*2.10. Multi-Photon Imaging*

CT26WT cells were seeded on a glass slide at a concentration of 1 <sup>×</sup> 105 cells/mL and cultured for 24 h. bi-HNTs were added to the cell culture plate (25 μg/mL) and continually incubated for 12 h. DiOC18(7) (DiR) (ThermoFisher Scientific) working solution was prepared by following the company provided protocol. Cells were washed with DPBS for 3 times, then the DiR working solution was added into cell culture well (1 μL/mL) and incubated for 2 h. Cells were then fixed by 4% paraformaldehyde (Sigma Aldrich, St. Louis, MO, USA) for imaging by an in-house assembled multi-photon microscopy. Three-dimensional images were acquired using a Vivo-2 upright, multiphoton microscope system (Intelligent Imaging Innovations, Inc. (3i), Denver, Colorado, USA) with GaAsP photomultipliers (Hamamatsu, Tokyo, Japan), a Pockels cell (Conoptics, Inc., Danbury, CT, USA) to control laser power, and an ELWD 40×/0.6 NA objective lens (Nikon Instruments, Inc., Tokyo, Japan). Excitation of fluorescence was provided by a Chameleon Vision-2 multiphoton laser (80 MHz, Coherent. Inc., Santa Clara, CA, USA) tuned to 850 nm. Slidebook 6 software (3i) (Conoptics, Inc., Danbury, CT, USA) was used to control the microscope and multiphoton laser systems and to acquire images. Images were scanned in three dimensions with x- and y-dimensions of 248 μm (1024 × 1024-pixel resolution), with z-steps of 2 μm (z stack) to produce z stacks of 36 and 46 μm to ensure that all cells were captured within the volume. Pixel dwell time was set to 2 μL with pixel averaging of 5/scan to acquire z stack images [17,18].
