Light sheet based imaging flow cytometry on a microfluidic platform

We propose a light sheet based imaging flow cytometry technique for simultaneous counting and imaging of cells on a microfluidic platform. Light sheet covers the entire microfluidic channel and thus omits the necessity of flow focusing and point scanning based technology. Another advantage lies in the orthogonal detection geometry that totally cuts‐off the incident light, thereby substantially reducing the background in the detection. Compared to the existing state‐of‐art techniques the proposed technique shows marked improvement. Using fluorescently‐coated Saccharomyces cerevisiae cells we have recorded cell counting with throughput as high as 2,090 cells/min in the low flow rate regime and were able to image the individual cells on‐the‐go. Overall, the proposed system is cost‐effective and simple in channel geometry with the advantage of efficient counting in operational regime of low laminar flow. This technique may advance the emerging field of microfluidic based cytometry for applications in nanomedicine and point of care diagnostics. Microsc. Res. Tech. 76:1101–1107, 2013. © 2013 Wiley Periodicals, Inc.     

MRT Letter: High resolution SEM imaging of nano‐architecture of cured urea‐formaldehyde resin using plasma coating of osmium

Nanoarchitecture of cured urea‐formaldehyde (UF) resins was examined with a field‐emission scanning electron microscope (FE‐SEM) after coating samples with osmium, which is considered to produce particles of considerably smaller size compared to other metal coatings used in SEM studies. This method enabled comparison of the nanoarchitecture of UF resins of low (1.0) and high (1.6) formaldehyde/urea (F/U) mole ratios to be made, based on imaging of extremely small size particles as part of UF resin architecture, not described before. Imaging revealed presence of relatively large globular particles (148.084–703.983 nm size range) as well as smaller substructures (28.004–39.604 nm size range) as part of the architecture of 1.0‐mole UF resin. Globular particles were also present in 1.6 mole UF resin, but of considerably smaller size (14.760–50.269 nm). The work presented demonstrates usefulness of osmium coating in unraveling the intricacies of the nanostructural organization of cured UF resins, prompting wider application of this immensely useful but grossly underutilized metal coating type in high resolution SEM examination of biological and materials samples. Microsc. Res. Tech. 76:1108–1111, 2013. © 2013 Wiley Periodicals, Inc.     

Atomic resolution imaging of beryl: an investigation of the nano‐channel occupation

Beryl in different varieties (emerald, aquamarine, heliodor etc.) displays a wide range of colours that have fascinated humans throughout history. Beryl is a hexagonal cyclo‐silicate (ring‐silicate) with channels going through the crystal along the c‐axis. The channels are about 0.5 nm in diameter and can be occupied by water and alkali ions. Pure beryl (Be₃Al₂Si₆O₁₈) is colourless (variety goshenite). The characteristic colours are believed to be mainly generated through substitutions with metal atoms in the lattice. Which atoms that are substituted is still debated it has been proposed that metal ions may also be enclosed in the channels and that this can also contribute to the crystal colouring. So far spectroscopy studies have not been able to fully answer this. Here we present the first experiments using atomic resolution scanning transmission electron microscope imaging (STEM) to investigate the channel occupation in beryl. We present images of a natural beryl crystal (variety heliodor) from the Bin Thuan Province in Vietnam. The channel occupation can be visualized. Based on the image contrast in combination with ex situ element analysis we suggest that some or all of the atoms that are visible in the channels are Fe ions.     

Compact non‐contact total emission detection for in vivo multiphoton excitation microscopy

We describe a compact, non‐contact design for a total emission detection (c‐TED) system for intra‐vital multiphoton imaging. To conform to a standard upright two‐photon microscope design, this system uses a parabolic mirror surrounding a standard microscope objective in concert with an optical path that does not interfere with normal microscope operation. The non‐contact design of this device allows for maximal light collection without disrupting the physiology of the specimen being examined. Tests were conducted on exposed tissues in live animals to examine the emission collection enhancement of the c‐TED device compared to heavily optimized objective‐based emission collection. The best light collection enhancement was seen from murine fat (5×–2× gains as a function of depth), whereas murine skeletal muscle and rat kidney showed gains of over two and just under twofold near the surface, respectively. Gains decreased with imaging depth (particularly in the kidney). Zebrafish imaging on a reflective substrate showed close to a twofold gain throughout the entire volume of an intact embryo (approximately 150 μm deep). Direct measurement of bleaching rates confirmed that the lower laser powers, enabled by greater light collection efficiency, yielded reduced photobleaching in vivo. The potential benefits of increased light collection in terms of speed of imaging and reduced photo‐damage, as well as the applicability of this device to other multiphoton imaging methods is discussed.     

MRT letter: A unified accelerated maximum likelihood technique for widefield,confocal, and super‐resolution 4Pi microscopy

We propose an algorithmic technique for accelerating maximum likelihood (ML) algorithm for image reconstruction in fluorescence microscopy. This is made possible by integrating Biggs–Andrews (BA) method with ML approach. The results on widefield, confocal, and super‐resolution 4Pi microscopy reveal substantial improvement in the speed of 3D image reconstruction (the number of iterations has reduced by approximately one‐half). Moreover, the quality of reconstruction obtained using accelerated ML closely resembles with nonaccelerated ML method. The proposed technique is a step closer to realize real‐time reconstruction in 3D fluorescence microscopy. Microsc. Res. Tech. 78:331–335, 2015. © 2015 Wiley Periodicals, Inc.     

Two‐photon laser scanning microscopy as a useful tool for imaging and evaluating macrophage‐, IL‐4 activated macrophage‐ and osteoclast‐based In Vitro degradation of beta‐tricalcium phosphate bone substitute material

Two‐photon microscopy is an innovative technology that has high potential to combine the examination of soft and hard tissues in vitro and in vivo. Calcium phosphates are widely used substitutes for bone tissue engineering, since they are degradable and consequently replaced by newly formed tissue. It is well known that osteoclasts are responsible for the resorption processes during bone remodelling. We hypothesize that also macrophages are actively involved in the resorption process of calcium phosphate scaffolds and addressed this question in in vitro culture systems by two‐photon laser scanning microscopy. Beta‐tricalcium phosphate specimens were incubated with (1) macrophages, (2) interleukin‐4 activated macrophages, and (3) osteoclasts for up to 21 days. Interestingly, macrophages degraded beta‐tricalcium phosphate specimens in an equivalent fashion compared to osteoclasts and significantly more than IL‐4 activated macrophages. An average of ～32% of the macrophages was partially filled with ceramic material while this was 18% for osteoclasts and 9% for IL‐4 activated macrophages. For the first time by applying two‐photon microscopy, our studies show the previously unrecognized potential of macrophages to phagocytose ceramic material, which is expected to have implication on osteoconductive scaffold design. Microsc. Res. Tech. 77:143–152, 2014. © 2013 Wiley Periodicals, Inc.     

Label‐free identification of the microstructure of rat spinal cords based on nonlinear optical microscopy

The spinal cord is a vital link between the brain and the body and mainly comprises neurons, glial cells and nerve fibres. In this work, nonlinear optical (NLO) microscopy based on intrinsic tissue properties was employed to label‐freely analyze the cells and matrix in spinal cords at a molecular level. The high‐resolution and high‐contrast NLO images of unstained spinal cords demonstrate that NLO microscopy has the ability to show the microstructure of white and grey matter including ventral horn, intermediate area, dorsal horns, ventral column, lateral column and dorsal column. Neurons with various sizes were identified in grey matter by dark spots of nonfluorescent nuclei encircled by cytoplasm‐emitting two‐photon excited fluorescence signals. Nerve fibres and neuroglias were observed in white matter. Besides, the spinal arteries were clearly presented by NLO microscopy. Using spectral and morphological information, this technique was proved to be an effective tool for label‐freely imaging spinal cord tissues, based on endogenous signals in biological tissue. With future development, we foresee promising applications of the NLO technique for in vivo, real‐time assessment of spinal cord diseases or injures.     

A 340/380 nm light‐emitting diode illuminator for Fura‐2 AM ratiometric Ca2+ imaging of live cells with better than 5 nM precision

We report the first demonstration of a fast wavelength‐switchable 340/380 nm light‐emitting diode (LED) illuminator for Fura‐2 ratiometric Ca²⁺ imaging of live cells. The LEDs closely match the excitation peaks of bound and free Fura‐2 and enables the precise detection of cytosolic Ca²⁺ concentrations, which is only limited by the Ca²⁺ response of Fura‐2. Using this illuminator, we have shown that Fura‐2 acetoxymethyl ester (AM) concentrations as low as 250 nM can be used to detect induced Ca²⁺ events in tsA‐201 cells and while utilising the 150 s switching speeds available, it was possible to image spontaneous Ca²⁺ transients in hippocampal neurons at a rate of 24.39 Hz that were blunted or absent at typical 0.5 Hz acquisition rates. Overall, the sensitivity and acquisition speeds available using this LED illuminator significantly improves the temporal resolution that can be obtained in comparison to current systems and supports optical imaging of fast Ca²⁺ events using Fura‐2.     

Automated detection of fluorescent cells in in‐resin fluorescence sections for integrated light and electron microscopy

Integrated array tomography combines fluorescence and electron imaging of ultrathin sections in one microscope, and enables accurate high‐resolution correlation of fluorescent proteins to cell organelles and membranes. Large numbers of serial sections can be imaged sequentially to produce aligned volumes from both imaging modalities, thus producing enormous amounts of data that must be handled and processed using novel techniques. Here, we present a scheme for automated detection of fluorescent cells within thin resin sections, which could then be used to drive automated electron image acquisition from target regions via ‘smart tracking’. The aim of this work is to aid in optimization of the data acquisition process through automation, freeing the operator to work on other tasks and speeding up the process, while reducing data rates by only acquiring images from regions of interest. This new method is shown to be robust against noise and able to deal with regions of low fluorescence.     

Immersion oil for high‐resolution live‐cell imaging at 37°C: optical and physical characteristics

The use of normal immersion oil, developed for 23°C, at 37°C greatly compromises both axial resolution and signal intensity. We developed and characterized an immersion oil for optimal performance in live‐cell imaging at 37°C. We quantify the improvements in resolution and intensity obtained when using the new oil instead of its standard 23°C counterparts.     

Flexible and stable optical parametric oscillator based laser system for coherent anti‐Stokes Raman scattering microscopy

The characteristics of a stable and flexible laser system based on a synchronously pumped optical parametric oscillator (OPO) is presented. This OPO can offer very stable operation with both ～1 ps and ～300 fs outputs over a broad wavelength range, i.e., 920–1200 nm. Combining the pump tuning with the OPO tuning, a total Raman range of 1900–5500 cm^?1 is accessible. For maximum spectral sensitivity, the CARS microsope based on the ps laser system is presented in detail. The lateral resolution of the microscope is diffraction limited to be about 390 nm. Fast wavelength switching (sub‐second) between two Raman vibrational frequencies, i.e., 2848 cm^?1 for C? H aliphatic vibrations and 3035 cm^?1 for C? H aromatic vibrations is presented as an example, although this also extends to other Raman frequencies. The possibility of obtaining a multimodal imaging system based on the fs laser system is also discussed. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.