LSFM variants and their associated illumination and detection optics.

The table lists the type of microscope, the illumination and detection optics-including NA where available and immersion type in parentheses-as well as the overall design architecture (e.g., rail carrier, cage system, etc.).

(a) Rendering of the detection arm elements. (b) Zemax Opticstudio layout and beam path of optimized illumination arm, where L1 is an f = 30 achromatic doublet, L2 is an f = 80 mm achromatic doublet, L3 is the f = 75 mm achromatic cylindrical doublet, and ILO is our TL20X-MPL illumination objective. (c) The simulated light-sheet beam profile in the xz plane at the focus of the illumination objective. (d) The cross-sectional profile through the center of the light-sheet beam profile in (c), where the FWHM of the light-sheet was found to be 0.382 µm.

(a) Rendering of the completed illumination arm baseplate, with an inset showing the dowel pin holes compatible with the Polaris mounting line from Thorlabs. (b) Overhead view of the imaging configuration of our system, where our detection objective and illumination objectives are placed orthogonal to each other and the sample is scanned diagonally in the space between them in the axial direction shown by the white dashed line. (c) Rendering of our sample mounting and translation system. Here, a piezo motor is mounted onto an angled adapter to allow precise translation over the diagonal region between the objectives. Our custom 5 mm coverslip sample holder is also featured, where the inset shows an exploded assembly of the holder.

(a) Experimental light-sheet beam profile at the focus (b) The center cross-section profile of (a), showing both raw data and a fitted curve with a FWHM of ∼0.415 µm. (c-e) Max intensity projections for an isolated 100 nm fluorescent bead in each of the 3 orthogonal planes (f) Gaussian-fitted distribution of the FWHM of beads imaged in a z-stack in each dimension both before (solid) and after (dashed) deconvolution.

Lateral maximum intensity projections of mouse embryonic fibroblasts (MEFs) fluorescently labeled with nuclei (cyan), tubulin (gray), actin (gold), and the Golgi apparatus (magenta).

(a) Maximum intensity projection of the full z-stack in the xy plane. (b–e) Individual channels corresponding to (a): (b) nuclei, (c) microtubules, (d) actin, and (e) Golgi apparatus. (f) Maximum intensity projection of a second z-stack in the xy plane. (g–j) Individual channels corresponding to (f): (g) DAPI, (h) microtubules, (i) actin, and (j) Golgi apparatus. Nuclei were labeled with DAPI, actin filaments with phalloidin, and both microtubules and the Golgi apparatus were stained using indirect immunofluorescence.

Function of the resonant galvo unit, where without dithering, objects in a sample can cast shadows.

Incorporation of dithering at a high speed over the image acquisition time effectively averages the effects of these shadows out of the final image.

(a) Depiction of the merit function criteria used in our tolerance analysis, where we observed how the beam profile in the perturbed instances changes in both size and position. (b) Schematic of the Polaris dowel pin mounting configuration when considering machining tolerances, where in a worst-case scenario the angle offset would be 1.454 degrees. (c) Nominal, best case, and worst-case beam profiles in xz for both coarse (+-0.005”, top row) and fine (+-0.002”, bottom row) machining tolerances.

Process of affixing a post to the baseplate, where one first places the post onto dowel pins inserted into the corresponding holes and then fixes the post to the baseplate with a screw.

(a) CAD rendering of our custom sample chamber, featuring three possible objective ports, each with two sets of O-rings to ensure a liquid-proof seal. (b) Top-down rendering of the traditional imaging configuration for the system, where the illumination and detection objectives are placed orthogonal to one another. (c) The second imaging configuration of the system used to image the beam itself, where the illumination objective is place directly in front of the detection objective.

General wiring diagram of the system showing all of the optoelectrical and optomechanical components used, and an inset showing how these components are wired into the NI DAQ.

Electrical pinouts used on National Instruments PCIe-6738 data acquisition card.

All analog and digital connections were made using a National Instruments SCB-68A shielded terminal block.

Detailed equipment list.

Prices are approximate and subject to change-often unpredictably due to economically self-defeating trade wars. *Indicates that the part was custom ordered from Thorlabs.

Approximate Cost.