A portable imaging system integrating four nonlinear optical imaging modalities (Fig. 5, A to C) was designed to collect label-free multimodal imaging data in the operating room during cancer surgeries. The laser source in this imaging system provided transform-limited 55-fs laser pulses at a 70-MHz repetition rate and with a spectral range of 1040 to 1100 nm, which can excite the four nonlinear optical processes with high efficiency and avoid the potential laser damage and photo bleaching in the tissue specimens. With this excitation spectral range, the emitted nonlinear optical signals from the four modalities were separately detected in different spectral windows (Fig. 5D), achieved by four optical bandpass filters. The color code for each imaging modality was chosen to represent the approximate wavelengths of the emitted nonlinear optical signals from the specimens. Using a pair of galvanometer scanning mirrors, a nonlinear optical image (800 by 800 pixels, 500 by 500 nm2 per pixel) was acquired within 80 s for each imaging modality. By sequentially switching between filters for each imaging modality, a complete set of images from a single field of view (FOV) (400 × 400 μm2) was acquired in approximately 5 min.

(A) A photograph of the compact and portable intraoperative label-free multimodal nonlinear imaging system. (B) Software interface of the imaging system. (C) System schematic. (D) Spectral range and display color of the four nonlinear optical imaging modalities. L, lens; GM, galvanometer-scanning mirror; DM, dichroic mirror; OBJ, objective; FW, filter wheel; PMT, photomultiplier tube. (Photo credit: Yi Sun, Biophotonics Imaging Laboratory, University of Illinois at Urbana-Champaign.)

To effectively observe EVs, the lateral resolution of THG imaging (~322 nm) was sufficiently high to visualize and resolve microvesicles (~500 to 1000 nm) and detect some exosomes (40 to 120 nm) (6). Under the spatial Nyquist sampling condition, the pixel size should be set below half of the THG imaging resolution (~161 nm) to assure sufficient sampling. However, because of some residual image jitter caused by inevitable cart vibration from mechanical elements and room equipment, acquiring images with small pixel sizes would considerably reveal vibration-induced artifacts, yield low image quality and fidelity, and increase image acquisition time. Therefore, the pixel size was somewhat compromised and set to 500 nm by 500 nm, which was still sufficient to visualize EVs as diffraction-limited bright points in the acquired images due to the strong THG signal they emitted. Furthermore, to assure the depth-resolved sectioning of EVs, the axial resolution of THG imaging in this imaging system was estimated to be 1.0 μm, which was later used to calculate the imaging volume and EV density.

The entire imaging system was housed in a compact and portable cart (90 cm by 90 cm by 120 cm, 90 kg) that was comparable in size and weight to other intraoperative equipment, such as intraoperative ultrasound, intraoperative optical coherence tomography, and anesthesia carts used within the intraoperative working environment. The system was able to be readily moved throughout the hospital and clinical environment and operated by a single person. The optical components were aligned in a robust way to withstand floor obstacles and vibrations during transportation, eliminating the need for realignment before image acquisition. The system was designed so all optical components and electronics were contained within the cart enclosure, and imaging was performed in an inverted microscope configuration where the specimen was simply placed on a clear glass window inlaid in the top surface of the cart, covered with a light-tight box-shaped cover, and imaged using an objective located within the cart and below the glass window. Because of the strictly controlled lighting conditions in the operating room, light concealment during imaging was considered a priority when the imaging system was designed and built, so as to obviate the need for the surgeon and staff to change lighting conditions during surgery and delay the procedure. Specifically, switching between modalities was automated using a motorized filter wheel, and focus adjustment was accomplished by a piezoelectric linear stage to eliminate the need to open the cart doors. As a result, noise from background light was minimized, and the laser beam was confined within the imaging cart for laser safety. On the other hand, necessary ventilation was maintained to reduce the thermal noise of the PMT (H7421-40, Hamamatsu Photonics K.K.). With the minimized background and thermal noise, the average signal-to-noise ratio (SNR) measured from the intraoperative THG images was approximately 19 ± 4 dB, sufficient to visualize the tissue structures.

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