Our work aims to advance knowledge on the relationship between trans work dynamics, local structure, and mechanical properties in far-from-equilibrium materials. Visualizing far-from-equilibrium materials, such as cytoskeleton networks requires an ability to image dense three-dimensional samples, with comparable image quality throughout the volume of the sample, while causing minimal damage via photo bleaching, a technically challenging task that fluorescence slit sheet microscopy is best suited for. Fluorescence light sheet microscope systems provide excellent optical sectioning capabilities, which allows for imaging of these thick three-dimensional samples for long time scales with minimal photo bleaching.
In particular, the single objective light sheet system we will present, is compatible with traditional slide mounted samples, making it a very versatile tool. The overwhelming majority of single objective light sheet guides, are written for users with extensive optics experience. To make the complex single objective light sheet style set up more accessible, this detailed guide provides steps that a user with an entry level optics course depth of knowledge can follow.
To begin, ensure that the microscope layout is prepared on the surface of the optical table with all the distances accurately measured. Then, mount the excitation laser on the table. Set two irises to the intended height of the laser, and use these irises to ensure that the beam is level and centered.
Position the translation stage, or TS one under the location of mirror one or M1.Use the pair of viruses set to the exact height to define the desired exit beam path, and guide the placement and alignment of each reflective element. Next, position M1 at the top of the TS one. Then, mount and align the dichroic over the table.
Similarly, mount the galvo, and align it with the irises. Once the alignments are made, position M two, then clamp M three to the table. Adjust the height and position until the beam is roughly centered on both frosted glass alignment discs.
Add supports to M three then. Next, start mounting lens one one the table. Adjust the tilt and lateral position until the beam is centered on the frosted glass plates above M three.
Position lens two, and check the collimation by using a mirror to bounce the beam onto a faraway surface. Use an index card or target to trace the beam, and ensure that the beam does not change in size. Then, adjust the X, Y position of the pinhole with the XY mount, and the axial distance with the one dimensional stage to maximize the transmission.
Adjust lens four axially to focus the excitation beam on the surface of the galvo, and arrange lens three onto the table, followed by SL1. Adjust the axial distance of SL1 to form a collimated telescope with lens four. Then possession TL one parallel to SL1.
Adjust the height of objective one on the cage system until the beam forms an airy disc on the ceiling. And then continue adjusting until the size of the disc is minimized. Place the square mirror on the sample stage of objective one, and adjust the mirror axially until the size of the beam profile is minimized after the dichroic.
Mount the alignment laser by sliding the cage rods into the empty holes of the two cage plates. Use one kinematic mirror mount, and one dropdown mirror to align the paths of the alignment and excitation beams. Position the square mirror axially on the sample stage of objective one to minimize the beam profile after the dichroic.
Insert SL2 and TL2 in the emission path at the respective distances, adjust the X, Y knobs in tilt of objective two so that the red alignment beam passes through the iris and frosted glass disc. Adjust the translation stage two until the beam forms a small airy disc on the surface, and then continue adjusting translation stage two to minimize the size of the airy disc. To optimize the galvo for tilt and variant scanning, press the FSK button on the waveform generator to select a triangle wave signal for the galvo, and set it to a low frequency such as one hertz.
Observe the alignment beam on the same faraway surface or wall. Mount objective three, about four to five millimeters in front of objective two at a zero degree angle, and adjust the height to match. Position a frosted glass alignment disc in the shared focal plane between SL2 and TL2, as measured with a ruler.
Once the emission light fills the back aperture of oh three, mount a frosted glass disc in the rough position of the camera sensor, and align the center of the disc to the emission light exiting oh three. Position TL3 behind objective three, and adjust the tilt to align the outgoing light with the frosted glass disc. Place the camera at the measured distance from the tube lens, and adjust objective three axially from the cage translation stage, until the hole is in focus on the camera.
Readjust objective three at a 30 degree angle from the optical axis of objective two, using the lines on the table as a guide. Remount the positive grid test target at the same axial height, and illuminate the grid with the bright field light. Sweep the in-focus portion of the field of view across the screen horizontally while the grid squares maintain a uniform size.
To align the oblique light sheet, position cylindrical or CL, so that the beam is focused into a horizontal sheet profile at the focal plane of CL3. Insert and position the slit in the vertical orientation at the focal plane between CL3 and lens three. At the camera sensor, confirm that the zero degree light sheet appears thin and vertical.
Using the motorized translation stage control, translate M1 toward the cylindrical lenses to angle the light sheet. Insert the pre-prepared rhodamine labeled micro tubule test sample onto the sample stage, and adjust it axially so that the dye is illuminated by the light sheet at five different depths between the center of the field of view, and the right side of the screen. Then, save each image.
Open the images in Fiji. For each image, using the line tool, draw a horizontal line from the center of the field of view to the center of the light sheet. Then, go to Analyze, followed by Plot profile, to calculate the angle of the light sheet above oh one.
After calibrating the instrument, mount the pre-prepared three-dimensional bead sample, and click the FSK button on the function generator to set a triangle wave. To find the sample, use the function generator to set the parameters starting from 20 millihertz frequency, 400 millivolts peak to peak amplitude, and 0.4 offset. Scroll in Z manually, until the sample plane is reached, and optimize the Z setting.
In the micromanager program, select an exposure time, and open the multi-dimensional acquisition window. Set the interval to 30, and use the count box to choose the number of frames. Once the parameters are set, record a time lapse for one full scan of the volume.
The volumetric scans of the reconstituted microtubial network, showed that the three dimensional structures grew dense toward the center, resulting in bright regions of fluorescence. In imaging planes near the cover slip, confocal microscopy resolved single filaments around the periphery of the astor, with additional background toward the center due to out of focus fluorescent signals from above. However, moving a few microns in Z, quickly reduced the quality of the images due to the out of focus dense sections of the astor.
The single plane illumination of the light sheet eliminated the out of focus signals, allowing comparable image quality between the planes.
