By further analysis of the infinite array equation, we arrive at an expression which describes the defocus positions where the principal sidelobes occur. Finally, we move on to show how rectangular arrays are predicted, by the infinite array equation, to outperform square arrays and we show examples of this.
We present a programmable array microscope (PAM) which uses a pair of
ferroelectric liquid crystal spatial light modulators (FLC SLMs). The
system is similar to a confocal imaging system called a direct-view
microscope (DVM), in which scanned aperture arrays are used to obtain
real-time confocal images. The PAM, unlike the DVM, allows arrays to
be scanned electronically rather than mechanically. In our system,
one SLM is placed in the source plane of a conventional microscope
system; the other is placed in the detector plane. Confocal aperture arrays
are displayed and scanned synchronously on the SLMs and confocal
imaging results. The resolution is improved when compared to a
similar previously presented system which employed a single SLM. We
present axial resolution measurements for a variety of array
dimensions and investigate the use of aperture correlation techniques
to improve the light throughput in the devices.
We describe a Programmable Array Microscope (PAM) system which is implemented using a single Ferroelectric Liquid Crystal Spatial Light Modulator in a double pass configuration. The SLM array is placed such that it is in both the source and detector planes of a confocal microscope. The pixels of the SLM are arranged to form an aperture array similar to the type found in confocal direct- view microscopes (DVMs). Among the advantages of the PAM system over DVM systems are a lack of moving parts, and complete control over the aperture function. We present optical sectioning curves taken using scanned grids of square apertures of varying number and spacing, showing how these parameters affect the confocal behavior. In particular, we demonstrate the effect which the finite contrast ratio of the SLM pixels has on the optical sectioning curve and introduce a simple theory which explains this effect. Finally, we show confocal images captured from test samples using the PAM system.
Using a new device which contains an array of microlenses whose focal lengths can be electrically varied, we have been able to control the input from one microlens to a single mode fiber using an applied voltage. For such a microlens array many closely-spaced focal spots can be generated in parallel, and electrically switched to address, potentially, an array of receiver fibers. We show how the particular switching characteristics of the device, whereby the lenses switch from diverging to converging, serves in turn to disperse light and to focus it into the fiber.
Confocal fluorescence imaging is widely used, particularly for biological applications, and also notably in direct-view microscopes. Recent work has compared the use of coherent and incoherent illumination sources on the optical sectioning characteristics of fluorescence direct-view microscopes. However this detailed comparison has been done in theory. This paper addresses the experimental aspects of using coherent light sources in fluorescence imaging using a range of finite- sized, multiple-aperture arrays. The experimental difficulties of choosing a suitable uniform, flat, fluorescent plane with a high quantum efficiency are considered. Axial response curves obtained with a fluorescent laser dye sample are presented.
Confocal photoluminescence imaging is an important tool in the investigation of recombination in semiconductors and in the characterization of material growth. This characterization is particularly important for II-VI wide band-gap semiconductors where the potential for blue-green lasers is being explored currently. To achieve room-temperature cw operation of these lasers over the multi-thousand hours necessary for commercialization, extremely low defect densities are required. The confocal microscope is used in this work to image photoluminescence from II-VI materials to characterize the defect formation and propagation within the quantum well region of the material. This imaging approach permits the degradation to be monitored in real time and over a large area in samples with low defect densities. The additional advantages of this set-up over a conventional microscope are, of course, the higher lateral resolution and narrow depth of field associated with a confocal microscope. While considerable effort has been focused on the degradation in these II-VI semiconductors, we have recently observed that annealing can occur simultaneously in the same sample when the material is exposed to intense optical excitation. Images of annealing and degradation of a range of II-VI samples will be presented to highlight these observations.
Experimental results are given for the optical sectioning characteristics of finite-sized, multiple-aperture arrays in brightfield direct-view microscopy. We also present a theoretical model for the brightfield direct-view microscope (DVM). This allows us to determine the optical sectioning strength for finite-sized, multiple-pinhole arrays with an arbitrary distribution of apertures. The theoretical model is modified, for experimental purposes, to take account of the presence of objective lens pupil shading. A comparison between experimental and theoretical results for the axial response of the DVM to a plane mirror specimen is presented. In particular, the effects of pinhole size, pinhole spacing and array geometry are investigated in detail with a veil to (1) achieving good optical sectioning characteristics and (2) maximizing the amount of light available for imaging. The implications of our results for practical systems as regards pinhole-array design and fabrication are also discussed.
We develop a theory for brightfield direct-view microscopy using finite-sized, multiple pinhole arrays in the source and detector planes. We show that the array geometry can be decoupled mathematically from the remainder of the optical system. In particular, we present an expression for the response to a point object and to plane reflector. While the theory applies to an arbitrary distribution of pinholes we consider in detail the case of square, hexagonal and interleaving Archimedean spiral arrays. We also consider the implications of the array configuration on both the optical sectioning strength and the light budget.
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