This method can help answer key questions in the optical microscopy field, such as how to take super-resolution images as well as FRET images using the same microscope. The main advantage of this technique is that we could combine three different imaging modules into one microscope, significantly reducing the overall cost. Visual demonstration of this method is critical as assembly of the excitation path is difficult to learn.
They require proper choice of parts and a sensitive optical alignments. To begin, install a data acquisition card through a PCI interface and use it to connect the lasers to the computer. Control the lasers'on and off behaviors by transistor-transistor logic output and their power adjustment by the analog output of this card.
Next, prepare a vibration-isolated optical table with mirrors and beam splitters as shown here and described in the accompanying text protocol. Combine the laser beams into a single-mode optical fiber by first mounting a fiber adaptor plate in a z-axis translation mount. Next, mount an achromatic doublet lens in a cage plate.
Use an extension rod to connect the adapter and lens to form a cage. Then, use one-inch thick optical posts to mount the cage on the optical table. Align the 647-nanometer laser by adjusting the components to gain maximum laser power output through the fiber.
Once the alignment of the first laser is done, temporarily install a pair of irises and align the rest of the lasers one by one. Check the alignment efficiency of each laser with a power meter. Be sure to leave one iris in front of the adapter plate to reduce the reflections of the lasers.
Next, design and install the magnification lens as described in the accompanying text protocol. In order to create the astigmatism effect necessary for extracting the z-coordinates of every single molecule, place a 3D lens with a 10-meter focal length into the cassette and insert it into the emission beam path. To minimize the vibrations during sequential multicolor epifluorescence imaging, use emission filters placed in a barrier filter wheel connected next to the microscope.
For simultaneous multicolor detection during single-molecule FRET experiments, place another filter set in an emission splitter. To set up for diffraction-limited imaging using epi-excitation, first adjust the excitation laser's incidental angle to epi mode in the illumination arm. Next, disengage the 3D lens, and insert the bypass cube into the emission splitter.
Then, insert the mag lens for broadened illumination. Once set up, take multi-channel, z-stack, and/or time lapsed images of your sample based on your desired results. To set up multicolor single-molecule detection of surface-immobilized molecules, first move the filter wheel to an empty position.
This will allow the lasers to pass through. Then, adjust the excitation lasers'incidental angle to the turf angle, and disengage both the mag and 3D lenses. Next, engage the three-channel mode in the emission splitter by first replacing the bypass cube with a calibration cube that allows all of the light to pass through all channels.
Then, turn on the camera under DIC and adjust the aperture of the emission splitter until three, fully separated channels appear on the screen. Turn the vertical-horizontal adjustment control knobs on the emission splitter, and roughly align the three channels. Next, turn off the camera and replace the calibration cube with the triple cube.
Place a sample of 100-nanometer multi-channel beads. Upon excitation at 488 nanometers, the 100-nanometer multi-channel beads emit different wavelengths of light, enabling three-channel alignment. Then, turn on the camera and the 488-nanometer laser, zoom in on one of the bright beads, and finally align the three channels by turning the adjustment control knobs again.
With the sample now in place, using a weak laser, navigate to an area with a reasonable spot density and adjust the laser power and exposure time to achieve acceptable signal-to-noise and photobleaching levels. Then use the imaging software to take time-lapse images. For super-resolution imaging, begin by inserting the 3D lens and remove the mag lens.
Then determine the optimal excitation laser's incidental angle to be the turf angle. In order to find the proper objective height for SR imaging, use DIC imaging to find the middle plane of the cells. Identify the plane by the height at which the cells become transparent.
Once the desired focal plane is determined, begin super-resolution imaging. While imaging, change the 405-nanometer laser's power to maintain a reasonable density of blinking-on spots. Begin imaging without violet laser power.
Count the number of blinking-on spots in a certain period and increase the violet laser power so that the number of blinking-on spots is kept above a user-defined counting threshold in the field of view. Analyze the data by detecting the centroids of each spot in the imaging frames and extract z-values for each spot from X and Y widths. Build a reconstructed image and visualize objects in 3D.
This microscope setup allows for flexible and reproducible switching between different imaging methods, including conventional epifluorescent imaging, single-molecule detection-based super-resolution imaging, and multicolor single-molecule detection. In order to reveal finer details in the molecular assembly, super-resolution microscopy combines thousands of images such as this one. These images are then reconstructed to generate a final super-resolution image.
Super-resolution techniques allow for high spatial resolution, providing details that can't be seen with other techniques. This is highlighted in the two images shown here. The super-resolution image shows the same bacterial regulatory RNase as the epifluorescence image but allows for single molecule detection.
SmFRET is another method capable of angstrom to nanometer resolution. Here, folded RNA molecules were labeled with a green donor dye and a red acceptor dye. Fluorescence intensity trajectories can be extracted from individual single molecules, generating FRET efficiency as a function of time.
Don't forget that working with lasers can be hazardous, and precautions such as wearing eye protection or lowering laser powers should always be taken where performing this procedure.
