Automatic and adaptive heterogeneous refractive index compensation for light-sheet microscopy

Optical tissue clearing has revolutionized researchers’ ability to perform fluorescent measurements of molecules, cells, and structures within intact tissue. One common complication to all optically cleared tissue is a spatially heterogeneous refractive index, leading to light scattering and first-order defocus. We designed C-DSLM (cleared tissue digital scanned light-sheet microscopy) as a low-cost method intended to automatically generate in-focus images of cleared tissue. We demonstrate the flexibility and power of C-DSLM by quantifying fluorescent features in tissue from multiple animal models using refractive index matched and mismatched microscope objectives. This includes a unique measurement of myelin tracks within intact tissue using an endogenous fluorescent reporter where typical clearing approaches render such structures difficult to image. For all measurements, we provide independent verification using standard serial tissue sectioning and quantification methods. Paired with advancements in volumetric image processing, C-DSLM provides a robust methodology to quantify sub-micron features within large tissue sections.

Gaussian light-sheet was laterally translated using excitation electro-tunable lens (ETL-1) (black lines with arrows). ETL-1 4 was placed at a telecentric position to the back aperture of the excitation objective by physically translating the optic along 5 the optical axis between the fiber coupler and the galvanometer mirrors until this position was determined. The physical 6 location of the light-sheet was altered in-plane or axially (blue line with arrows) using the two-dimensional scanning unit.

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The unit consisted of the scanning mirrors, scan lens (f scan = 70 mm), and tube lens (f tube = 200 mm). The position of the 8 detection plane was altered using a 4f relay system containing the detection electro-tunable lens (ETL-2). The optimal 9 position of the objective and tube lens were determined for each objective by calculating L:

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Where is the entrace pupil of the tube lens, is the exit pupil of the detection objective (DO), F 2 is the focal length 13 of the tube lens, and is the image field diameter of the detector.

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The maximum relay lens focal length (f relay = 300 mm) that can be used while maintaining the full NA of the detection 16 objective was determined in a similar manner by calculating f relay,max :

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Where is the entrance pupil of the ETL placed in the imaging arm and is the exit pupil of the detection 20 objective. ETL-2 is placed on an xyz translation stage to ensure that the optical axial precisely aligns with the middle of the lens and that the optic is located at the correct plane within the 4f system.

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terms at a given wavelength (), refractive index (n), and the smallest distance that can be resolved by the detector (e).

Supplementary Note 2 89
Effective displacement of the detection electro-tunable lens (ETL-2). Because the excitation and detection arm of the C-

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In theory, these equations and the response curve of the ETL-2 can be used to encode the required hardware settings for 96 any given sample RI. However, we find imaging specific calibration planes and fine-tuning the hardware is the best 97 approach to ensure the focal plane of the detection arm coincides with the axial location of the light-sheet because the 98 index of refraction, n, in this equation is not constant.

00 Supplementary Note 3 01
Detailed initial cleared tissue digital scanned light-sheet microscopy (C-DSLM) alignment. After C-DSLM construction and 02 alignment of the scanning mirrors and scan lens, it is necessary to align the 4f configuration to ensure that the detection 03 arm is truly telecentric and directed through the center of the detection electro-tunable lens (ETL-2). We perform 04 alignment using two calibration samples, a grid target for coarse alignment and a microsphere bead sample suspended in 05 agar for fine alignment.

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We place a grid target (Edmund Optics) with spacing of 1 mm between lines at the working distance of the detection 09 objective and back illuminate it with a light emitting diode (LED) white-light source. We then manually displace the grid 10 target using the automated stage, refocusing using ETL-2, and compare the magnification grid using the correct pixel size

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Using a multi-band emission filter (Semrock), we locate an area of isolated 0. has cooled, the sample is mounted onto the RSSM, and the focal position of the light-sheet is set identically for both the 46 488, 532, and 640 nm lasers using ETL-1. Ensuring that the cuvette surfaces are perpendicular to both the excitation and 47 detection objective is critical for this step and we utilize the fine manual adjustment of the rotation stage to minimize the 48 distortion in the light-sheet before proceeding to imaging.

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Using a multi-band emission filter (Semrock), we locate an area of isolated microspheres and apply a slow sinusoidal 51 signal (0.25 Hz) to ETL-2 over a range large enough to axially traverse the size of the 15 m FocalCheck microspheres.

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We utilize the two steering mirrors to ensure the fluorescence in the 488 nm channel forms a perfect ring around the 53 fluorescence in the 640 nm channel. We similarly iterate for each pair of 488, 532, and 640 nm lasers.

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Finally, we manually calibrate an image stack and scan through the entire focal range of ETL-2 for each color to check the 56 calibration over the entire range. This procedure is repeated until chromatic aberrations are minimized across the full 57 range of the focal stack.