3D adaptive optics in a light sheet microscope.

We report on a single plane illumination microscope (SPIM) incorporating adaptive optics in the imaging arm. We show how aberrations can occur from the sample mounting tube and quantify the aberrations both experimentally and computationally. A wavefront sensorless approach was taken to imaging a green fluorescent protein (GFP) labelled transgenic zebrafish. We show improvements in image quality whilst recording a 3D "z-stack" and show how the aberrations come from varying depths in the fish.

Early stage dental caries detection using near infrared spatial frequency domain imaging.

Early stage dental caries can be remineralized without the need for "drill-and-fill" treatments that are more invasive and less permanent. However, early stage caries lesions typically present as a white spot on a white background, resulting in many lesions only being identified after they have developed beyond the point of remineralization as cavities. We present a spatial frequency domain imaging technique to characterize the optical properties of dental tissue. This technique enables different dental tissue types (healthy enamel, healthy dentin and damaged or demineralized enamel) to be easily distinguished from one another and allows quantification of the reduced scattering coefficients of dental tissue. The use of near-infrared light at 850 nm allows high depth penetration into the tissue and suppression of absorption effects, ensuring only changes in the reduced scattering coefficient that result directly from demineralization of enamel are observed and simplifying the analysis method. This technique provides a tool to both guide the attention of dentists to areas of interest and potential demineralization, and to provide longitudinal quantified assessments to monitor caries lesion behaviour over time.

Characterization of natural carious lesions by fluorescence spectroscopy at 405-nm excitation wavelength.

We aim to characterize natural caries enamel lesions by fluorescence spectroscopy. Sixty human samples with natural noncavitated caries lesions on smooth surfaces were selected and classified into three groups: dull, shiny, and brown lesions. All the samples were analyzed externally at the natural surface and after hemisectionig internally at the center of the lesion. The lesions were excited with a 405-nm InGaN diode laser and the fluorescence was collected with a single grating spectrometer. Four emission bands (455, 500, 582, and 622 nm) are identified in both sound and carious regions. The area under each emission band is correlated with the total area of the four bands for the sound and carious regions. The detected fluorescence from natural and cut surfaces through the caries lesions is not statistically different for the shiny and dull lesion, but is different [analysis of variance (ANOVA) (p<0.05)] for brown lesion at all emission bands. At the 405-nm excitation wavelength, the area of the fluorescence bands at 455 and 500 nm differ statistically for natural carious lesions and sound tissue.

Creating permanent 3D arrangements of isolated cells using holographic optical tweezers.

We report the creation of permanent 3D configurations of cells, at predefined positions, within a gelatin matrix. The technique used holographic optical tweezers to manipulate individual E. coli within a solution comprising monomer precursors. The matrix was then set and after the laser beam was removed, we were able to demonstrate that the structures remained intact for many days. We were also able to demonstrate that, in the presence of appropriate nutrients, the E. coli survived within the gelatin matrix for several days. The technique could have a number of potential future applications, including the arrangement of a variety of different cell types in complex architectures, as motifs for promoting tissue differentiation and growth within the field of cell engineering.

A preliminary investigation of a spectroscopic technique for the diagnosis of natural caries lesions.

OBJECTIVE: To report the use of spectroscopic analysis of dental fluorescence excited with a blue InGaN laser diode operating at 405 nm. METHOD: The spectra resulting from three classifications of smooth surface non-cavitated caries lesions (dull, shiny, brown) with 20 samples in each group were examined using the ratio of integrated fluorescence intensity in two spectral bands. RESULTS: All lesions demonstrated spectra which were significantly different from sound tooth structure. As expected, the 'brown' lesions demonstrated a significantly different spectral profile from the two white spot lesion classifications. Dull and shiny lesions had significantly different spectral measurements when examining the ratio of the integrated fluorescence in spectral bands between 480-500 and 620-640 nm. CONCLUSION: This method has application for detection of dental caries as well as demonstrating potential application to evaluate lesions which may represent different degrees of caries activity.

Using two photon microscopy to quantify enzymatic reaction rates on polymer beads.

Two-photon fluorescence microscopy was introduced as a tool to assess enzyme accessibility and to quantify enzyme reactions rates on solid supports.

Enamel erosion and prevention efficacy characterized by confocal laser scanning microscope.

The aim of this study was to evaluate the erosion-inhibiting effect of two toothpastes on the development of erosion-like lesions, by a confocal laser scanning microscope (CLSM). Forty human enamel blocks were divided into five groups (n = 8), in accordance to evaluate the GC MI Paste Plus and Oral B with stannous fluoride, applied as slurries and associated with toothbrush. Specimens were submitted to an erosion challenge from citric acid (0.5%, pH = 2.8), for 5 min, six times a day, alternating in artificial saliva immersions. Reference group was not exposed to treatment. Part of specimens (Groups 02 and 03) was exposed twice daily just to slurries, for 2 min, therefore specimens from Groups 04 and 05 were also abraded, for 30 s. The enamel surfaces were morphological characterized using CLSM images, with mineral loss being measured using the resulting 3D images referenced to an un-challenged portion of the sample. Step values were compared using the one-way ANOVA test. CLSM was shown to be a viable, noncontact, and simple technique to characterize eroded surfaces. The statistical difference in the step size was significant between the groups (P = 0.001) and using multiple comparisons a statistically significant protective effect of toothpastes was shown when these were applied as slurries. Although groups submitted to tooth brush showed mineral loss similar to reference control group, due to the damages of abrasion associated.

Single cell and subcellular measurements of intracellular Ca²âº concentration.

Increases in bulk average cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) are derived from the combined activities of many Ca(2+) channels. Near (<100 nm) the mouth of each of these channels the local [Ca(2+)](c) rises and falls more quickly and reaches much greater values than occurs in the bulk cytoplasm. Even during apparently uniform, steady-state [Ca(2+)] increases large local inhomogeneities exist near channels. These local increases modulate processes that are sensitive to rapid and large changes in [Ca(2+)] but they cannot easily be visualized with conventional imaging approaches. The [Ca(2+)] changes near channels can be examined using total internal reflection fluorescence microscopy (TIRF) to excite fluorophores that lie within 100 nm of the plasma membrane. TIRF is particularly powerful when combined with electrophysiology so that ion channel activity can be related simultaneously to the local subplasma membrane and bulk average [Ca(2+)](c). Together these techniques provide a better understanding of the local modulation and control of Ca(2+) signals.

A miniaturised integrated biophotonic point-of-care genotyping system.

This paper reports the development of a novel genotyping device specifically designed for point-of-care applications. As the results of the human genome project are applied to clinical practice there is an increasing requirement for simple to operate high-speed, potentially low-cost genotyping devices for use in the clinic. The aim of such devices is not to specifically detect a full gene sequence but to monitor the presence of specific Single Nucleotide Polymorphisms (SNPs). The instrument is designed to fulfil this specific clinical requirement. Using a FRET-based assay the instrument completes a full PCR process and then performs a melting point test to determine the exact SNPs present in the sample. Results are presented in which the instrument produces results within 18 min based upon saliva samples provided by the patient. The paper also reports successful results both with purified DNA samples and saliva-based samples which were taken from subjects after experiments deliberately aimed at confusing the instrument.

Two-photon fluorescence excitation microscopy to assess transscleral diffusional pathways in an isolated perfused bovine eye model.

PURPOSE: To assess the feasibility of using two-photon microscopy to study the pattern of diffusion through the sclera of a tracer (tazarotenic acid [TA]). METHODS: Polyvinyl alcohol films containing 1% tazarotenic acid (PVA-TA) were applied to the equatorial sclera of isolated perfused bovine eyes. Two-photon microscopy (TPM) was used to determine the lateral spread and depth of penetration of TA in the sclera over time. Protein and collagen binding were determined, and calibration standards were prepared by TPM imaging at 10 µm depth in scleral samples that had been immersed for 24 hours in solutions of TA of 0.7, 7.0, or 70 µg/mL. RESULTS: TA was weakly bound to collagen and sclera (<55%) but strongly bound to plasma protein (95%). In perfused eyes, 50 minutes after PVA-TA application, peak fluorescence in the sclera was detected at a 210-µm depth. By 85 minutes after application of the PVA-TA film, fluorescence had disappeared from surface layers of the sclera and was at maximum at 250 to 290 µm. Penetration of the tracer followed the track of scleral collagen bundles rather than that of the proteoglycan ground substance between collagen bundles. CONCLUSIONS: TPM can image in real time the progressive diffusion of TA from its source in a PVA-TA film applied to the equatorial sclera of the isolated perfused bovine eye and follow its subsequent penetration deeper into the sclera. The data suggest that lateral spread and deeper penetration of the test compound occurred along the course of scleral collagen bundles. Imaging was possible to a depth of 340 µm, the average thickness of the human equatorial sclera.