Development of Planar Illumination Strategies for Solving Mysteries in the Sub-Cellular Realm

Abstract

Optical microscopy has vastly expanded the frontiers of structural and functional biology, due to the non-invasive probing of dynamic volumes in vivo. However, traditional widefield microscopy illuminating the entire field of view (FOV) is adversely affected by out-of-focus light scatter. Consequently, standard upright or inverted microscopes are inept in sampling diffraction-limited volumes smaller than the optical system's point spread function (PSF). Over the last few decades, several planar and structured (sinusoidal) illumination modalities have offered unprecedented access to sub-cellular organelles and 4D (3D + time) image acquisition. Furthermore, these optical sectioning systems remain unaffected by the size of biological samples, providing high signal-to-noise (SNR) ratios for objective lenses (OLs) with long working distances (WDs). This review aims to guide biologists regarding planar illumination strategies, capable of harnessing sub-micron spatial resolution with a millimeter depth of penetration.

Keywords: axially swept light sheet; lattice light sheet; light sheet microscope; oblique plane illumination; single-molecule localization light sheet; sub-voxel resolving technique; super-resolution.

Figure 1

LSM beam profile characteristics. (A) LSM single lobe and multilobe top-view and side-view intensity distributions [84]. (B) Gaussian light sheet hyperbolic beam profile, D = lens diameter, Zr = Rayleigh length, w0 = beam waist, f = focal length of objective lens [83].

Figure 2

Illustration of ASLSM. (A) Schematic of ASLSM. Sample is illuminated with a laser from the y-direction and the fluorescence is taken perpendicular from a camera. L_R is the Rayleigh length of the beam. The axial position of the beam is swept in the y-direction with a 2D array of pixels to create an FOV image. (B) Image of fluorescently labeled collagen with NA = 0.8. (C) Image (B) with high optical sectioning. (D) Image of fluorescently labeled collagen with NA = 0.29. (E) Image of (D) with high optical sectioning. (B–E) Scale bars are 10 µm [87].

Figure 3

Two illustrations of OPM. (A) The light sheet (blue) is from the primary objective lens illuminated at an oblique angle. (B) Traditional OPM—Primary objective lens fluorescence is replicated when secondary objective lens crosses with tertiary objective lens. (C) Optimal microscope resolution (from α to β) created when light moves from a low refractive index, η₁, to a high refractive index, η₂. (A–C) [14] (D) Zebrafish tail at 24 hours post-fertilization with mCherry-labeled histones. (E) Side projection of light sheets entering sample at 45 degrees. (F) Example regions where (left) View 1 has better image quality, whereas (right) View 2 has better image quality. (G) Fusion of View 1 and View 2 with better image quality. (H) Time-lapse of dorsomedial tail. (I) Spatio-temporal image of a cell division. (D–I) [95].

Figure 4

Illustration of how aberration laser beams (ALBs) were produced and propagated in free space and turbulence after passing through an incoming excitation Gaussian beam into a diffractive optical element (DOE) [77].

Figure 5

This figure shows separated muscle fibers (examples are shown with green arrowheads) and vascular endothelial cells (examples are shown with magenta arrowheads) that express DsRed-CLTA. The upper right corner shows 75 µm by 99 µm by 41 µm of the tail of a developing zebrafish, whereas the bottom right corner shows a graphical depiction of clathrin puncta in the endothelial cells [98]. Scale bars are 10 µm.

Figure 6

Visualization of neurons on Tg:thy1-GFP-M mouse brain. (a) 4× imaging on benchtop SPIM with a voxel size of 1.625µm × 1.625µm × 6 µm. (b) 4× imaging with multiview SPIM with a voxel size of 1.625µm × 1.625µm × 1.625µm. (c) 4× imaging with Mars-SPIM with a reconstructed voxel size of 0.41µm × 0.41µm × 0.41µm [104].