Simultaneous Label-Free Autofluorescence Multi-Harmonic Microscopy

Simultaneous label-free autofluorescence multi-harmonic microscopy, or SLAM microscopy, captures unique information in biological samples by measuring non-linear interactions with light. In our lab, we use this technique to study life and disease at the microscopic scale. We observe a shift toward label-free multimodal imaging, offering richer insights into cellular and tissue structures and dynamics, while avoiding toxicity and enabling complimentary contrasts and correlations across the modalities.

A state-of-the-art label-free nonlinear microscopes acquire one signal at a time, risking misalignment, photo damage and a lot of concurrent processes. The SLAM overcomes these issues by enabling a strictly coregistered multimodal contrast, ensuring reliable spatial and temporal data. This technique enables simultaneous excitation and detection of four channels in a multiphoton microscope.

Using photonic crystal fiber to broaden pulses for SHG, THG, and two-and three-photon fluorescence. SLAM captures multiplexed metabolic structural and dynamic cell signaling information without alignment artifacts or exogenous labels, enabling standardized quantitative tissue analysis. Its applications span fundamental biology, translational research, and biomarker discovery in oncology, neurodegeneration, and tissue regeneration.

To begin, use a spectrometer or optical spectrum analyzer to measure the laser after the parabolic mirror and verify the supercontinuum generation of the photonic crystal fiber. Rotate the halfway plate after the polarizing beam splitter to optimize the width of the supercontinuum. Turn on the microscopy autocorrelator.

In the software, set the detector to the internal detector and adjust the Scan Range to 5 picoseconds to capture the full width of the pulse autocorrelation. Unblock the laser and align the autocorrelator to the beam exiting the pulse shaper until a signal appears. Ensure that shutter 2 is closed.

Then apply the appropriate immersion media, such as water or oil, to the objective lens. Place the autocorrelator external detector on the microscope stage and position it directly above the objective. Now turn on the scanning galvanometer mirrors to set their voltage to 0.

Open the shutter and use a fluorescent target to confirm that the beam reaches the external detector. Set the autocorrelator detector to be the external detector. Then adjust the stage position until the signal appears.

Adjust the polynomial coefficients of the pulse shaper to iteratively minimize the pulse width measured by the autocorrelator. As the pulse width decreases, reduce the scan range accordingly to improve the accuracy and visualization of the autocorrelation function. Ensure that the laser input to the photonic crystal fiber is blocked and turn on the laser.

Allow the laser to warm up for 30 minutes. Verify that the beam path is free of obstructions, then place an optical power meter in the beam path just before the photonic crystal fiber. Press the Control button to open shutter 1 and measure the input power.

Close shutter 1 again. Move the power meter to the output of the photonic crystal fiber after the parabolic mirror. Open shutter 1 and use the input three-axis stage to maximize the output power.

Let the photonic crystal fiber warm up for 10 minutes then maximize the output power again using the three-axis stage and record the transmission of the PCF. Move the power meter after the neutral density filter and rotate the filter to adjust the laser power reaching the sample. Multiply the measured power by the transmittance of the remaining optics to determine sample power.

Now, turn on the galvanometers and stage. Launch the acquisition software and enter the desired imaging parameters. Place the sample on the stage using the same immersion media that was applied earlier to the objective lens.

Then turn off the room lights and close the curtains to prevent ambient light from entering. Next, switch on the detector power supplies and amplifiers. Shut the light box surrounding the microscope stage.

Use the electronic control switches to manually open the detector shutters 3-6. In the acquisition software, press the Click to Start Acquisition option to begin SLAM imaging. Adjust the microscope focus using the stage controller knob until the sample is clearly visible.

Use the joystick on the microscope stage controller to find the desired field of view. Once positioned, click Stop and Reset. Under the Saving tab, toggle Save Data to Yes.

Then press on the Click to Start Acquisition option and wait for the imaging to complete. To manipulate the sample during imaging, close the detector shutters and use a flashlight or headlamp for illumination. After adjustments, return to the imaging setup.

To end image acquisition, close the detector shutters, turn off the detectors, and only then switch on the room lights. Block the laser using shutter 1, then turn off the laser. Turn off the galvanometers and stage and close the acquisition software.

Dispose of the tissue sample appropriately and clean the workstation. A properly cleaved photonic crystal fiber showed a flat end face perpendicular to its axis and a clean face. Poorly cleaved photonic crystal fibers showed angled cuts, chipped faces, and pronounced scratches or debris on the surface.

Improper handling or contamination resulted in visible dirt and particulate buildup around the fiber surface obscuring the air hole pattern. A burned core with darkened regions indicated a damaged photonic crystal fiber at the end of its life. The supercontinuum spectrum spanned approximately 975-1, 175 nanometers with a flat top and distinct spectral modulations.

Pulse autocorrelation after compression showed a symmetrical profile with a narrow full-width half-maximum, indicating good pulse shaping. Properly functioning supercontinuum sources produced strong second and third harmonic signals. A narrowed supercontinuum resulted in loss of third harmonic signal intensity while retaining the second harmonic signal.

Absence of pulse shaping drastically reduced all nonlinear signals, yielding a faint and low contrast image. Successful SLAM imaging of ex vivo kidney tissue showed distinct nephron structures with multiple strong optical signals, including third harmonic generation and NADPH fluorescence. Renal interstitial collagen fibers were identifiable via strong second harmonic generation signals.

The redox ratio image indicated elevated aerobic mitochondrial metabolism in the proximal tubules of the renal cortex compared to the medulla. A second redox ratio map confirmed spatial variation in oxidative and glycolytic metabolism, reinforcing differential energy usage within the tissue.