Our method allows us to visualize single molecules with a 40 times larger image area than conventional methods, and has a greatly reduced background signal. Unlike other techniques our approach uses a single objective lens, does not require a special sample chamber, and it is easily compatible with commercial microscope systems. After gathering the necessary components start building the system.
Here, several elements are already in position on the bench. Mounted on an aluminum block is a microscope body with a Piezo stage and sample holder. Also on the bench are three lasers and optical elements to combine the beams.
The combined beams are coupled to a single mode fiber as efficiently as possible. As a final preliminary step, assemble a collimated light source in a cage system. This has a temporary coherent light source connected to a single mode fiber, a fiber adapter, achromatic lens and iris.
Begin work with the detection path. First, place the collimated light source in the microscope body's objective port. Adjust the mirror beneath the objectives so the output beam is roughly horizontal and aligned with the bench's holes.
Insert a dichroic mirror in the beam path and reflect the beam by 90 degrees. Add mirrors to allow adjustment of the transmitted beam's path to an sCMOS camera. Use the mirrors to ensure the beam hits the center of the chip.
Next, insert a 300-milliliter focal length tube lens about a focal length from the camera. Remove the collimated light source from the microscope body. Then adjust the position of the tube lens in order to set its focal plane.
Stop the adjustments when the camera clearly resolves the pattern on the ceiling. The detection path is depicted in this schematic. Note the addition of a multi-band pass filter before the tube lens for multicolor fluorescence imaging.
Return to the microscope body. There, reinstall the collimated light source in the objective holder. After the dichroic mirror, place a fold-mirror in the path of the reflected beam to redirect it by 90 degrees.
Remove the collimated light source from the microscope body and place it nearby. Next, insert a 400-millimeter focal length lens at about a focal length from the mirror. At the microscope body install the objective lens.
Then adjust the position of the mirror along the optical axis. Observe the ceiling above the objective and stop when a perfect Airy disk pattern is formed there. Now remove the objective and reinstall the collimated light source with an open iris.
Determine where along the beam path the beam is smallest at about 400 millimeters away from the mirror. Once the point is identified, mount a mirror there, a conjugated image plane. The state of the set up at this point appears in this schematic.
The mirror just added is labeled M5.Insert another mirror to reflect the beam 90 degrees and mount a 150-millimeter focal length lens beyond it. The lens should be 150 millimeters from the conjugated image plane. Next, temporarily take away the first lens in the excitation beam path.
With this configuration find the focal position of the second lens. Place a single axis galvo mirror at this focal point as a conjugated back focal plane. Supply zero volts to the galvo mirror and rotate its holder so that it reflects the beam 90 degrees.
Next along the beam path, place a fold-mirror that reflects the beam at 90 degrees. Place the lens 100 millimeters along the beam path from the conjugated back focal plane. Once again, remove the collimated light source from the microscope mount.
Add another 100-millimeter collimation lens with a fiber adapter and single mode fiber along with an iris. Then use the iris, fiber and lens to send a collimated beam through the imaging system. Next, insert a cylindrical lens of focal length 400 millimeters just before the last collimation lens in the beam path.
Use it to focus the beam. Insert a 50-millimeter cylindrical lens 450 millimeters in front of the first. This is a schematic representation of the final setup with both the detection and excitation paths.
The output of the three lasers is coupled into the path by the single mode fiber. For testing prepare a 3D hydrogel sample. This one consists of 20-nanometer crimson beads mixed with hydrogel.
At the setup place the hydrogel sample in the sample holder. For sample excitation turn on the 638-nanometer laser and adjust its power to less than one milliwatt. Set up the camera control software in internal trigger mode and capture video.
At this point the galvo motor has zero volts applied. Position the camera so that the image is at the center of the lens. Next, adjust the mirror that is the conjugate image plane.
Rotate the horizontal knob on the mirror in order to achieve a highly-inclined illumination. Record the tile image as in the sample fluorescence image of 3D hydrogel with 20-nanometer beads. The scale bar is 20 micrometers.
Proceed after setting up hardware and software to sweep the galvo mirror. This is the galvo mirror sweep software for the setup. Arrange for full field-of-view imaging by setting Vmin to minus 500 millivolts.
Then set Vmax to 500 millivolts. Next, switch to the camera acquisition software. In Trigger Mode select External.
Under the LightScan PLUS drop-down menu select Down Next, click Scan Speed Control. Certain control parameters can now be set. Under Window Height enter 180 rows.
Under Exposure enter 28 milliseconds. When done, return to the mirror control software. In the mirror control software switch on 3D stacks ON.Specify the number of stacks and the step size.
Click take video to start recording images. This is an example of Epi imaging of single-stranded labeled DNA. By contrast, the highly-inclined swept tile technique produced this image.
The HIST image shows less background compared with the Epi image. The DNA is in a 3D hydrogel, and the excitation wavelength is 638 nanometers. Here is an Epi image of labeled eukaryotic translation elongation factor 2.
The HIST image has improved signal-to-background ratio. It also has more uniform illumination. Both have the same illumination power of 7.5 milliwatts.
The imaging speeds were both 2.5 frames per second. It is important to constantly keep a high incline angle, and synchronize the sweeping mirror with the camera readout. This guarantees a high signal-to-background ratio during image acquisition.
We expect that HIST microscopy will benefit several applications including super-resolution imaging at deeper imaging depths and high-throughput gene expression profiling in thick tissue slices.
