Martine, S. Mandel, M. Words: 31, Fiction from A. Kersel, Jamie Lackey, Margret A. Words: 36, Chet Williamson is the featured writer! Words: 33, Damien Angelical Walters is our featured writer! Her story, All The Pretty Cages, is of outsiders and freedom. We talk to her about writing, her favorites and what is coming up next! Kristi DeMeester is our featured writer. She brings her new story, "Abide with Me" and we talk to her about Georgia, "December Skin" and her upcoming short fiction collection.
Kevin Lucia talks Quiet Horror in the final installment of Horror Words: 29, Featuring A. We talk about writing, Canada, and her Women To Read posts. Kevin Lucia's Horror , Faith-ful Horror, he talks about faith, horror, and how the two weave together. Words: 35, Featuring Livia Llewellyn.
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What happens when secrets are revealed that the human mind cannot comprehend? Words: 46, Double Issue! Our featured writer is Gene O'Neill. Words: 28, The June-ish issue of LampLight is here.
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Kelli Owen brings us part 3 of her serial Novella, Wilted Lilies. Cryo-electron tomography is used for obtaining the three-dimensional structure information. Vitrification of biological specimens in liquid nitrogen LN2 temperature has been proved to be a powerful technique to preserve the native structures of macromolecules either in vitro or in situ. In combination with the most recent hardware and software breakthroughs, cryo-fixed macromolecule complexes isolated from cells can be determined at near atomic resolution using the single particle reconstruction method Ma et al.
However, it is still very challenging to identify and determine the structures of interested macromolecules in situ with cryo-electron microscopy cryo-EM Lucic et al. The major hurdle lies in the fact that many different kinds of macromolecules are crowded within the cell and therefore are hard to be distinguished from each other simply by their shapes revealed in cryo-EM Bauerlein et al.
Furthermore, the large dimension of a cell and a rather small viewing area of EM at a high magnification render it more difficult to localize relatively sparse molecules in a specific cellular structure environment Plitzko et al. Light microscopy LM , more specifically, fluorescence microscopy FM is well developed to label specific molecules and localize them in a large viewing area with a resolution of several hundreds of nanometers. The advantages of specific labeling and localization in LM and high spatial resolution in EM can be combined, leading to the development of a powerful technique as the correlative light and electron microscopy CLEM Anderson et al.
CLEM technique was first reported in the early s for adenovirus study with conventional chemical fixation EM specimen preparation Godman et al. While the chemical fixed specimens are still the majority of targets investigated by CLEM Hellstrom et al. FM imaging of samples at room temperature could be done with any typical FM instrument. FM can have better resolution with high-powered, oil-immersion lenses with large numerical apertures. After FM imaging, the sample needs to be frozen for further cryo-EM imaging. Various factors can be introduced to the specimen between the LM imaging and specimen vitrification, causing more difficulties for accurate correlation.
Therefore, it works better for specimen that is relatively stable and the fluorescently marked structure would not change in the seconds to minutes before vitrification. The sample is plunge frozen or cryo-sectioned first, then the grid is visualized under LM and EM sequentially with little disturbance of its structure, thus making the correlation more consistent. Special specimen stages designed for the related instruments are essential to preserve the cryo-fixed specimens Li et al.
While the first type of CLEM method provides convenience of use and better resolution of LM, the second type guarantees more accurate correlation and is more favored Briegel et al. For the second type of CLEM method, two different designs are commercially available. One is the integrated Agronskaia et al. The experiments efficiency is greatly increased without repeated grid transfers Agronskaia et al. Arnold et al. Different image processing software packages have been developed for high-precision correlation.
The study of virus-infected or transfected mammalian cells by using Leica Cryo system has been reported. Here we present a detailed process for the application of the CorrSight system in combination with a cryo-TEM. We used plunge-frozen technique to fix the biological specimen and cryo-electron tomography cryo-ET for obtaining the three-dimensional 3D structure information.
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GiG R3. The vitrified specimen on cryo-EM grids was stored in LN2 dewars for further examination. Cryo shuttle and transfer box assembly for CorrSight. A The display of cryo shuttle components and transfer box. Lid and cylinders are used for fixing Autogrids. B — D The process of assembly. Cryo shuttle is assembled after mounting Autogrids on cryo shuttle 1 , putting cylinders 2 and then putting lid 3 on the grids as the numbers correspond to the sequence illustrated in B. The assembled cryo shuttle in C is eventually loaded into transfer box in D.
The steps 2 — 4 should all be performed at LN2 temperature. The use of the cryo module of CorrSight. A Purging and cooling of the cryo module chamber before LM imaging. B The overhead view of cryo module chamber.
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There are locations with similar shape of transfer box and cryo shuttle. The screw of cryo shuttle should be facing toward the user. The right sample numbered 1 would be viewed as the left one in Maps software, while the left sample numbered 2 would be viewed as the right one in Maps software. The acquisition of multichannel LM images with Maps software. A The samples 1 and 2 correspond to the ones described in Fig. B Layer of sample 1.
Each sample can have a layer which may contain one low magnification grid map and many high-magnification fluorescence square images numbered in sequence.
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Check the intactness of carbon film, flatness of grid, and thickness of vitreous ice for the Autogrids Fig. Screen the cryo grids to choose the best one called finder grid for further image acquisition. The correlation of LM and EM grid map. More than 60 squares are multichannel imaged and labeled with sequence number, which corresponds to the number in Fig. Note : Turn on the air compressor only at the beginning of the multichannel image acquisition and turn it off before the cryo sample transfer to prevent severe vibration of CorrSight.
Once the imaging is completed, the cryo samples should be unloaded and put back in the LN2 tank immediately. After the image acquisition, put the cryo shuttle back to the LN2 storage tank. Overlaid multichannel images can be adjusted and saved manually for EM correlation. Within a good grid, more than 60 squares could be imaged and marked Figs. This is the step for rough and quick screening of squares. Find squares with interested fluorescence signal and label the locations within the overlaid images Fig.
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A Acquisition of montaged EM atlas of grid and square. The acquisition usually starts from the stage center and stage movement is used. The conversion matrix between the LM and EM grid maps should be determined, which can be derived from two reference positions, i. For each reference position, the coordinate in the LM map is obtained by using any graphic software, while the coordinate in the EM map is obtained and converted to the corresponding EM stage position via a precalibrated matrix between the camera and stage by using AutoEMation.
The correlation of LM and EM square images and target identification. B Zooming in on the area highlighted in A. Three vesicles can be identified and marked by asterisks in red , blue , and yellow , respectively, which are close to the fluorescence signal, highlighted by yellow circle.
D Zooming in on the area highlighted in C. The target can be located via correlation by means of AutoEMation and verified with the three neighboring vesicles identified accordingly. F Tomogram slice of the target after tomographic data collection and reconstruction. Similarly, the conversion matrix between the LM and EM square maps should be determined, which can be derived from two reference positions, i. With the help of the conversion matrix, the coordinates of fluorescence target in the LM map Fig.
The third step is the verification of the target particles at a much higher magnification to ensure they are suitable for cryo-ET data collection. The target particles selected from the above two steps may not be suitable for cryo-ET data collection as images acquired at the above magnification cannot provide enough details.
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Therefore, it is worth imaging the target particles at a much higher magnification i. Select file format and output folder. Set the focus and tracking areas away from the exposure area.
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