csiLSFM combines light-sheet fluorescence microscopy and coherent structured illumination for a lateral resolution below 100 nm

Abstract

Light-sheet-based fluorescence microscopy (LSFM) features optical sectioning in the excitation process. It minimizes fluorophore bleaching as well as phototoxic effects and provides a true axial resolution. The detection path resembles properties of conventional fluorescence microscopy. Structured illumination microscopy (SIM) is attractive for superresolution because of its moderate excitation intensity, high acquisition speed, and compatibility with all fluorophores. We introduce SIM to LSFM because the combination pushes the lateral resolution to the physical limit of linear SIM. The instrument requires three objective lenses and relies on methods to control two counterpropagating coherent light sheets that generate excitation patterns in the focal plane of the detection lens. SIM patterns with the finest line spacing in the far field become available along multiple orientations. Flexible control of rotation, frequency, and phase shift of the perfectly modulated light sheet are demonstrated. Images of beads prove a near-isotropic lateral resolution of sub-100 nm. Images of yeast endoplasmic reticulum show that coherent structured illumination (csi) LSFM performs with physiologically relevant specimens.

Keywords: LSFM; SIM; SPIM; light sheet; structured illumination.

Fig. 1.

csi of a fluorescent specimen with counterpropagating light sheets. (A) Two microscope objective lenses are used to illuminate the specimen and at least one microscope objective lens collects the fluorescence light. (B) The two coherent counterpropagating light sheets interfere in the specimen and generate the structured illumination pattern in the focal plane of the detection lens. (C) The interference of two complementarily tilted counterpropagating light sheets generates a structured illumination pattern of the same spatial frequency that is rotated around the optical axis of the detection system.

Fig. 2.

Flexible control of light sheets. (A and B) Top and side views of one arm of the illumination system. The scanning mirror is used to direct the light sheet along two axes as indicated by the dashed lines. (C) Top view of three objective lenses, which lie in a single plane. The two illumination lenses are centered on the common focal point of three lenses and rotated by an angle of ∼25° relative to the focal plane of the detection lens. (D) Top view of the two light sheets. The two scanning mirrors are used complementarily to guide the two light sheets along different angles (52°, 25°, and 0°). (E) Side view of two light sheets recorded through the front window of the specimen chamber. The chamber is filled with 50 μM Rose Bengal in PBS buffer to visualize the fluorescence emission generated by the light sheets with a 543-nm HeNe laser (25-LGP-193–230, Melles Griot). Two light sheets are rotated by an angle of 45° relative to the normal axis of the focal plane. The top view of the directions of two light sheets in E is identical to the image in the middle of D.

Fig. 3.

Flexible control of interference pattern. For demonstration purposes, the scanning mirrors are tilted to direct the coherent beams into the detection objective lens and thus on the camera. (A) Three images demonstrate the generation of different spatial periods in the illumination pattern by changing the relative angle θ_x (defined in Fig. 2) of the scanning mirrors. The interference pattern is observable within a certain angular range, i.e., as long as the beams pass both the illumination and detection objective lenses. (B) Three images show the effect of changing the angle θ_y (defined in Fig. 2) and thus the pattern orientation angles of 49°, 24°, and 157°. (C) Three images show the interference pattern of two light sheets with pattern orientation angles of 0°, 49°, and 133°. The spatial period of the interference pattern is 301 nm. (Scale bars, 1 μm wide.)

Fig. 4.

High-resolution image with counterpropagating light sheets. (A) Conventional wide-field (summedWF), deconvolved wide-field (decWF), and SIM (SIall) images. The beads are embedded in 1.5% low-melt agarose. (B) Magnified images of the red dashed boxes in A clearly show the resolution improvement in the SIM (SIall) image. (Scale bars, 1 µm wide.)

Fig. 5.

Resolution, image quality, and illuminated area at different intersection angles. Conventional wide-field image (summedWF), SIM image (SIall), and the power spectra of the SIM image (PS_SIall). The drawings overlaying the spectra indicate the extended frequency information in the SIall images and the achievable rotation angle of the pattern. The orange circles represent the optical transfer function (OTF) region, whose radius is 2 N.A.∕λ (N.A. = 1.0, λ = 515 nm). The white arrows represent the corresponding illuminated areas. The top view illustrates the configuration of two light sheets at 0° pattern orientation. The intersection angles of the 0° pattern in A, B, and C are 73.4°, 99.8°, and 180°, respectively, which result in pattern periods of 307, 240, and 183 nm, respectively.

Fig. 6.

High-resolution images of GFP-tagged ER structure within live yeast cells embedded in 1.5% low-melt agarose. (A) Maximum-intensity projections of conventional wide-field (summedWF), deconvolved wide-field (decWF), and SIM (SIall) image stacks as well as the bright-field (BF) image of a live yeast cell. (B) Four planes were chosen to allow a more detailed comparison between the summedWF, decWF, and the SIall image. The detailed structure of the ER, which appears to be quite blurry in both summedWF and decWF images, is resolved in the SIM images. The image stack consists of 50 planes with an axial spacing of 200 nm. (Scale bars, 1 μm wide.) (C) The line profiles of the SIM (SIall) image show the ER structure, which cannot be observed in either the summedWF or the decWF image.