Journal: eLife

Article Title: Expansion-assisted selective plane illumination microscopy for nanoscale imaging of centimeter-scale tissues

doi: 10.7554/eLife.91979

Figure Lengend Snippet: ( a ) Schematic of the ExA-SPIM system. Light enters the system from the laser combiner and is reflected by mirror M1. A cylindrical lens focuses the light in one dimension onto the surface of a tunable lens, which is magnified onto the back focal plane of the excitation through a 1.5×relay consisting of lenses L1 and L2 and mirrors M2 and M3. The excitation objective is oriented vertically and dipped into a liquid immersion chamber. The tunable lens is conjugated to the back focal plane of the excitation objective to enable axial sweeping. A pair of galvo mirrors is used in tandem to translate the position of the light sheet in z (along the optical axis of the detection objective). The detection objective is oriented horizontally. A beam splitter is removed from the lens and replaced with approximately 35 mm of water. A large-format CMOS camera captures images from the detection lens, at a back focusing distance of 50 cm. ( b ) The field of view of the system is 10.6×8.0 mm (13.3 mm diagonal), which is digitized by the camera into a 151-megapixel (MP) image with 0.75 µm/px sampling. Although the optical resolution of the detection lens is ~1.0 µm, the sampling limited resolution based on the Nyquist criterion is ~1.5 µm. The large field of view dramatically reduces the need for tiling. For example, a 3× expanded mouse brain can be captured in only 15 tiles. Representative images of a three expanded mouse brain are shown with a 1 cm scale bar. ( c ) The PSF for 561 nm excitation is shown in the xy , xz , and yz planes. The mean and standard deviation of the lateral and axial full-width half-maximum are shown as a function of x and y position across the full field of view. ( d ) The field curvature and distortion of the system as a function of field position is shown for different wavebands. The field curvature is <2.5× the depth of field (DoF) for all wavebands. This performance is better than ‘Plan’ specified life sciences objectives . ( e ) The relative signal-to-noise ratio (rSNR) of the VP-151MXCMOS camera and an Orca Flash V3 sCMOS camera as a function of imaging speed. The VP-151MX camera provides equivalent SNR at nearly twice the imaging speed.

Article Snippet: A bead phantom (see point spread function measurement) was used to compare the sensitivity of the large-format CMOS camera and a scientific CMOS camera (Orca Flash V3, Hamamatsu).

Techniques: Sampling, Standard Deviation, Imaging

Journal: eLife

Article Title: Expansion-assisted selective plane illumination microscopy for nanoscale imaging of centimeter-scale tissues

doi: 10.7554/eLife.91979

Figure Lengend Snippet: ( a–c ) A traditional cleared-tissue SPIM system uses standard 100 mW or less lasers, a scientific CMOS camera, and life sciences objectives with higher NA and <10 mm working distance. In theory, these systems can image an entire cleared mouse brain at 500 nm or less resolution without any physical cutting. However, this would require 400+ individual image tiles and high camera framerates, which is problematic for techniques such as axial sweeping. (bd) By comparison, the ExA-SPIM system uses 1000+ mW lasers, a large-format CMOS camera, and electronics metrology lenses with a moderate NA and ~35 mm working distance. After expanding a mouse brain 3×, the system is still capable of imaging the entire brain in only 15 tiles.

Article Snippet: A bead phantom (see point spread function measurement) was used to compare the sensitivity of the large-format CMOS camera and a scientific CMOS camera (Orca Flash V3, Hamamatsu).

Techniques: Comparison, Imaging