Fluorescence is the property in which certain molecules (fluorochromes) emit fluorescent radiation when excited by a laser beam or exposed to near‐infrared light (NIR) at specific wavelengths. Once the light energy is absorbed by the fluorochrome's organic molecules, an excitation of delocalized electrons from ground state to a higher energy level occurs. Upon return from the excited singlet state to the ground state, energy is emitted in the form of photons, reaching the observer's eye as fluorescence of a specific wavelength.
Fluorescence image‐guided surgery (FIGS) is a medical imaging technique that uses fluorescence to detect properly labeled structures during surgery. Its purpose is to guide the surgical procedure and provide the surgeon a real‐time view of the operating field. When compared to other medical imaging modalities, FIGS is cheaper and superior in terms of resolution and number of molecules detectable. As a drawback, penetration depth is usually very poor (100 microns) in the visible wavelengths, but it can reach up to 1–2 cm when excitation wavelengths in the near infrared are used. FIGS is performed using imaging devices with the purpose of providing real‐time simultaneous information from color reflectance images (bright field) and fluorescence emission. One or more light sources are used to excite and illuminate the sample. Light is collected using optical filters that match the emission spectrum of the fluorophore. Imaging lenses and digital cameras are used to produce the final image [20–22].
Indocyanine green dye (ICG) was developed for near‐infrared (NIR) photography by Kodak Research Laboratories in 1955 and introduced for clinical use in 1956. Several clinical applications comprised the use of ICG, such as cirrhotic liver resection.
New successful applications of ICG were very recently published. For example, the use of this dye for assistance during a hepatic metastasectomy allows to identify superficially located colorectal liver metastases and in some patients it was possible to localize small lesions otherwise undetectable by typical procedures.
ICG is recognized to be relatively free of adverse effects when injected into the bloodstream and has been used in veterinary medicine extensively. Once excited with a specific wavelength of light, ICG becomes fluorescent, and this phenomenon can be detected using specific filter scopes and cameras, and then displayed on a screen. Some specific areas with ICG accumulating abilities can be seen, which would not occur with a normal white light endoscopic imaging system. ICG rapidly binds to plasma proteins after its intravascular injection, resulting in minimal leakage to the interstitial compartment.
The major limitation in FIGS is the availability of clinically approved fluorescent dyes. Indocyanine Green has been widely used as a nonspecific agent to detect sentinel lymph nodes (SLN) during surgery.
ICG‐NIR is classified as a nontargeted dye. This means that following administration, the pharmacokinetics of the molecule are known in detail, and this defines whether a probe is a good imaging agent for the observation of a given target according to its biodistribution, excretion pathways, accumulation patterns, etc. In general, nontargeted probes show poor differentiation between malignant and healthy tissue boundaries. A major area of research in fluorescence imaging is the identification and approval of new targeted dyes, which will offer better tissue differentiation at a reasonable cost.
Future trends in fluorescence imaging will depend on the research and development of antibody binding and specific tissue‐binding dye abilities. This step will expand the opportunities for targeted oncologic surgery and specific planning for complete tissue resection. Most of these specific fluorophore molecules rely on NIR fluorescence properties, and their ability to bind to receptors on specific cell membranes or extracellular matrix enzymes. They can efficiently recognize the intended molecular target and selectively accumulate on the malignancy sites, thereby rendering dramatic improvements in signal‐to‐background ratio over nontargeted probes.
The complete system can be switched from standard white light mode to NIR/ICG mode, and vice versa, simply by foot‐pedal control, or with camera head buttons on the new generation 4K NIR/ICG cameras (RUBINA® – KARL STORZ). When used in NIR/ICG fluorescence mode, the technology can either offer images in blue or green, which enhances the image in highly perfused and fluorescent areas (blue mode), to more intense fluorescence and detailed delineation against surrounding tissue (green mode) [20–22]. RUBINA cameras (KARL STORZ) present also other NIR/ICG advantages such as Monochromatic Fluorescence which enables black and white imaging mode for better operating contrast, sharper edges, and brightness (Figure 3.9a).
Videoendoscopic oncologic surgery can benefit from NIR technology, since ICG is used to map neoplastic tissues and their lymphatic drainage. Consequently, mapped lymph nodes are surgically removed, allowing for subsequent histopathological analysis. Sentinel lymph node mapping is now enhanced with the use of the Fluorescence Intensity Map, since it achieves an improved distinction between lymph nodes and surrounding lymphatic pathways.
The recent introduction of Intensity Map enables the surgeon to interpret the grade of fluorescence displayed on the screen and correlate that with an intensity fluorescence scale, throughout the procedure, thus resulting in improved perception of tissue marking over surgical time. Overlaid images of white light and NIR/ICG are combined and processed with intelligent analysis software. With a specialized CCU, miniscule fluorescence tonal differences not visible to the human eye are detected and highlighted by a color scale mapping system.
Ranging from blue tones (the weakest fluorescence signal) to yellow/orange (the strongest fluorescence signal), color mapping maximizes early detection of lymphatic circulation, and SLN. The color grading system also positively influences the distinction of SLN from subsequent lymph nodes, which can be very helpful in surgical decision making.
The standard endoscopic imaging systems provide the surgeon with indirect monocular views of the operative field, denying the operator the binocular depth cues that provide a sense of stereopsis. The loss of binocular vision in a two‐dimensional (2D) display causes visual misperceptions – mainly loss of depth perception, adding to the surgeon's fatigue.
One of the biggest challenges for laparoscopic surgeons is hand–eye coordination within a 3D scene observed on a 2D display. Experienced surgeons learn to use monocular depth cues such as light and shade, relative size of objects, object interposition, texture gradient, aerial perspective, and motion parallax instead of stereovision. Using these cues, all laparoscopic operations can be accomplished; however, time and accuracy may be lost as these techniques do not completely compensate for loss of stereoscopic depth perception [23–32].
The 3D HD view with enhanced haptic feedback makes laparoscopic surgery safer and more intuitive. It improves hand–eye coordination and surgical precision, with the use of all conventional instruments. The 3D view provides increased depth perception and more accurate measurement of the dimensions of the anatomical spaces, enhancing the skills of the laparoscopic surgeon to manipulate tissues, dissect, design surgical strategies, and place intracorporeal sutures. Studies have reported less strain on the surgeon when using 3D rather than 2D vision [33–38].
Recent technological advances have led to sophisticated high‐resolution systems and light polarizing glasses that are lighter and more comfortable. A dual‐optical scope is connected to a two‐chip imaging system that transmits two pictures to a stereoscopic screen. When the surgeon wears polarized 3D glasses, the two images are merged by the brain into one single image that gives the perception of depth.
Stereoscopic vision improves accuracy in laparoscopic skills for novices. Several studies demonstrated a marked reduction in the number of repetitions and errors.
Few 3D systems are used yet in veterinary practice, but in the near future as popularity increases and prices are reduced, availability will increase, likely resulting in improved performance of laparoscopic surgical procedures. Nevertheless, when choosing between 3D and ECE (enhanced contact endoscopy) or NIR/ICG, the latter two currently stand as a more rational, cost‐effective options for small animal endoscopy and MIS [39–43].