Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy

Our protocol presents a multimodal, nonlinear, optical microscopy and applies it to perform chemically-specific imaging of gold nanoparticles in cancer cells. The main advantage of our imaging system is that it can be used to review the biomolecular contrast of cellular structures and gold nanoparticles in a multimodal manner. To begin, switch on the plugs of all the equipment, except the remote control box.

Then, switch on the back of the box and press Start Operation after the remote light goes blue on the control box. Afterward, turn on the PC of the laser scanning microscope and run the confocal microscope software. Then, check the light path of the microscope using the computer software and control the focus through the touch screen or the remote control knobs or the computer software.

Select the desired image settings in the software, including Zoom, the Image Size in pixels, and pixel dwell time, while ensuring that the pixel dwell time is greater than the integration time. Place a droplet of distilled water on top of the water immersion objective that focuses the laser beams into the sample and between the condenser and the sample for imaging. Then, adjust the height of the condenser to approximately one millimeter above the sample to collect the maximum amount of light and move the SRS detector placed on a movable mount out of the beam path to focus on the sample using white light.

Using the software settings, select the pump beam and change the laser wavelength to 802 nanometers for CH vibrations and 898 nanometers for amide I peak, accounting for large changes in the Raman shift. To achieve small adjustments in the Raman shift, scan the delay stage in the SFTRU unit as described in the manuscript. For the hyperspectral data set, select the Trigger tab, click on Start by ttl trigger on, and then select Trigger every frame.

Next, in the Series tab, set the time series on and the Z series off. Furthermore, set the number of frames in the time series to match the number of steps in the ATM software. Following, go to the Run tab in the ATM software and set the delay stage positions for CH at 90.25 and 92.25, whereas for the amide I region at 89 and 91 in millimeters, corresponding to the start and stop position of the hyperspectral scan, while ensuring that the number of steps matches the number of frames in the computer software.

After checking the correct settings, start the hyperspectral stack in the computer software by first going to Acquire and then clicking on Start scan. Then, in the ATM software, click on Start, which initiates the collection of a series of images with incremental increases in the delay stage position between the selected start and stop positions. Then, take a time series of images at different delay stage positions to generate a hyperspectral scan.

Using the analog unit, send and receive ttl signals to and from the SFTRU system to synchronize image acquisition and movement of the delay stage. For switching between imaging in the CH vibrational region to the amide I vibrational region, change the pump wavelength in the laser software from 802 nanometers to 898 nanometers. Further, change the Stokes dispersion setting in the ATM software from 30 millimeters to 5 millimeters.

Adjust the lower knob on the mirror mount to make a small adjustment to the mirror in the pump beam dispersion pathway inside the SFTRU box by changing the mirror angle with an incremental amount. To validate the chemical selectivity of the polystyrene microspheres, hyperspectral stimulated Raman scattering and spontaneous Raman spectra were recorded. The spectra were identical except for the difference observed in the relative intensity.

The stimulated Raman scattering imaging of 4T1 cancer cells was performed at 2, 852, 2, 930 and 2, 968 wave numbers, corresponding to the carbon-hydrogen stretching vibrational band of lipid, protein, and DNA biomolecules. Multimodal imaging of 4T1 cancer cells dosed with gold nanoparticles was performed to investigate the nanoparticle distributions in cancer cells. The acquired stimulated Raman scattering images were obtained for CH2 and CH3 channel, LysoTracker fluorescent probes, and off-resonance stimulated Raman scattering channel.

The most important thing to remember is to change the vibrational region, the pump reference, in the laser software and also the Stokes dispersion setting during the protocol. This system can be easily switched from femtosecond to picosecond region without affecting optical alignment. This capability allows us to perform multiphoton coherent Raman scattering and pump probe spectroscopy and their applications.

This multimodal imaging platform provides new insights into nanomedicine and paves the way for molecular imaging of cells, tissues, and gold nanoparticles.