Adaptive optics microscopy for fine imaging of live plant cells

Live imaging using optical microscopes enables observation of live cells, tissues, and organisms, and is vital for biological research. The discovery of fluorescent proteins¹ and the development of fluorescent probes² has allowed us to monitor the dynamics of molecules of interest by combining fluorescent probes in live cells with detection using fluorescent microscopes.³ However, live cells and tissues are composed of complex structures with different refractive indices, where the amplitude of light is disturbed and the wavefront is distorted. Thus, images degrade with increasing observation depth inside live tissues. Plant cells refract light more strongly than those of animals, and even within a single cell, images are strongly degraded.^4–6

A solution to this problem is adaptive optics (AO), which was first developed for astronomy to correct the wavefront distortion caused by atmospheric turbulence, and therefore enabled fine imaging of astronomical objects using ground-based telescopes.^{7, 8} AO requires a reference light source, such as a guide star, and consists of three main components: a wavefront sensor, a control unit, and a spatial light modulator. First, the wavefront sensor enables measurement of the distorted wavefront of the light from the reference light source. Based on this measurement, the control unit drives the modulator to correct the distortion in the wavefront. This process is repeated as a “closed-loop” control, and the wavefront is kept close to flat. The application of AO to microscopy for fine live imaging has recently become an active research focus,^9–12 and is the subject of our work.

Currently, most observation targets of AO microscopes are animal cells and tissues. In contrast, our research target is plants. Thus, we sought to develop an AO microscope for live imaging of plant cells. We used leaves of the moss Physcomitrella patens as a sample because its single cell layer is suitable for optical analyses.¹³ Additionally, it has a strong ability to induce stem cells, which is interesting for biological research.^14–16

First, we analyzed the optical properties of plant cells to determine the specifications of the AO microscope.⁶ We observed the cells using a phase-contrast microscope to measure the phase retardation in live plant cells,¹⁷ and we noted strong white-to-black contrast in chloroplasts: see Figure 1(a). This indicates that chloroplasts disturb the wavefront of the light: Figure 1(b) is the bright-field image for reference. We also observed the point spread function (PSF) using 390nm fluorescent beads attached to the leaf: see Figure 1(c). We obtained clear images of beads attached at the objective lens (top) side of the plant cells: see Figure 1(d). In contrast, when we observed beads attached to the opposite (bottom) side of cells, the bead images were severely degraded: see Figure 1(e). However, the degradation was much milder in the region with only cell walls (no chloroplasts or other cellular components): see Figure 1(f). This was due to plasmolysis, the contraction of the protoplasm (nucleus and cytoplasm). These findings indicate that chloroplasts are the main source of image degradation in plant cells. Consequently, we focused on correction of the phase aberration caused by chloroplasts as the primary specification for our AO microscope: see Figure 2(a). We generally use a 60× water-immersion objective lens (NA=1.2) to observe structures in plant cells. When this lens is used to observe the deep side of the cell, the distance between the focal plane and the layer of chloroplasts is typically 15μm. The chloroplast layer—indicated by the arrow in Figure 1(c)—is seen as the ca. 60μm-diameter circle, where the ray from a single point at the focal plane passes through before reaching the objective lens. The typical diameter of chloroplasts, which are spheroidal, is around 5μm. This suggests that 12×12 elements can be effective for the correction of the base spatial frequency of the aberration caused by chloroplasts. We therefore used a 12×12-element deformable mirror (AOK1-UP01, Thorlabs) for our microscope. We also excited a chloroplast using the spot of a green laser through the deformable mirror. We used this autofluorescence spot as the reference guide source, similar to the laser guide star in astronomical AO.^18–20 Closed-loop operation of the AO microscope improved the convergence of the green laser and sharpened the autofluorescence spot. Notably, we successfully obtained diffraction-limited images of the chloroplast located at the deep side of the plant cell: see Figure 2(b, c).

Figure 1. Optical property analyses of plant cells. (a) Phase-contrast and (b) bright-field images of a Physcomitrella leaf cell (the arrowhead indicates a chloroplast). (c) Schematic model of the point spread function analysis using a Physcomitrella leaf with fluorescent beads attached. The model represents the experiment used to obtain the blurred image in (e). The arrow indicates the chloroplast layer that disturbs the wavefront and is the focal plane of the images in (a, b). The leaf cell is shown larger than the actual size. (d–f) Images of fluorescent beads attached at the top (d), and bottom (e, f) sides of the Physcomitrella leaf cell from the objective lens. A normal leaf (d, e) or a plasmolyzed leaf (f) was used. Brightness of the left panels in (e, f) was adjusted to that of (d). Bars: 20μm (a, b) and 2μm (d–f).

Figure 2. Correction of the chloroplast image with our adaptive optics (AO) microscope. (a) Configuration of the AO microscope. DeM: Deformable mirror. BS: Beam splitter. WFS: Wavefront sensor. CCD: Charge-coupled device. (b, c) Images of a chloroplast located at the deep side of a plant cell without correction (b) or with AO (c). Bar: 5μm.

In summary, light is strongly disturbed when it goes through plant cells, and images degrade even in one live plant cell. We developed AO microscopy that corrects the disturbed light and enables fine imaging of live plant cells. One of the limitations of current AO is the narrow field of correction. To expand this, astronomy developers have studied 3D AO, such as multi-object or multi-conjugate AO.^21–23 AO microscopes are useful for advancing the research and development of 3D AO because of their compact and inexpensive optical systems. In future, we plan to conduct research using 3D AO to expand the correction field of our AO microscope for fine wide-field live imaging of plant cells.

This research was performed by Yutaka Hayano, Shin Oya (National Astronomical Observatory of Japan), Yasuhiro Kamei, Takashi Murata, Shigenori Nonaka, and Mitsuyasu Hasebe (National Institute for Basic Biology). We are grateful for their comments on the article. This research is supported by the National Institutes of Natural Sciences Program for Cross-Disciplinary Study and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (ID: 24113521, 50579290, 50579290, 26282128).