imaging set-up and volumetric scans for cytoskeleton network visualization

Design and Building of a Customizable, Single-Objective, Light-Sheet Fluorescence Microscope for the Visualization of Cytoskeleton Networks

Imaging Set-Up and Volumetric Scans for Cytoskeleton Network Visualization

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 TS1, under the location of mirror 1, or M1.Use the pair of irises 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 TS1. 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 M2 then clamp M3 to the table. Adjust the height and position until the beam is roughly centered on both frosted glass alignment discs.Add supports to M3 then. Next, start mounting lens 1 on the table. Adjust the tilt and lateral position until the beam is centered on the frosted glass plates above M3.Position lens 2 and check the collimation by using a mirror to bounce the beam onto a far away 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 4 axially to focus the excitation beam on the surface of the galvo and arrange lens 3 onto the table, followed by SL1.Adjust the axial distance of SL1 to form a columated telescope with lens 4, then position TL1 parallel to SL1. Adjust the height of objective 1 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 1 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 1 to minimize the beam profile after the dichroic. Insert SL2 and TL2 in the emission path at the respective distances. Adjust the XY knobs and tilt of objective 2 so that the red alignment beam passes through the iris and frosted glass disc.Adjust the translation stage 2 until the beam forms a small airy disc on the surface, and then continue adjusting translation stage 2 to minimize the size of the airy disc. To optimize the galvo for tilt in 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 1 hertz. Observe the alignment beam on the same far away surface or wall.Mount objective 3 about 4-5 millimeters in front of objective 2 at a 0 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 O3, 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 O3.Position TL3 behind objective 3 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 3 axially from the cage translation stage until the hole is in focus on the camera. Readjust objective 3 at a 30 degree angle from the optical axis of objective 2 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 lenses, or CL, so that the beam is focused into a horizontal sheet profile at the focal plane of CL3. Insert and position a slit in the vertical orientation at the focal plane between CL3 and lens 3.At the camera sensor, confirm that the 0 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 microtubule 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 01. 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 megahertz 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 multidimensional 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 microtubule 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 Astar 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 Astar. The single plane illumination of the light sheet eliminated the out-of-focus signals, allowing comparable image quality between the planes.

Research

JoVE Journal

Biology

Customizable Fluorescence Microscope to Visualize Cytoskeleton Networks

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