Proposed sample preparation procedure for biological particles under humidity-controlled conditions. In this scheme, samples are mounted on a thin membrane covering the pinhole of a sample-mounting disk. The details of steps 1–3 are described in the text.
(a) Schematic illustration of the sample preparation chamber. For clarity, the components of the chamber are colored differently. The main chamber acts as a platform to perform steps 1–2 in Figure 1 and is filled by moist air from a HUM-1 generator. In the pincette box, a pincette (1), which holds a custom-made sample-mounting disk (2) (see also panel (b)), is sandwiched by the lower (3) and upper (4) parts of the pincette box. Both the lower and upper parts, which are attached using rubber magnet sheets (5) have a 22-mm square-shaped cover glass (drawn as cyan square plate) used as a view port and for light illumination, respectively. Moist air from the HUM-1 generator passes through the small tunnel (6) in the lower part. The 22-mm cover glass in the sample loader acts as a workstation to pick up sample particles. The chamber lid also has a cover glass to allow light illumination. The two sponge plates (7) prevent the diffusion of moist air from the main chamber and enable the smooth movement of the microcapillary (8) attached to the micro-manipulator. The microcapillary moves approximately 15 mm from the sample droplet to the sample-mounting disk. The two small yellow cubes (9) indicate the positions of the SHT75 sensors. The L-shaped plate (10) is used to cover the pincette box when removing the box from the main chamber. (b) Microscopic view of a custom-made sample-mounting disk of 3-mm diameter and 35-μm thickness. The magnified view of its ellipsoid-shaped hole with a long axis of 300 μm is also presented. (c) A photograph of the assembled sample preparation chamber fixed on the sample stage of a microscope. The scale bar in the photograph is 20 mm. (d) Photograph of the pincette box set on the flash-cooling device immediately prior to plunging the pincette into liquid ethane.
RH-dependent change in the size of a droplet of sample buffer for a chloroplast particle set in the main chamber. The inset photographs demonstrate the variation in the size of the droplet at the times and RHs indicated by the arrows. The dotted circles show the diameter of a 12-μm droplet, as measured prior to starting the RH control. The scale bars represent 5 μm. The solid and dotted lines above the panel indicate the constant and variable RH stages, respectively. Small fluctuations in RH of approximately 0.5%rh were observed during the temperature variation of the experimental room caused by the air conditioner.
(a) Series of photographs taken during the first step (step 1; Fig. 1) in the preparation of a spinach chloroplast sample under a dim green light. In each photograph, the position of the targeted chloroplast particle is indicated by a white arrow. From left to right, the images show aspiration, transfer, contact with the carbon membrane, and setting of the particle. The scale bar represents 20 μm. (b) Photographs of a chloroplast particle in step 2 of the preparation procedure. The upper image was taken just after exposure of the sample to an atmosphere of 93.5%rh, and the lower image was captured after 7 min of exposure. Excess sample buffer around the particle was drawn off using the tip of the micro-injector. The size of the x-ray beam is indicated by the dotted circles, and the scale bars represent 10 μm. (c) Diffraction pattern of the flash-frozen chloroplast particle at 66 K. The pattern shown is a CCD image of 128 × 128 pixels. The spatial frequency of 2.8 μm−1 at the edge corresponded to a resolution of 181.6 nm/pixel. Central missed area of 21 × 17 pixels was initially approximated with a diffraction pattern calculated from optical microscope image. The x-ray intensity was approximately 1 × 108 photons/14 μmϕ at the sample position. (d) Comparison of a projection image of the targeted chloroplast particle (upper panel), which was phase-retrieved from the diffraction pattern in panel (c), with a microscopic image (lower) taken prior to the flash-cooling of the particle. The scale bar is 2.5 μm. A phase-retrieval calculation composed of the 1000 HIO iteration with reset of the support area by SW in every 20 cycles and 100 iteration of HIO. The γ value29 of the best five phase-retrieval calculations was less than 0.029.
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