Fluorescence image-guided surgery | |
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Specialty | oncology (surgery) |
Fluorescence guided surgery (FGS), also called fluorescence image-guided surgery, or in the specific case of tumor resection, fluorescence guided resection, is a medical imaging technique used to detect fluorescently labelled structures during surgery. [1] Similarly to standard image-guided surgery, FGS has the purpose of guiding the surgical procedure and providing the surgeon of real time visualization of the operating field. When compared to other medical imaging modalities, FGS is cheaper and superior in terms of resolution and number of molecules detectable. [2] As a drawback, penetration depth is usually very poor (100 μm) in the visible wavelengths, but it can reach up to 1–2 cm when excitation wavelengths in the near infrared are used. [3]
FGS 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 (CCD or CMOS) are used to produce the final image. Live video processing can also be performed to enhance contrast during fluorescence detection and improve signal-to-background ratio. In recent years a number of commercial companies have emerged to offer devices specializing in fluorescence in the NIR wavelengths, with the goal of capitalizing upon the growth in off label use of indocyanine green (ICG). However commercial systems with multiple fluorescence channels also exist commercially, for use with fluorescein and protoporphyrin IX (PpIX).[ citation needed ]
Fluorescence excitation is accomplished using various kind of light sources. [4] Halogen lamps have the advantage of delivering high power for a relatively low cost. Using different band-pass filters, the same source can be used to produce several excitation channels from the UV to the near infrared. Light-emitting diodes (LEDs) have become very popular for low cost broad band illumination and narrow band excitation in FGS. [5] Because of their characteristic light emission spectrum, a narrow range of wavelengths that matches the absorption spectrum of a given fluorophore can be selected without using a filter, further reducing the complexity of the optical system. Both halogen lamps and LEDs are suitable for white light illumination of the sample. Excitation can also be performed using laser diodes, particularly when high power over a short wavelength range (typically 5-10 nm) is needed. [6] In this case the system has to account for the limits of exposure to laser radiation. [7]
Live images from the fluorescent dye and the surgical field are obtained using a combination of filters, lenses and cameras. During open surgery, hand held devices are usually preferred for their ease of use and mobility. [8] A stand or arm can be used to maintain the system on top of the operating field, particularly when the weight and complexity of the device is high (e.g. when multiple cameras are used). The main disadvantage of such devices is that operating theater lights can interfere with the fluorescence emission channel, with a consequent decrease of signal-to-background ratio. This issue is usually solved by dimming or switching off the theater lights during fluorescence detection. [9]
FGS can also be performed using minimally invasive devices such as laparoscopes or endoscopes. In this case, a system of filters, lenses and cameras is attached to the end of the probe. [10] Unlike open surgery, the background from external light sources is reduced. Nevertheless, the excitation power density at the sample is limited by the low light transmission of the fiber optics in endoscopes and laparoscopes, particularly in the near infrared. Moreover, the ability of collecting light is much reduced compared to standard imaging lenses used for open surgery devices. FGS devices can also be implemented for robotic surgery (for example in the da Vinci Surgical System). [11]
The major limitation in FGS is the availability of clinically approved fluorescent dyes which have a novel biological indication. Indocyanine green (ICG) has been widely used as a non-specific agent to detect sentinel lymph nodes during surgery. [12] ICG has the main advantage of absorbing and emitting light in the near infrared, [3] allowing detection of nodes under several centimeters of tissue. Methylene blue can also be used for the same purpose, with an excitation peak in the red portion of the spectrum. [13] First clinical applications using tumor-specific agents that detect deposits of ovarian cancer during surgery have been carried out. [14]
The first uses of FGS dates back to the 1940s when fluorescein was first used in humans to enhance the imaging of brain tumors, cysts, edema and blood flow in vivo. [15] In modern times the use has fallen off, until a multicenter trial in Germany concluded that FGS to help guide glioma resection based upon fluorescence from PpIX provided significant short-term benefit. [16]
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, than the absorbed radiation. A perceptible example of fluorescence occurs when the absorbed radiation is in the ultraviolet region of the electromagnetic spectrum, while the emitted light is in the visible region; this gives the fluorescent substance a distinct color that can only be seen when the substance has been exposed to UV light. Fluorescent materials cease to glow nearly immediately when the radiation source stops, unlike phosphorescent materials, which continue to emit light for some time after.
Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.
Fluorescence spectroscopy is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light; typically, but not necessarily, visible light. A complementary technique is absorption spectroscopy. In the special case of single molecule fluorescence spectroscopy, intensity fluctuations from the emitted light are measured from either single fluorophores, or pairs of fluorophores.
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds.
Laser-induced fluorescence (LIF) or laser-stimulated fluorescence (LSF) is a spectroscopic method in which an atom or molecule is excited to a higher energy level by the absorption of laser light followed by spontaneous emission of light. It was first reported by Zare and coworkers in 1968.
Plate readers, also known as microplate readers or microplate photometers, are instruments which are used to detect biological, chemical or physical events of samples in microtiter plates. They are widely used in research, drug discovery, bioassay validation, quality control and manufacturing processes in the pharmaceutical and biotechnological industry and academic organizations. Sample reactions can be assayed in 1-1536 well format microtiter plates. The most common microplate format used in academic research laboratories or clinical diagnostic laboratories is 96-well with a typical reaction volume between 100 and 200 µL per well. Higher density microplates are typically used for screening applications, when throughput and assay cost per sample become critical parameters, with a typical assay volume between 5 and 50 µL per well. Common detection modes for microplate assays are absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization.
Fluorescein angiography (FA), fluorescent angiography (FAG), or fundus fluorescein angiography (FFA) is a technique for examining the circulation of the retina and choroid using a fluorescent dye and a specialized camera. Sodium fluorescein is added into the systemic circulation, the retina is illuminated with blue light at a wavelength of 490 nanometers, and an angiogram is obtained by photographing the fluorescent green light that is emitted by the dye. The fluorescein is administered intravenously in intravenous fluorescein angiography (IVFA) and orally in oral fluorescein angiography (OFA). The test is a dye tracing method.
A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed.
A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.
A spectrofluorometer is an instrument which takes advantage of fluorescent properties of some compounds in order to provide information regarding their concentration and chemical environment in a sample. A certain excitation wavelength is selected, and the emission is observed either at a single wavelength, or a scan is performed to record the intensity versus wavelength, also called an emission spectrum. The instrument is used in fluorescence spectroscopy.
Two-photon excitation microscopy is a fluorescence imaging technique that is particularly well-suited to image scattering living tissue of up to about one millimeter in thickness. Unlike traditional fluorescence microscopy, where the excitation wavelength is shorter than the emission wavelength, two-photon excitation requires simultaneous excitation by two photons with longer wavelength than the emitted light. The laser is focused onto a specific location in the tissue and scanned across the sample to sequentially produce the image. Due to the non-linearity of two-photon excitation, mainly fluorophores in the micrometer-sized focus of the laser beam are excited, which results in the spatial resolution of the image. This contrasts with confocal microscopy, where the spatial resolution is produced by the interaction of excitation focus and the confined detection with a pinhole.
Molecular imaging is a field of medical imaging that focuses on imaging molecules of medical interest within living patients. This is in contrast to conventional methods for obtaining molecular information from preserved tissue samples, such as histology. Molecules of interest may be either ones produced naturally by the body, or synthetic molecules produced in a laboratory and injected into a patient by a doctor. The most common example of molecular imaging used clinically today is to inject a contrast agent into a patient's bloodstream and to use an imaging modality to track its movement in the body. Molecular imaging originated from the field of radiology from a need to better understand fundamental molecular processes inside organisms in a noninvasive manner.
A fluorometer, fluorimeter or fluormeter is a device used to measure parameters of visible spectrum fluorescence: its intensity and wavelength distribution of emission spectrum after excitation by a certain spectrum of light. These parameters are used to identify the presence and the amount of specific molecules in a medium. Modern fluorometers are capable of detecting fluorescent molecule concentrations as low as 1 part per trillion.
The DyLight Fluor family of fluorescent dyes are produced by Dyomics in collaboration with Thermo Fisher Scientific. DyLight dyes are typically used in biotechnology and research applications as biomolecule, cell and tissue labels for fluorescence microscopy, cell biology or molecular biology.
Fundus photography involves photographing the rear of an eye, also known as the fundus. Specialized fundus cameras consisting of an intricate microscope attached to a flash enabled camera are used in fundus photography. The main structures that can be visualized on a fundus photo are the central and peripheral retina, optic disc and macula. Fundus photography can be performed with colored filters, or with specialized dyes including fluorescein and indocyanine green.
Indocyanine green (ICG) is a cyanine dye used in medical diagnostics. It is used for determining cardiac output, hepatic function, liver and gastric blood flow, and for ophthalmic and cerebral angiography. It has a peak spectral absorption at about 800 nm. These infrared frequencies penetrate retinal layers, allowing ICG angiography to image deeper patterns of circulation than fluorescein angiography. ICG binds tightly to plasma proteins and becomes confined to the vascular system. ICG has a half-life of 150 to 180 seconds and is removed from circulation exclusively by the liver to bile.
Photoacoustic microscopy is an imaging method based on the photoacoustic effect and is a subset of photoacoustic tomography. Photoacoustic microscopy takes advantage of the local temperature rise that occurs as a result of light absorption in tissue. Using a nanosecond pulsed laser beam, tissues undergo thermoelastic expansion, resulting in the release of a wide-band acoustic wave that can be detected using a high-frequency ultrasound transducer. Since ultrasonic scattering in tissue is weaker than optical scattering, photoacoustic microscopy is capable of achieving high-resolution images at greater depths than conventional microscopy methods. Furthermore, photoacoustic microscopy is especially useful in the field of biomedical imaging due to its scalability. By adjusting the optical and acoustic foci, lateral resolution may be optimized for the desired imaging depth.
Fluorescence imaging is a type of non-invasive imaging technique that can help visualize biological processes taking place in a living organism. Images can be produced from a variety of methods including: microscopy, imaging probes, and spectroscopy.
Indocyanine green angiography (ICGA) is a diagnostic procedure used to examine choroidal blood flow and associated pathology. Indocyanine green (ICG) is a water soluble cyanine dye which shows fluorescence in near-infrared (790–805 nm) range, with peak spectral absorption of 800-810 nm in blood. The near infrared light used in ICGA penetrates ocular pigments such as melanin and xanthophyll, as well as exudates and thin layers of sub-retinal vessels. Age-related macular degeneration is the third main cause of blindness worldwide, and it is the leading cause of blindness in industrialized countries. Indocyanine green angiography is widely used to study choroidal neovascularization in patients with exudative age-related macular degeneration. In nonexudative AMD, ICGA is used in classification of drusen and associated subretinal deposits.
Infracyanine green (IFCG) is a cyanine dye used in medical diagnostics especially in ophthalmology. Unlike Indocyanine green (ICG) it is an iodine free dye.
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