Bullseye Analysis: A Fluorescence Microscopy Technique to Detect Local Changes in Intracellular Reactive Oxygen Species (ROS) Production.

Reactive oxygen species (ROS) are naturally produced compounds that play important roles in cell signaling, gene regulation, and biological defense, including involvement in the oxidative burst that is central to the anti-microbial actions of macrophages. However, these highly reactive, short-lived radical species also stimulate cells to undergo programmed cell death at high concentrations, as well as causing detrimental effects such as oxidation of macromolecules at more moderate levels. Imaging ROS is highly challenging, with many researchers working on the challenge over the past 10-15 years without producing a definitive method. We report a new fluorescence microscopy-based technique, Bullseye Analysis. This methodology is based on concepts provided by the FRAP (Fluorescence Recovery after Photobleaching) technique and refined to evidence the spatiotemporal production of ROS, and the subsequent consequences, on a subcellular scale. To exemplify the technique, we have used the ROS-reporter dye, CellROX, and the ROS-inducing photosensitizer, LightOx58, a potent source of ROS compared with UV irradiation alone. Further validation of the technique was carried out using differing co-stains, notably Mitotracker and JC-1.

Integrated fiber optic spectrally resolved downwelling irradiance sensor for pushbroom spectrometers.

We present an integrated fiber optic spectrally resolved downwelling irradiance sensor for pushbroom hyperspectral imagers. The system comprises of a cosine corrector and custom fiber patch cables, collecting the ambient light in a large solid angle and feeding it directly to the entrance slit of the spectrometer. The system enables simultaneous measurement of downwelling and upwelling irradiance using the main hyperspectral camera sensor. As a demonstration, the spectral reflectance of a soil sample was measured with a RMSE of 8.4%, a significant improvement on the RMSE of 54% found without correction. At a weight of approximately 10 grams, this system provides a substantial weight saving over standalone incident light sensing instruments.

Freeform based hYperspectral imager for MOisture Sensing (FYMOS).

We present FYMOS, an all-aluminum, robust, light weight, freeform based, near infrared hYperspectral imager for MOisture Sensing. FYMOS was designed and built to remotely measure moisture content using spectral features from 0.7-1.7µm integrating an InGaAs sensor. The imaging system, operating at F/2.8, is based on the three-concentric-mirror (Offner) spectrograph configuration providing a spectral resolution of 8 nm optimized for broad spectral coverage with sufficient resolution to make assessments of water levels. To optimize the optical performance, whilst minimizing weight and size, the design incorporates a bespoke freeform blazed grating machined on a commercial 5 axis ultra precision diamond machine. We achieve a 30% improvement on the RMS wavefront error in the spatial and spectral fields compared to a conventional Offner-Chrisp design with similar aperture and the monolithic Primary/Tertiary mirror eases the manufacturing assembly whilst minimizing weight. We demonstrate the performance of FYMOS by measuring the evaporation rate of water on a soil sample and results are processed with a physical multilayer radiative transfer model (MARMIT) to estimate the mean water thickness.

Mitochondrial ATP production provides long-range control of endothelial inositol trisphosphate-evoked calcium signaling.

Endothelial cells are reported to be glycolytic and to minimally rely on mitochondria for ATP generation. Rather than providing energy, mitochondria in endothelial cells may act as signaling organelles that control cytosolic Ca2+ signaling or modify reactive oxygen species (ROS). To control Ca2+ signaling, these organelles are often observed close to influx and release sites and may be tethered near Ca2+ transporters. In this study, we used high-resolution, wide-field fluorescence imaging to investigate the regulation of Ca2+ signaling by mitochondria in large numbers of endothelial cells (∼50 per field) in intact arteries from rats. We observed that mitochondria were mostly spherical or short-rod structures and were distributed widely throughout the cytoplasm. The density of these organelles did not increase near contact sites with smooth muscle cells. However, local inositol trisphosphate (IP3)-mediated Ca2+ signaling predominated near these contact sites and required polarized mitochondria. Of note, mitochondrial control of Ca2+ signals occurred even when mitochondria were far from Ca2+ release sites. Indeed, the endothelial mitochondria were mobile and moved throughout the cytoplasm. Mitochondrial control of Ca2+ signaling was mediated by ATP production, which, when reduced by mitochondrial depolarization or ATP synthase inhibition, eliminated local IP3-mediated Ca2+ release events. ROS buffering did not significantly alter local Ca2+ release events. These results highlight the importance of mitochondrial ATP production in providing long-range control of endothelial signaling via IP3-evoked local Ca2+ release in intact endothelium.

Cellular localisation of structurally diverse diphenylacetylene fluorophores.

Fluorescent probes are increasingly used as reporter molecules in a wide variety of biophysical experiments, but when designing new compounds it can often be difficult to anticipate the effect that changing chemical structure can have on cellular localisation and fluorescence behaviour. To provide further chemical rationale for probe design, a series of donor-acceptor diphenylacetylene fluorophores with varying lipophilicities and structures were synthesised and analysed in human epidermal cells using a range of cellular imaging techniques. These experiments showed that, within this family, the greatest determinants of cellular localisation were overall lipophilicity and the presence of ionisable groups. Indeed, compounds with high log D values (>5) were found to localise in lipid droplets, but conversion of their ester acceptor groups to the corresponding carboxylic acids caused a pronounced shift to localisation in the endoplasmic reticulum. Mildly lipophilic compounds (log D = 2-3) with strongly basic amine groups were shown to be confined to lysosomes i.e. an acidic cellular compartment, but sequestering this positively charged motif as an amide resulted in a significant change to cytoplasmic and membrane localisation. Finally, specific organelles including the mitochondria could be targeted by incorporating groups such as a triphenylphosphonium moiety. Taken together, this account illustrates a range of guiding principles that can inform the design of other fluorescent molecules but, moreover, has demonstrated that many of these diphenylacetylenes have significant utility as probes in a range of cellular imaging studies.

Tandem fluorescence and Raman (fluoRaman) characterisation of a novel photosensitiser in colorectal cancer cell line SW480.

The development of new imaging tools, molecules and modalities is crucial to understanding biological processes and the localised cellular impact of bioactive compounds. A small molecule photosensitiser, DC473, has been designed to be both highly fluorescent and to exhibit a strong Raman signal in the cell-silent region of the Raman spectrum due to a diphenylacetylene structure. DC473 has been utilised to perform a range of novel tandem fluorescence and Raman (fluoRaman) imaging experiments, enabling a thorough examination of the compound's cellular localisation, exemplified in colorectal cancer cells (SW480). This multifunctional fluoRaman imaging modality revealed the presence of the compound in lipid droplets and only a weak signal in the cytosol, by both Raman and fluorescence imaging. In addition, Raman microscopy detected the compound in a cell compartment we labelled as the nucleolus, whereas fluorescence microscopy did not detect the fluoRaman probe due to solvatochromatic effects in a local polar environment. This last finding was only possible with the use of tandem confocal Raman and fluorescence methods. By following the approach detailed herein, incorporation of strong Raman functional groups into fluorophores can enable a plethora of fluoRaman experiments, shedding further light on potential drug compound's cellular behaviour and biological activity.

Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium.

Agonist-mediated signaling by the endothelium controls virtually all vascular functions. Because of the large diversity of agonists, each with varying concentrations, background noise often obscures individual cellular signals. How the endothelium distinguishes low-level fluctuations from noise and decodes and integrates physiologically relevant information remains unclear. Here, we recorded changes in intracellular Ca(2+) concentrations in response to acetylcholine in areas encompassing hundreds of endothelial cells from inside intact pressurized arteries. Individual cells responded to acetylcholine with a concentration-dependent increase in Ca(2+) signals spanning a single order of magnitude. Interestingly, however, intercellular response variation extended over 3 orders of magnitude of agonist concentration, thus crucially enhancing the collective bandwidth of endothelial responses to agonists. We also show the accuracy of this collective mode of detection is facilitated by spatially restricted clusters of comparably sensitive cells arising from heterogeneous receptor expression. Simultaneous stimulation of clusters triggered Ca(2+) signals that were transmitted to neighboring cells in a manner that scaled with agonist concentration. Thus, the endothelium detects agonists by acting as a distributed sensing system. Specialized clusters of detector cells, analogous to relay nodes in modern communication networks, integrate populationwide inputs, and enable robust noise filtering for efficient high-fidelity signaling.-Wilson, C., Saunter, C. D., Girkin, J. M., McCarron, J. G. Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium.

Age decreases mitochondrial motility and increases mitochondrial size in vascular smooth muscle.

KEY POINTS: Age is proposed to be associated with altered structure and function of mitochondria; however, in fully-differentiated cells, determining the structure of more than a few mitochondria at a time is challenging. In the present study, the structures of the entire mitochondrial complements of cells were resolved from a pixel-by-pixel covariance analysis of fluctuations in potentiometric fluorophore intensity during 'flickers' of mitochondrial membrane potential. Mitochondria are larger in vascular myocytes from aged rats compared to those in younger adult rats. A subpopulation of mitochondria in myocytes from aged, but not younger, animals is highly-elongated. Some mitochondria in myocytes from younger, but not aged, animals are highly-motile. Mitochondria that are motile are located more peripherally in the cell than non-motile mitochondria. ABSTRACT: Mitochondrial function, motility and architecture are each central to cell function. Age-associated mitochondrial dysfunction may contribute to vascular disease. However, mitochondrial changes in ageing remain ill-defined because of the challenges of imaging in native cells. We determined the structure of mitochondria in live native cells, demarcating boundaries of individual organelles by inducing stochastic 'flickers' of membrane potential, recorded as fluctuations in potentiometric fluorophore intensity (flicker-assisted localization microscopy; FaLM). In freshly-isolated myocytes from rat cerebral resistance arteries, FaLM showed a range of mitochondrial X-Y areas in both young adult (3 months; 0.05-6.58 µm(2) ) and aged rats (18 months; 0.05-13.4 µm(2) ). In cells from young animals, most mitochondria were small (mode area 0.051 µm(2) ) compared to aged animals (0.710 µm(2) ). Cells from older animals contained a subpopulation of highly-elongated mitochondria (5.3% were >2 µm long, 4.2% had a length:width ratio >3) that was rare in younger animals (0.15% of mitochondria >2 µm long, 0.4% had length:width ratio >3). The extent of mitochondrial motility also varied. 1/811 mitochondria observed moved slightly (∼0.5 µm) in myocytes from older animals, whereas, in the younger animals, directed and Brownian-like motility occurred regularly (215 of 1135 mitochondria moved within 10 min, up to distance of 12 µm). Mitochondria positioned closer to the cell periphery showed a greater tendency to move. In conclusion, cerebral vascular myocytes from young rats contained small, motile mitochondria. In aged rats, mitochondria were larger, immobile and could be highly-elongated. These age-associated alterations in mitochondrial behaviour may contribute to alterations in cell signalling, energy supply or the onset of proliferation.

Advancing Age Decreases Pressure-Sensitive Modulation of Calcium Signaling in the Endothelium of Intact and Pressurized Arteries.

Aging is the summation of many subtle changes which result in altered cardiovascular function. Impaired endothelial function underlies several of these changes and precipitates plaque development in larger arteries. The endothelium transduces chemical and mechanical signals into changes in the cytoplasmic calcium concentration to control vascular function. However, studying endothelial calcium signaling in larger arteries in a physiological configuration is challenging because of the requirement to focus through the artery wall. Here, pressure- and agonist-sensitive endothelial calcium signaling was studied in pressurized carotid arteries from young (3-month-old) and aged (18-month-old) rats by imaging from within the artery using gradient index fluorescence microendoscopy. Endothelial sensitivity to acetylcholine increased with age. The number of cells exhibiting oscillatory calcium signals and the frequency of oscillations were unchanged with age. However, the latency of calcium responses was significantly increased with age. Acetylcholine-evoked endothelial calcium signals were suppressed by increased intraluminal pressure. However, pressure-dependent inhibition of calcium signaling was substantially reduced with age. While each of these changes will increase endothelial calcium signaling with increasing age, decreases in endothelial pressure sensitivity may manifest as a loss of functionality and responsiveness in aging.

Pressure-dependent regulation of Ca2+ signalling in the vascular endothelium.

KEY POINTS: Increased pressure suppresses endothelial control of vascular tone but it remains uncertain (1) how pressure is sensed by the endothelium and (2) how the vascular response is inhibited. This study used a novel imaging method to study large numbers of endothelial cells in arteries that were in a physiological configuration and held at normal blood pressures. Increased pressure suppressed endothelial IP3 -mediated Ca(2+) signals. Pressure modulated endothelial cell shape. The changes in cell shape may alter endothelial Ca(2+) signals by modulating the diffusive environment for Ca(2+) near IP3 receptors. Endothelial pressure-dependent mechanosensing may occur without a requirement for a conventional molecular mechanoreceptor. ABSTRACT: The endothelium is an interconnected network upon which haemodynamic mechanical forces act to control vascular tone and remodelling in disease. Ca(2+) signalling is central to the endothelium's mechanotransduction and networked activity. However, challenges in imaging Ca(2+) in large numbers of endothelial cells under conditions that preserve the intact physical configuration of pressurized arteries have limited progress in understanding how pressure-dependent mechanical forces alter networked Ca(2+) signalling. We developed a miniature wide-field, gradient-index (GRIN) optical probe designed to fit inside an intact pressurized artery that permitted Ca(2+) signals to be imaged with subcellular resolution in a large number (∼200) of naturally connected endothelial cells at various pressures. Chemical (acetylcholine) activation triggered spatiotemporally complex, propagating inositol trisphosphate (IP3 )-mediated Ca(2+) waves that originated in clusters of cells and progressed from there across the endothelium. Mechanical stimulation of the artery, by increased intraluminal pressure, flattened the endothelial cells and suppressed IP3 -mediated Ca(2+) signals in all activated cells. By computationally modelling Ca(2+) release, endothelial shape changes were shown to alter the geometry of the Ca(2+) diffusive environment near IP3 receptor microdomains to limit IP3 -mediated Ca(2+) signals as pressure increased. Changes in cell shape produce a geometric microdomain regulation of IP3 -mediated Ca(2+) signalling to explain macroscopic pressure-dependent, endothelial mechanosensing without the need for a conventional mechanoreceptor. The suppression of IP3 -mediated Ca(2+) signalling may explain the decrease in endothelial activity as pressure increases. GRIN imaging provides a convenient method that gives access to hundreds of endothelial cells in intact arteries in physiological configuration.

From structure to function: mitochondrial morphology, motion and shaping in vascular smooth muscle.

The diversity of mitochondrial arrangements, which arise from the organelle being static or moving, or fusing and dividing in a dynamically reshaping network, is only beginning to be appreciated. While significant progress has been made in understanding the proteins that reorganise mitochondria, the physiological significance of the various arrangements is poorly understood. The lack of understanding may occur partly because mitochondrial morphology is studied most often in cultured cells. The simple anatomy of cultured cells presents an attractive model for visualizing mitochondrial behaviour but contrasts with the complexity of native cells in which elaborate mitochondrial movements and morphologies may not occur. Mitochondrial changes may take place in native cells (in response to stress and proliferation), but over a slow time-course and the cellular function contributed is unclear. To determine the role mitochondrial arrangements play in cell function, a crucial first step is characterisation of the interactions among mitochondrial components. Three aspects of mitochondrial behaviour are described in this review: (1) morphology, (2) motion and (3) rapid shape changes. The proposed physiological roles to which various mitochondrial arrangements contribute and difficulties in interpreting some of the physiological conclusions are also outlined.

Mitochondrial motility and vascular smooth muscle proliferation.

OBJECTIVE: Mitochondria are widely described as being highly dynamic and adaptable organelles, and their movement is thought to be vital for cell function. Yet, in various native cells, including those of heart and smooth muscle, mitochondria are stationary and rigidly structured. The significance of the differences in mitochondrial behavior to the physiological function of cells is unclear and was studied in single myocytes and intact resistance-sized cerebral arteries. We hypothesized that mitochondrial dynamics is controlled by the proliferative status of the cells. METHODS AND RESULTS: High-speed fluorescence imaging of mitochondria in live vascular smooth muscle cells shows that the organelle undergoes significant reorganization as cells become proliferative. In nonproliferative cells, mitochondria are individual (≈ 2 µm by 0.5 µm), stationary, randomly dispersed, fixed structures. However, on entering the proliferative state, mitochondria take on a more diverse architecture and become small spheres, short rod-shaped structures, long filamentous entities, and networks. When cells proliferate, mitochondria also continuously move and change shape. In the intact pressurized resistance artery, mitochondria are largely immobile structures, except in a small number of cells in which motility occurred. When proliferation of smooth muscle was encouraged in the intact resistance artery, in organ culture, the majority of mitochondria became motile and the majority of smooth muscle cells contained moving mitochondria. Significantly, restriction of mitochondrial motility using the fission blocker mitochondrial division inhibitor prevented vascular smooth muscle proliferation in both single cells and the intact resistance artery. CONCLUSIONS: These results show that mitochondria are adaptable and exist in intact tissue as both stationary and highly dynamic entities. This mitochondrial plasticity is an essential mechanism for the development of smooth muscle proliferation and therefore presents a novel therapeutic target against vascular disease.

Evaluation of cataract formation in fish exposed to environmental radiation at Chernobyl and Fukushima.

Recent studies apparently finding deleterious effects of radiation exposure on cataract formation in birds and voles living near Chernobyl represent a major challenge to current radiation protection regulations. This study conducted an integrated assessment of radiation exposure on cataractogenesis using the most advanced technologies available to assess the cataract status of lenses extracted from fish caught at both Chernobyl in Ukraine and Fukushima in Japan. It was hypothesised that these novel data would reveal positive correlations between radiation dose and early indicators of cataract formation. The structure, function and optical properties of lenses were analysed from atomic to millimetre length scales. We measured the short-range order of the lens crystallin proteins using Small Angle X-Ray Scattering (SAXS) at both the SPring-8 and DIAMOND synchrotrons, the profile of the graded refractive index generated by these proteins, the epithelial cell density and organisation and finally the focal length of each lens. The results showed no evidence of a difference between the focal length, the epithelial cell densities, the refractive indices, the interference functions and the short-range order of crystallin proteins (X-ray diffraction patterns) in lens from fish exposed to different radiation doses. It could be argued that animals in the natural environment which developed cataract would be more likely, for example, to suffer predation leading to survivor bias. But the cross-length scale study presented here, by evaluating small scale molecular and cellular changes in the lens (pre-cataract formation) significantly mitigates against this issue.

Subplasma membrane Ca2+ signals.

Ca(2+) may selectively activate various processes in part by the cell's ability to localize changes in the concentration of the ion to specific subcellular sites. Interestingly, these Ca(2+) signals begin most often at the plasma membrane space so that understanding subplasma membrane signals is central to an appreciation of local signaling. Several experimental procedures have been developed to study Ca(2+) signals near the plasma membrane, but probably the most prevalent involve the use of fluorescent Ca(2+) indicators and fall into two general approaches. In the first, the Ca(2+) indicators themselves are specifically targeted to the subplasma membrane space to measure Ca(2+) only there. Alternatively, the indicators are allowed to be dispersed throughout the cytoplasm, but the fluorescence emanating from the Ca(2+) signals at the subplasma membrane space is selectively measured using high resolution imaging procedures. Although the targeted indicators offer an immediate appeal because of selectivity and ease of use, their limited dynamic range and slow response to changes in Ca(2+) are a shortcoming. Use of targeted indicators is also largely restricted to cultured cells. High resolution imaging applied with rapidly responding small molecule Ca(2+) indicators can be used in all cells and offers significant improvements in dynamic range and speed of response of the indicator. The approach is technically difficult, however, and realistic calibration of signals is not possible. In this review, a brief overview of local subplasma membrane Ca(2+) signals and methods for their measurement is provided.

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.

Improved method for kinetic studies in microreactors using flow manipulation and noninvasive Raman spectrometry.

A novel method has been devised to derive kinetic information about reactions in microfluidic systems. Advantages have been demonstrated over conventional procedures for a Knoevenagel condensation reaction in terms of the time required to obtain the data (fivefold reduction) and the efficient use of reagents (tenfold reduction). The procedure is based on a step change from a low (e.g., 0.6 µL min(-1)) to a high (e.g., 14 µL min(-1)) flow rate and real-time noninvasive Raman measurements at the end of the flow line, which allows location-specific information to be obtained without the need to move the measurement probe along the microreactor channel. To validate the method, values of the effective reaction order n were obtained employing two different experimental methodologies. Using these values of n, rate constants k were calculated and compared. The values of k derived from the proposed method at 10 and 40 °C were 0.0356 ± 0.0008 mol(-0.3) dm(0.9) s(-1) (n = 1.3) and 0.24 ± 0.018 mol(-0.1) dm(0.3) s(-1) (n = 1.1), respectively, whereas the values obtained using a more laborious conventional methodology were 0.0335 ± 0.0032 mol(-0.4) dm(1.2) s(-1) (n = 1.4) at 10 °C and 0.244 ± 0.032 mol(-0.3) dm(0.9) s(-1) (n = 1.3) at 40 °C. The new approach is not limited to analysis by Raman spectrometry and can be used with different techniques that can be incorporated into the end of the flow path to provide rapid measurements.

Impact of wavefront distortion and scattering on 2-photon microscopy in mammalian brain tissue.

Two-photon (2P) microscopy is widely used in neuroscience, but the optical properties of brain tissue are poorly understood. We have investigated the effect of brain tissue on the 2P point spread function (PSF2p) by imaging fluorescent beads through living cortical slices. By combining this with measurements of the mean free path of the excitation light, adaptive optics and vector-based modeling that includes phase modulation and scattering, we show that tissue-induced wavefront distortions are the main determinant of enlargement and distortion of the PSF2p at intermediate imaging depths. Furthermore, they generate surrounding lobes that contain more than half of the 2P excitation. These effects reduce the resolution of fine structures and contrast and they, together with scattering, limit 2P excitation. Our results disentangle the contributions of scattering and wavefront distortion in shaping the cortical PSF2p, thereby providing a basis for improved 2P microscopy.

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.

Full spectrum fluorescence lifetime imaging with 0.5 nm spectral and 50 ps temporal resolution.

The use of optical techniques to interrogate wide ranging samples from semiconductors to biological tissue for rapid analysis and diagnostics has gained wide adoption over the past decades. The desire to collect ever more spatially, spectrally and temporally detailed optical signatures for sample characterization has specifically driven a sharp rise in new optical microscopy technologies. Here we present a high-speed optical scanning microscope capable of capturing time resolved images across 512 spectral and 32 time channels in a single acquisition with the potential for ~0.2 frames per second (256 × 256 image pixels). Each pixel in the resulting images contains a detailed data cube for the study of diverse time resolved light driven phenomena. This is enabled by integration of system control electronics and on-chip processing which overcomes the challenges presented by high data volume and low imaging speed, often bottlenecks in previous systems.

Non-invasive analysis in micro-reactors using Raman spectrometry with a specially designed probe.

An optical interface has been designed to maximise the sensitivity and spatial resolution required when Raman spectrometry is used to monitor a reaction in a micro-reactor, revealing advantages over a conventional commercial probe. A miniature aspheric lens was shown to be better than microscope objectives to focus the probing laser beam onto the sample. The diameters of the exciting and collection optical fibres were also shown to have a significant influence on sensitivity and the signal-to-background ratio, with 62.5 microm diameter 0.28 numerical aperture (NA) fibres found to be best for analysis of liquids in the 150 microm deep channel in the micro-reactor used. With a spectral measurement time of 2 s, it was shown that the probe could monitor the progress of an esterification reaction in real time and quickly optimise the reagent flow rates. The fast response time revealed features related to short-term pump instabilities and micro-reactor rheology effects that would not have been identified without rapid real-time measurements.