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MicroscopyInnovations


its hard-to-reach areas, as well as the spinal cord. The electronics of Quartet


allowing connection and disconnection of the fibers in an awake mouse, with minimal stress to the animal. Applications of Quartet


neuronal micro-circuitries forming and changing under various conditions. For example, does a drug candidate for Alzheimer’s improve the formation of micro-circuitries that are responsible for memory formation and recall?


™ do not sit on the head of the animal, ™ include the study of


Refl ective Imaging Improves Spatiotemporal Resolution and Collection Effi ciency in Light Sheet Microscopy


National Institute of Biomedical Imaging and Bioengineering Developers: Yicong Wu and Hari Shroff


This new method uses reflection to improve the spatiotemporal resolution and collection efficiency of light sheet microscopy. The improvement is a result of introducing additional specimen views that carry extra information about the sample.


The sample is deposited on a reflective coverslip, instead of a conventional glass coverslip. The reflection (a) introduces an additional light sheet that increases the fluorescence emission rate, improving speed; (b) introduces multiple extra virtual views of the specimen that enhance axial resolution as the views are normally not “seen” by the microscope; and (c) increases collection efficiency since fluorescence that is usually “lost” to the coverslip can now be captured. In addition to this concept, new deconvolution methods were invented that combine the extra information gleaned from reflection and reduce out-of-focus fluorescence that would otherwise contaminate images.


Reflective coverslips are commercially available and are easily substituted for conventional coverslips, so the method can be straightforwardly applied to existing light sheet microscopes. The method can also be used to generate higher numerical aperture (NA) views that would normally be difficult to obtain.


The utility of the method has been demonstrated on a dual-view inverted selective plane illumination microscope (diSPIM), achieving an effective quadrupling of speed relative to single-view microscopy and a four-fold enhancement in collection efficiency. These advantages allowed us to image calcium flux at an unprecedented volumetric rate (~3 volumes/s), allowing us to “catch” transients in rapidly moving worm embryos. We also demonstrated the improvement in volumetric resolution and collection efficiency that would occur at higher NA, obtaining high-resolution datasets within single cells and worm embryos.


2018 September • www.microscopy-today.com


Fluorescence microscopy is an enabling method in cell biology because of the superb specificity it affords. Light sheet fluorescence microscopy is a further advance that much better uses the available photon budget compared to a confocal or widefield fluorescence microscope. However, the usefulness of any fluorescence microscope is limited by the available photon signal. By capturing more views of the specimen without introducing additional delay, the reflective cover slip fundamentally improves on light sheet fluorescence microscopy, extending the value of the limited photon budget by improving resolution and information content within the images.


MUSE Microscopy University of California Davis Health


Developers: Richard Levenson, Farzad Fereidouni, and Stavros Demos


MUSE microscopy (microscopy with ultraviolet surface excitation) is based on two properties of 280 nm UV light: (1) UV light in this range penetrates tissue specimens to a depth of no more than about 10 microns, a thickness only slightly greater than that of a


standard histology slide tissue section; and (2) UV light at this wavelength can excite, and cause to emit in the visible range, a large variety of inexpensive fluorescent stains that can mimic (and extend) the tissue specificity of hematoxylin and eosin (H&E) stain. The low penetration depth limits excitation to a thin layer and removes the usual requirement for tissue sectioning. Using color conversion algorithms, the resulting fluorescent images can be converted, in real time, to resemble that seen with conventional pathology microscopy. T e design is based on UV LEDs, conventional glass (not UV-transparent) objectives, and color cameras. T ere is no need for lasers, emission or excitation fi lters, or the computa- tional input required by other methods. Sample preparation is straightforward, consisting of a brief (seconds) stain of tissue and a brief wash.


Light microscopy for tissue diagnostics, the industry standard, typically relies on thin-sectioned formalin-fixed specimens and absorbance-based stains (such as H&E) that are useful for general morphological visualizations. But those stains provide only a limited color gamut that reveals little about the molecular constitution of the sample. Moreover, thin-cut specimens on slides represent a narrow and flat planar slice of a complicated 3D sample. MUSE overcomes both of these limitations. First, a number of rapidly applied fluorescent tissue stains can generate multiple and potentially useful color signals that convey more information than is available with H&E. Secondly, since MUSE views the surfaces


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