Combining optical trapping in a microfluidic channel with simultaneous micro-Raman spectroscopy and motion detection.

Since their invention by Ashkin optical tweezers have demonstrated their ability and versatility as a non-invasive tool for micromanipulation. One of the most useful additions to the basic optical tweezers system is micro-Raman spectroscopy, which permits highly sensitive analysis of single cells or particles. We report on the development of a dual laser system combining two spatial light modulators to holographically manipulate multiple traps (at 1064nm) whilst undertaking Raman spectroscopy using a 532nm laser. We can thus simultaneously trap multiple particles and record their Raman spectra, without perturbing the trapping system. The dual beam system is built around micro-fluidic channels where crystallisation of calcium carbonate occurs on polymethylmethacrylate (PMMA) beads. The setup is designed to simulate at a microscopic level the reactions that occur on items in a dishwasher, where permanent filming of calcium carbonate on drinking glasses is a problem. Our system allows us to monitor crystal growth on trapped particles in which the Raman spectrum and changes in movement of the bead are recorded. Due to the expected low level of crystallisation on the bead surfaces this allows us to obtain results quickly and with high sensitivity. The long term goal is to study the development of filming on samples in-situ with the microfluidic system acting as a model dishwasher

Multi-wavelength, all-solid-state, continuous wave mode locked picosecond Raman laser

We demonstrate the operation of a cascaded continuous wave (CW) mode-locked Raman oscillator. The output pulses were compressed from 28 ps at 532 nm down to 6.5 ps at 559 nm (first Stokes) and 5.5 ps at 589 nm (second Stokes). The maximum output was 2.5 W at 559 nm and 1.4 W at 589 nm with slope efficiencies up to 52%. This technique allows simple and efficient generation of short-pulse radiation to the cascaded Stokes wavelengths, extending the mode-locked operation of Raman lasers to a wider range of visible wavelengths between 500 - 650 nm based on standard inexpensive picosecond Nd:YAG oscillators

Use of fiber optic technology to measure the effects of anesthesia on luciferase reaction kinetics

In vivo bioluminescent imaging (BLI) is a sensitive and reliable technique for studying gene expression, although experiments must be controlled tightly to obtain reproducible and quantitative measurements. The luciferase reaction depends on the availability of the reaction substrate, oxygen, and ATP, the distribution of which can vary markedly in different tissues. Here we used in vivo fiber optic technology, combined with stereotaxis-assisted surgery, to assess luciferase reaction kinetics in response to 2 anesthetic regimens, isoflurane and ketamine–xylazine. Transgenic rats that expressed luciferase under the control of the human prolactin promoter were used as a model organism. Anesthesia had a marked effect on luciferase reaction kinetics. The rise time to peak emission differed by 20 min between isoflurane and ketamine–xylazine. Optical imaging using a charge-coupled–device camera confirmed this delay. These results demonstrate that different anesthetics can have substantial effects on luciferase reaction kinetics and suggest that the timing of image acquisition after substrate injection should be optimized in regard to experimental conditions and the tissues of interest

Quantitative High Dynamic Range Beam Proling for Fluorescence Microscopy

Modern developmental biology relies on optically-sectioning uorescence microscope techniques to produce non-destructive in-vivo images of developing specimens at high resolution in three dimensions. As optimal performance of these techniques is reliant on the three-dimensional (3-D) intensity prole of the illumination employed, the ability to directly record and analyze these proles is of great use to the uorescence microscopist or instrument builder. Though excitation beam proles can be measured indirectly using a sample of uorescent beads and recording the emission along the microscope detection path, we demonstrate an alternative approach where a miniature camera sensor is used directly within the illumination beam. Measurements taken using our approach are solely concerned with the illumination optics as the detection optics are not involved. We present a miniature beam proling device and high dynamic range ux reconstruction algorithm that together are capable of accurately reproducing quantitative 3-D ux maps over a large focal volume. Performance of this beam proling system is veried within an optical test bench and demonstrated for uorescence microscopy by proling the low NA illumination beam of a single plane illumination microscope. The generality and success of this approach showcases a widely- exible beam amplitude diagnostic tool for use within the life sciences

Comparison of closed loop and sensorless adaptive optics in widefield optical microscopy

We report on a closed loop widefield adaptive optics, optical microscopy system in which the feedback signal is provided by backscattered light from the sample acting as a guide star. The improvement in imaging performance is compared to an adaptive optics system controlled via an image optimisation routine commonly described as sensorless adaptive optics. The samples viewed were imaged without fluorescence to ensure that photobleaching and other potential variations did not affect the comparisons in system performance though the method is equally applicable for fluorescence microscopy. The closed loop system is self-optimising for different areas of the sample, using a common reference wavefront, with the accuracy of the loop being limited by variation across the sub-aperture images induced by guide star elongation. Optimisation using an image sharpness metric gives slightly sharper images but takes significantly longer. We thus believe that both wavefront sensor based closed loop AO and metric based optimisation have a role to play in AO for microscopy and that the method of backscattered light as a guide star has a great potential in the application of AO, particularly to optical coherence tomography

Macular Ganglion Cell Inner Plexiform Layer Thickness in Glaucomatous Eyes with Localized Retinal Nerve Fiber Layer Defects

Purpose: To investigate macular ganglion cell–inner plexiform layer (mGCIPL) thickness in glaucomatous eyes with visible localized retinal nerve fiber layer (RNFL) defects on stereophotographs. Methods: 112 healthy and 149 glaucomatous eyes from the Diagnostic Innovations in Glaucoma Study (DIGS) and the African Descent and Glaucoma Evaluation Study (ADAGES) subjects had standard automated perimetry (SAP), optical coherence tomography (OCT) imaging of the macula and optic nerve head, and stereoscopic optic disc photography. Masked observers identified localized RNFL defects by grading of stereophotographs. Result: 47 eyes had visible localized RNFL defects on stereophotographs. Eyes with visible localized RNFL defects had significantly thinner mGCIPL thickness compared to healthy eyes (68.3 ± 11.4 μm versus 79.2 ± 6.6 μm respectively, P<0.001) and similar mGCIPL thickness to glaucomatous eyes without localized RNFL defects (68.6 ± 11.2 μm, P = 1.000). The average mGCIPL thickness in eyes with RNFL defects was 14% less than similarly aged healthy controls. For 29 eyes with a visible RNFL defect in just one hemiretina (superior or inferior) mGCIPL was thinnest in the same hemiretina in 26 eyes (90%). Eyes with inferior-temporal RNFL defects also had significantly thinner inferior-temporal mGCIPL (P<0.001) and inferior mGCIPL (P = 0.030) compared to glaucomatous eyes without a visible RNFL defect. Conclusion: The current study indicates that presence of a localized RNFL defect is likely to indicate significant macular damage, particularly in the region of the macular that topographically corresponds to the location of the RNFL defect

The three-peat challenge : business as usual, responsible agriculture, and conservation and restoration as management trajectories in global peatlands

FundingThis work was supported by the Natural Environment Research Council [grant numbers NE/X015238/1; NE/ V006444/1; NE/V018760/1], the Royal Geographical Society (RBEA 02.21), the Royal Society (RGS\R2\202229), and Growing Health (BB/X010953/1) BBSRC Institute Strategic Programme.Peer reviewedPublisher PD

Multi-photon attenuation-compensated light-sheet fluorescence microscopy

We thank the UK Engineering and Physical Sciences Research Council for funding (grants EP/P030017/1 and EP/R004854/1), the European Union’s Horizon 2020 Framework Programme (H2020) (675512, BE-OPTICAL), the Danish Council for Independent Research (DFF FTP grant 7017-00021), and the Otto Mønsted Foundation (grant 19-70-0109).Attenuation of optical fields owing to scattering and absorption limits the penetration depth for imaging. Whilst aberration correction may be used, this is difficult to implement over a large field-of-view in heterogeneous tissue. Attenuation-compensation allows tailoring of the maximum lobe of a propagation-invariant light field and promises an increase in depth penetration for imaging. Here we show this promising approach may be implemented in multi-photon (two-photon) light-sheet fluorescence microscopy and, furthermore, can be achieved in a facile manner utilizing a graded neutral density filter, circumventing the need for complex beam shaping apparatus. A “gold standard” system utilizing a spatial light modulator for beam shaping is used to benchmark our implementation. The approach will open up enhanced depth penetration in light-sheet imaging to a wide range of end users.Publisher PDFPeer reviewe