A new tool for cryo-electron microscopy

By | 07/09/2022

Cryo-electron Microscope Developed for Simultaneous Stem, SEM Imaging and Its Application to Biological Samples


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Jiro Usukura*1, Akihiro Narita*1, Tomoharu Matsumoto*1, Eiji Usukura*1, Takeshi Sunaoshi*two, Yusuke Tamba*ii, Yasuhira Nagakubo*ii, Junzo Azuma*2, Takashi Mizuo*2, Kazutaka Nimura*2, Masako Osumi*3, Ryuichiro Tamochi*2, Yoichi Ose*2

*ane
School of Scientific discipline, Nagoya University
*2
Hitachi High-Tech Corporation
*3
Nihon Women’s Academy

Introduction

Cryo-electron microscopy (cryo-EM), which has get gradually popular in recent years, is used exclusively herein for unmarried-particle analysis of purified proteins and viruses in their native state. Single-particle analysis (a kind of image processing) has thus far been applied to purified molecules stained negatively. All the same, negative staining is basically air-drying, even if the staining solution encompasses a molecule and its surroundings quickly, and thus it is difficult to entirely forestall structural changes. Moreover, the images obtained vary depending on the penetration of the staining solution. In cryo-EM, withal, purified protein molecules are chop-chop frozen and observed without whatsoever further treatment, which allows even biological samples to be observed at applied EM resolution. This is a crucial point, because conventional EM training techniques such every bit freeze-etching replicas or ultra-thin sections incur a reduction of resolution during pre-treatment. Nonetheless, raw protein samples are extremely vulnerable to irradiation damage past electron beams; where even for observations at liquid-nitrogen temperature, the samples are reportedly damaged at a beam irradiation of approximately 20 electrons/Åii. This implies that raw poly peptide samples must exist observed under an illumination of one–iii electrons/Å2. Though challenging, this feat is made possible by the contempo evolution of high-sensitivity cameras based on straight detection of electrons (DD cameras). Further, single-particle analysis does not require the crystallization of protein molecules, simply allows structural analysis in dispersed systems. The highest spatial resolution recently obtained from single-particle analysis is 3Å (Meyerson
et al.,
2016), which is approaching the resolution of X-ray crystallography. Moreover, it should exist noted that these analytical results were obtained for samples in a frozen country shut to their native state. Meanwhile, cryo-EM is not only useful for single-particle analysis, simply is as well an extremely powerful tool for conventional structural observations. Indeed, cryo-EM observations of frozen sections take successfully revealed the molecular structure of functioning organelles, ribosomes, and the desmosomes that mediate adhesion between cells (Al-Amoudi
et.al.,
2004). In the futurity, cryo-EM will get an essential tool for pathology of cells. Recently, cryo-EM has been used for structural assay of the infection procedure of the influenza virus in cells (Fontana & Steven, 2015). In full general, for agreement life phenomena, information technology is very important to know the structure of proteins, their complexes in a functioning state and their spatial construction, and how such proteins are disposed inside a cell. Cryo-EM is clearly an essential tool for this purpose. Unfortunately, however, cryo-EM is not sufficiently widespread in Japan compared to Europe and the Usa. Instead, cryo-EM observations of fresh tissue and cells depend on preprocessing equipment such every bit quick freezers, cryo-microtomes and other pregnant investments—on the gild of 700 million yen in total—which is likely the primary factor preventing the more than widespread use of cryo-EM. However, if these instruments are required for futurity progress in the life sciences, there is no choice but to equip laboratories with them. Cryo-instrumentation is now rapidly beingness facilitated in leading European and American universities, and the development of cryo-systems including preprocessing instruments has occurred almost entirely in the Eu, while Japanese contributions are disappointingly few and far between. Although we are something of a latecomer to the marketplace, nosotros have now developed a new cryo-EM that detects both transmission and surface images simultaneously in the frozen country. Herein we study the features and applications of this new musical instrument.

Purpose

Nosotros aim to develop a cryo-EM that everybody can use easily in a wide range of scientific fields with an economical budget. In detail, a low-voltage cryo-EM (scanning transmission electron microscope, or STEM) was developed based on a scanning electron microscope (SEM: SU 9000) equipped with a STEM detector to capture SEM and STEM images simultaneously. Additionally, the sample grooming method is also important and should be developed together with the EM. Indeed, if the samples are not candy in an appropriate manner, the EM will neglect to realize its total capability, even if designed to the most exacting performance standards, and the reliability of the observed results will be impaired as well. Thus, the development of a cryo-EM and the development of the sample preparation appear inseparable. Therefore, we developed a device for cell-membrane removal (i.east., unroofing) to facilitate observations of the membrane cytoskeleton.

Features of the New Cryo-EM

Because our enquiry project was supported by government funds, we should develop the about powerful cryo-EM in the world. All the same, a 300 kV cryo-EM has already been offered on a commercial basis that is, to some degree, user-friendly and also has produced world-tape data. Although this instrument is extremely expensive every bit its disadvantage, the development of a great bargain of elemental engineering science would be necessary to exceed this microscope, which is not feasible to reach with the funding nosotros currently receive from the Japan Agency for Medical Research and Development (AMED). This is the primary reason nosotros chose not to target atomic resolution in cryo-EM development. Nevertheless, the depression-accelerating-voltage (i.eastward., only 30 kV) cryo-EM developed in this report is capable of simultaneous Stem and SEM measurements, which offers several prominent advantages and may come to occupy a highly competitive position inside EM technologies. For case, considering images of frozen biological samples in conventional cryo-EM typically exhibit extremely low contrast at the focal point, images are usually made by shifting the focus to the underside of the sample to yield a phase contrast (i.e., Fresnel contrast), which results in a subtract of the original resolution of the EM. To perform single-particle analysis of the images obtained in this way, both a correction of the contrast transfer function (CTF) and the collection of a big number of images are needed. However, the new cryo-EM adult in this project allows manual images to be formed in the Stalk optical organisation. In Stem, images are obtained past scanning an extremely focused electron beam over the sample, and therefore images are a bitmap of electrons transmitted through the sample. Thus, the paradigm obtained is not formed past an objective lens and does non suffer as much from the CTF. Every bit a matter of grade, images were observed at the focal point without any reduction of the resolution. Thus, the new cryo-EM developed herein is particularly useful for unmarried-particle analysis, where it yields adept results with a relatively small number of images compared to conventional cryo-EM in transmission electron microscopy (TEM) style. Some other major divergence between the new Stem cryo-EM and conventional TEM is the method of image detection. Whereas TEM requires a photographic camera to capture the image, STEM requires but a scintillator to capture electrons. This distinction may become meaningful and increasingly important in the forthcoming development of EM. Today, the complementary metallic-organic-semiconductor (CMOS)-based DD cameras installed in cryo-TEMs offering a much better functioning, just these cameras are very expensive (nearly 100 million yen per unit of measurement). The scintillators installed in Stem, withal, capture electrons passing through the sample (i.e., position sensitive), and thus the sensitivity should be sufficient to count electrons. For example, if information technology proves possible, applying a thick coating of practiced fluorescent textile may attain the high sensitivity required for single-electron detection, whereupon images of quality comparable to that of DD cameras could be obtained at vastly lower cost. The feasibility of such a detector depends on future study.

Because the total size of the cryo-EM developed in this project is meaty, and indeed is the same equally the Hitachi SEM (SU 9000) used equally a development base machine, it was not necessary to create a special electron microscopy room just to simply install it in the corner of the laboratory. We approximate that many researchers in the life sciences would be satisfied with this cryo-EM, considering with it single-particle assay is easily feasible with a small number of images fifty-fifty at 1 nm resolution, and the fine structure in the cell can be observed in the native state at the same resolution. Nosotros thus aimed to develop a cryo-EM with the higher up advantages.

Details of Research and Development

Cryo-transfer holder

Of all the elemental technologies encompassed by our development plan, the most crucial component was the development of a cryo-transfer holder. Approximately 2/3 of the holder arm was formed by a double-walled vacuum pipe that was nearly entirely filled with liquid nitrogen, which increased the cooling rate of the specimen holder. We also added a vacuum system to release the gas from the liquid nitrogen in the pipe, and we tested the effect of converting the liquid nitrogen into slush nitrogen in what was the world’due south kickoff endeavour to use slush nitrogen in a cryo-transfer holder. In this manner, nosotros succeeded ultimately in reaching temperatures as low every bit −190°C. Moreover, nosotros optimized the incoming and approachable flow paths for liquid nitrogen to better the speed and efficiency of cooling. Practical observation of samples was carried out while maintaining the specimen holder at −180°C. In the present state of the cryo-EM, information technology was difficult to discover specimens for extended periods at temperatures lower than −185°C because the images drifted with the temperature fluctuations. This drift is owing to an influx of heat into the cryo-transfer holder from the contact region between the holder and the phase, which is positioned at the tip of the holder. Based on this observation, we expect that the development of a non-contact phase may reduce image drift to allow long-duration observations.

Fig. 1 CAD diagram of the prototype cryo-transfer holder. The inset (upper left) shows an enlarged view of the sample holder at the tip.

Fig. 1 CAD diagram of the epitome cryo-transfer holder. The inset (upper left) shows an enlarged view of the sample holder at the tip.

Anti-contamination trap

Considering the sample in a cryo-EM is held at an extremely low temperature, a contamination trap cooled to a temperature below that of the sample is needed to prevent contagion of the samples. In our organization, the arm portion of the trap was besides comprised of a pipe in which liquid nitrogen may be introduced at a point almost its tip. We also added a gas-exhaust arrangement and succeeded in cooling the trap to −200°C by converting the liquid nitrogen into a slush. The shape of the tip of the trap was designed to surround the sample region at the tip of the cryo-transfer holder. In addition, by cooling the buffer tank of the microscope, nosotros accomplished a vacuum strength in the sample bedchamber of two × 10−half-dozen
Pa, to be compared to the conventional strength of 7 × 10−six
Pa. These improvements succeeded in almost entirely preventing the reformation of ice crystals on the sample.

Fig. 2 CAD diagram of anti-contamination trap. The use of slush nitrogen succeeded in cooling to −200°C. The shape of the tip of the trap was designed to surround the tip of the sample holder (the sample region).

Fig. 2 CAD diagram of anti-contagion trap. The employ of slush nitrogen succeeded in cooling to −200°C. The shape of the tip of the trap was designed to surroundings the tip of the sample holder (the sample region).

Development of cell-membrane removal (i.e., unroofing) device

As mentioned previously, even high-performance observation instruments cannot achieve their full performance if the sample is processed incorrectly, and in such cases 1 cannot expect to obtain reliable measurement results: The evolution of the instrument itself and of techniques for preparing samples truly are two sides of the same money. Unfortunately, preprocessing techniques for cryo-EM observations is another area in which Nippon lags behind Europe and The states. The commonly-used “orthodox” methods for cryo-EM ascertainment; i.eastward., the cryo-section of rapidly frozen samples; was developed in Europe. This process involves a collection of preprocessing instruments with a full toll on the order of 100 meg yen. Nosotros have therefore adopted the unroofing method for ascertainment of the membrane cytoskeleton instead of using the orthodox frozen-department method, where unroofing means mechanical removal of the cell membrane. In this instance, the membrane cytoskeleton attached on the ventral cell membrane became observable in cryo-EM afterwards removal of the dorsal cell membrane. We have improved and redeveloped the technique of ultrasonic unroofing conventionally used as a preprocessing step for freeze-etching replica methods. In this approach, soluble components in the jail cell are washed away so as to increase the contrast of the cytoskeleton and other organelles. Samples candy in this fashion exhibited a number of unexpected advantages, including panoramic observations of the cytoplasmic surface of the cell membrane. Nonetheless, the commercially bachelor probe-type ultrasonic generator used without modification produced an output power that was besides stiff to properly regulate the unroofing, and its excessive output power led to sure problems with reproducibility. For example, when applied to cells cultured on a carbon support film on EM grids, the cells were completely destroyed and removed. An appropriate output power for unroofing was found to exist approximately ane Due west or less. In dissimilarity, the output power of commercially bachelor probe-blazon ultrasonic generators is typically 50 W or more than, and it is hard to command it to under five W. Therefore, nosotros customized an ultrasonic generator with an output ability of five W or less, with an choice to precisely regulate the power below 1 W. Our prison cell-membrane removal (unroofing) apparatus thus consisted of (ane) this custom-fabricated low-output-power ultrasonic generator, together with (two) a position controller to bring the ultrasonic probe near the sample accurately, and (3) a stereo optical microscope to notice the process of jail cell-membrane removal (Figure 3). With this apparatus, the removal of the prison cell membrane became extremely uncomplicated and easy. We likewise found this tool to be useful in preparing samples not but for cryo-EM but as well for other types of observation instruments. In detail, although it was difficult to observe the intracellular fine construction with atomic force microscopy (AFM), unroofing enabled u.s.a. to view the supra molecular structure inside of the jail cell for the first time in h2o (Usukura
et al.,
2016). Figure 5 shows an AFM epitome of the intra-cellular fine structure prepared using the apparatus nosotros adult, wherein molecular structure analysis of the individual actin filaments in the cell is likewise possible.

Fig. 3 The prototype cell-membrane removal (i.e., unroofing) apparatus constructed herein, as viewed from the front (left) and at short distance from an angle (right). The apparatus consists of a low-output-power probe-type ultrasonic generator (indicated by an arrow) a position controller for this probe, a stereo microscope, and a horizontal illuminator (indicated by an asterisk).

Fig. 3 The image jail cell-membrane removal (i.e., unroofing) apparatus constructed herein, every bit viewed from the front (left) and at brusque distance from an angle (correct). The apparatus consists of a low-output-power probe-type ultrasonic generator (indicated by an arrow) a position controller for this probe, a stereo microscope, and a horizontal illuminator (indicated by an asterisk).

Fig. 4 Phase-contrast optical microscopic image of cells after unroofing. Membrane cytoskeletons (stress fibers) are visible above the center of cell membranes (indicated by “U” symbols). The region marked “PU” indicates cells from which the cell membrane was partially removed.

Fig. 4 Phase-contrast optical microscopic image of cells afterward unroofing. Membrane cytoskeletons (stress fibers) are visible above the center of prison cell membranes (indicated by “U” symbols). The region marked “PU” indicates cells from which the cell membrane was partially removed.

Fig. 5 (A) AFM image showing the structure of the membrane undercoat of NRK culture cells. (B) Enlargement of the boxed area of A. The short periodicity of the actin filaments is clearly observed. “CL” indicates clathrin coat. “CV” indicates caveola. Asterisks indicate smooth-surfaced endoplasmic reticulum. (Sci. Rep., 6: 27472, 2016)

Fig. v (A) AFM prototype showing the structure of the membrane undercoat of NRK culture cells. (B) Enlargement of the boxed area of A. The short periodicity of the actin filaments is clearly observed. “CL” indicates clathrin coat. “CV” indicates caveola. Asterisks bespeak smooth-surfaced endoplasmic reticulum. (Sci. Rep.,
six: 27472, 2016)

Application to Biological Sciences

The simultaneous cryo-Stem and cryo-SEM ascertainment of membrane cytoskeletons in their native state, which was an original goal of this evolution project, has been achieved. Indeed, such ascertainment has become near routine (Figure 6). Because soluble components in a prison cell are washed away upon unroofing, the cytoskeleton could exist observed with sufficient dissimilarity on focus. Surprisingly, ribosomes and endoplasm reticulum remained partially intact after unroofing, and were observed well together with the membrane cytoskeleton (Effigy 7). In particular, many organelles such as mitochondria, polish endoplasmic reticulum (ER), rough ER and ribosomes were detected with extremely high contrast, despite the presence of partially unroofed cells in which many soluble components still remained. A fortunate and unexpected finding was that regions of thickness estimated at 200 nm or more could be clearly observed. We are unsure whether or not this is a feature of the STEM optical organisation, only information technology became evident that the Stalk is capable of describing intracellular fine structures even at 30 kV accelerating voltage. This represents a crucial first step toward the evolution of new types of Ems in the future.

Fig. 6 Simultaneously recorded cryo-STEM (left) and cryo-SEM (right) images (for an unfixed sample). Initially, the sample is fully embedded in ice and the SEM image is flat owing to the ice coating. As the electron-beam irradiation induces a temperature increase, a portion of the ice sublimates, yielding the structure seen in the figure at right.

Fig. 6 Simultaneously recorded cryo-Stem (left) and cryo-SEM (right) images (for an unfixed sample). Initially, the sample is fully embedded in ice and the SEM image is flat owing to the ice blanket. As the electron-beam irradiation induces a temperature increase, a portion of the ice sublimates, yielding the structure seen in the figure at right.

Fig. 7 Cryo-EM images of unroofed cell without chemical fixation. The cytoskeleton consisting of actin filaments and microtubules is observed well together with smooth endoplasmic reticulum (ER), rough ER and ribosomes in spite of the presence of unroofed cells.

Fig. 7 Cryo-EM images of unroofed cell without chemical fixation. The cytoskeleton consisting of actin filaments and microtubules is observed well together with smooth endoplasmic reticulum (ER), crude ER and ribosomes in spite of the presence of unroofed cells.

Another of import finding was derived from the application research carried out under this development projection. As discussed above, the objective lens is non directly involved in image germination in Stalk eyes, which is optimal for single-particle analysis. This advantage is especially prominent in our developed depression-voltage cryo-EM installed with a cold-field emission electron gun, which generates a very focused electron beam (0.34 nm in diameter). Indeed, the single-particle analysis of the actin filaments and molecular construction calculated from only xx images obtained by our 30 kV STEM exhibited a much higher resolution than that obtained by conventional 100 kV TEM (Figure 8). In single-particle assay using an adenovirus stained in the conventional way, xviii images were sufficient to obtain the molecular structure of the virus at 5 nm resolution (Figure nine). Because the images were obtained without CTF correction and with high contrast on focus, the molecular structure was determined more than precisely than in the conventional fashion past single-particle analysis using a pocket-sized number of images. This fact suggests the possibility of
in situ
single-particle analysis revealing the existent molecular structure in a prison cell, but not purified molecules.

Fig. 8 Comparison of cryo-EM developed here vs. standard TEM. From negative-stained images (A) we performed threedimensional reconstruction via single-particle analysis, taking into account the helical symmetry. (B) 30 kV STEM image captured with new cryo-EM described herein, and (C) 100 kV image captured with a standard TEM. Upon fitting to an atomic coordinate model (Oda et al., 2009), the three-dimensional image reconstructed from the 100 kV standard TEM image exhibited some regions of disagreement, while the three-dimensional image reconstructed from the STEM images obtained with our new system exhibit excellent agreement.

Fig. viii Comparing of cryo-EM developed here vs. standard TEM. From negative-stained images (A) we performed iii-dimensional reconstruction via single-particle analysis, taking into account the helical symmetry. (B) 30 kV STEM prototype captured with new cryo-EM described herein, and (C) 100 kV image captured with a standard TEM. Upon fitting to an atomic coordinate model (Oda
et al.,
2009), the iii-dimensional epitome reconstructed from the 100 kV standard TEM epitome exhibited some regions of disagreement, while the three-dimensional prototype reconstructed from the Stem images obtained with our new arrangement exhibit splendid agreement.

Fig. 9 Eighteen images of adenovirus particles captured with new cryo-EM developed in this paper (upper) and reconstruction based on these images (lower).

Fig. nine Xviii images of adenovirus particles captured with new cryo-EM developed in this paper (upper) and reconstruction based on these images (lower).

Application to Materials Sciences

Although the new 30 kV cryo-EM with cryo-transfer holder developed herein was primarily to observe frozen samples of living cells, cryo-EM seems to be a useful tool for materials science because of its remarkable reduction of irradiation harm to the samples. In fact, a cryo-holder, though non a cryo-transfer holder, has been used in materials science to reduce irradiation damage, wherein the cooling of the cryo-holder is initiated afterwards being placed into the microscope. This delayed cooling of the cryo-holder is not a problem in the case of metallic samples; simply in materials that include water, ice crystals are formed upon cooling and break the fine structure. Consequently, cryo-holders are not useful for materials including water. Instead, biological samples and materials including water must be rapidly frozen to produce baggy (not-crystalline) ice, and and then the frozen sample must be transferred into the cryo-EM while maintaining its frozen state using the cryo-transfer holder. The germination of crystalline ice is a crucial trouble in EM report on liquid substances. We take recently succeeded in observing nano-bubbles (gas bubbles approximately fifty–100 nm in size) formed in water (Figure ten). These could possibly exist observed with conventional cryo-TEM systems as well, but the ability of our organisation to capture simultaneous Stem and SEM images offers proof that these bubbles actually practice exist in the water. This is a useful new application of the cryo-EM that we have adult. Strong future need of this cryo-EM is anticipated in areas such as cosmetics, pharmaceuticals, foodstuffs and soft materials, and the research establish of soft cloth sections is anticipated in future.

Fig. 10 Nanobubbles formed in water, as imaged using the new system developed herein. These observations were made by pouring water into holes in a QUANTIFOIL membrane and rapidly freezing; the STEM (left) and SEM (right) images were obtained simultaneously. Bubbles are indicated by arrows. The bubbles frequently appear dark in the STEM image.

Fig. x Nanobubbles formed in water, as imaged using the new system developed herein. These observations were fabricated past pouring water into holes in a QUANTIFOIL membrane and rapidly freezing; the Stalk (left) and SEM (right) images were obtained simultaneously. Bubbles are indicated by arrows. The bubbles frequently announced night in the Stalk image.

Concluding Remarks

We used an SEM (SU 9000, Hitachi High-Tech) installed with a STEM detector every bit a base of operations for the evolution of a new cryo-EM. However, information technology would be desirable to design an instrument based on a STEM cadre with added capabilities for high-operation SEM. To retain the prototype quality of conventional EM in the cryo-EM, we promise to increase the scan speed to prevent drifting associated with cooling. A decade agone, high-resolution AFM imaging required 20 min per frame, just today speeds of 10 s per frame are becoming increasingly standard. In contrast, SEM scan speeds have remained unchanged (ane min per frame) for some xxx years. A method of increasing scan speed in SEM is to increase the probe current, but this increases the risk of sample harm. We hope to design new cryo-EM systems capable of producing high-magnification, loftier-quality images past combining the newest drift-correction techniques with increased sensitivity of the detector system.

Acknowledgements

This work was supported by the Medical Enquiry and Development Programs Focused on Technology Transfer (Evolution of Avant-garde Measurement and Analysis Systems) of the Nippon Bureau for Medical Research and Development (AMED).

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