Super-Resolution Imaging and Shared Management: A Protocol for Confocal Microscopy with Multiplex Detection

Our research focuses on developing accessible super-resolution imaging methods to visualize dynamic subcellular structures, overcoming the diffraction limit of conventional microscopy. The super-resolution method face phototoxicity, complex operation, and specialized preparation. This protocol reduces these barriers through confocal-based multiplex detection.

To begin, power on the microscope main unit, the laser launch module, the LED light source, and the control computer. Launch the microscope operating software. From the main interface, select GFP, then choose turn the reflected light off.

Navigate to the Acquisition panel. From the objective lens dropdown menu, select the appropriate objective lens. For live cell imaging, 45 to 60 minutes before imaging, activate the environmental chamber and the active focus stabilization system.

After the system is stabilized, verify the stability using the chamber sensors. Apply immersion oil on the objective's front lens without introducing bubbles. Place the prepared sample on the microscope stage.

Raise it carefully until the coverslip contacts the immersion oil. Next, open the acquisition software. Go to the Acquisition tab and select create a new experiment.

Now, add imaging tracks for the fluorophores used. For each track, set the confocal pinhole diameter to one air unit. To optimize the laser power, select the first channel and switch to live scanning mode.

Set the initial laser power to a low value, then set detector gain to a moderate level within its linear range. After that, increase the laser power gradually until the target structures are clearly visible above the background in the live image. Set the pixel size using the software's Nyquist sampling button to calculate it automatically.

Next, set the pixel time. Now, select the sequential scan mode between tracks to minimize crosstalk. Click Snap to acquire a single image.

Save the image with relevant metadata. For consistent settings across optical sections, confirm that all imaging parameters are configured on a single optimal focal plane before defining the Z-stack. Open the Acquisition tab and select the Z-stack mode.

Define the volume of interest by using a fast speed live scan to focus first on the bottommost structure of interest, then click Set Last or Bottom. Carefully adjust focus up to the topmost structure of interest and click Set First or Top. The software will then display the total depth.

The Z-stack size is set to meet the Nyquist sampling criterion. Use the Optimal button to calculate it automatically. After configuring the imaging parameters for each channel, increase laser power or gain slightly for weak signals compared to a single two-dimensional image.

Start the acquisition to collect the entire image stack. For visualization, use the microscope's native processing module. Generate maximum intensity projections to view all in focus signals in a single two-dimensional image.

Then perform volume rendering using the softwares built-in algorithms to generate a three-dimensional representation. Adjust the brightness, opacity, and lighting as needed. Subsequently, generate orthogonal XZ and YZ cross-sections to inspect axial extent and registration.

Multicolor fluorescence imaging of fixed COS-7 cells showed that the nuclei, mitochondria, and microtubules were clearly labeled with minimal overlap, confirming effective sequential scanning and optimized detection. The maximum intensity projection integrates signals across all optical sections, highlighting the spatial distribution of fluorescently labeled structures. A three-dimensional reconstruction provides a depth aware view of the sample architecture.

Time-lapse imaging of live Hep 3B cells with EGFP tagged mitochondria is compared across modes. Confocal frames across time points show clear structures, but gradual signal loss. Standard super resolution or SR frames show higher resolution, but increased photo-bleaching over time.

Fast super resolution or SR-4Y images preserve stable fluorescence even at later time points. The graph of mean EGFP fluorescence intensity over 100 time points demonstrates that the SR-4Y mode maintained the most stable signal. Imaging performance in fixed COS-7 cells was compared across modes, and super-resolution modes provided clearer structural detail than confocal.

Measuring the point spread function using 100 nanometer fluorescent beads showed that Airyscan SR with subsequent joint deconvolution improved both lateral and axial resolution compared to Airyscan SR alone. This protocol allows researchers to study organelle morphology, cytoskeleton dynamics, and molecule colocalization in both fixed and live cells at approximately 120 nanometer resolution. The most critical consideration of this protocol is meticulous sample preparation using high-quality cover glass and optimized staining to ensure signal integrity for super-resolution processing.

Following this procedure, researchers can perform quantitative image analysis, such as measuring signal-to-noise ratio or colocalization, using dedicated the software like ImageJ.