Introduction to fluorescence imaging
Fluorescence is a phenomenon where substance that has absorbed certain wavelength of light emits it back at another wavelength. These substances are called fluorophores or fluorescent dyes. The emitted light has a longer wavelength than the absorbed one since some energy of the photon is lost in the process. The difference in wavelengths between absorbed and emitted light, called Stokes shift, allows very low background for fluorescence detection by separating it from the excitation light. The process of fluorescence generation is illustrated by the Jablonski diagram on the right.
Fluorescence is generated in a cyclic process, meaning that single fluorophore can be repeatedly excited to generate fluorescence until irreversible damage called photobleaching at some point occurs. This makes fluorescence detection a very sensitive detection method, 1000 times more sensitive than UV-VIS absorbance. It is also possible to monitor multiple targets or processes simultaneously by using several fluorescent probes each with unique optical characteristics.
Fluorescence detection is heavily utilized method in biosciences for applications like next-generation sequencing, fluorescence microscopy, flow cytometry, and in vitro plate readers. It has also important role in clinical use, such as in endoscopy, theranostics, fluorescence-guided surgery and ophthalmology.
Several fluorescent agents have been approved for fluorescence based diagnosis & intraoperative imaging:
| Dye generic name | Countries | Excitation | Detection | Indications | ||
| Indocyanine Green (ICG) | Worldwide | 800 nm | Near-infrared (820 nm) | Multiple uses: lymphatic mapping, tissue perfusion, visualization of biliary ducts and blood veins, retinal angiography |
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| Fluorescein | Worldwide | 490 nm | Green fluorescence (525 nm) | Fluorescein angiography or angioscopy (ophthalmology) | ||
| Methylene Blue | Worldwide | 665 nm | 688 nm | Endoscopic polypectomy, chromoendoscopy, lymphatic drainage | ||
| 5-ALA Hydrochloride (converted to Pp-IX) |
Worldwide | 400 – 410 nm | Red fluorescence (620 – 710 nm) |
Intraoperative visualization of high-grade gliomas, non-muscle invasive bladder cancer cystoscopy (Japan) | ||
| Hexaminolevulinate (HAL) | Worldwide | 360 – 450 nm | Red fluorescence (635 nm) | Blue light fluorescence cystoscopy for non-muscle invasive bladder cancer |
Endoscopy
Endoscopy is widely used minimally invasive technique to diagnose and treat diseases. Endoscope is a thin and flexible tube that has a small camera typically at the tip of the scope that is guided through the body’s opening such as mouth or rectum to the target site. Common endoscopy types include colonoscopy (large intestine), cystoscopy (bladder), bronchoscopy (lungs), and gastroscopy (stomach). Endoscopy is particularly important tool in oncology to detect and remove tumors or premalignant lesions that have potential to become tumorous.
Fluorescence detection in endoscopy
Endoscopic imaging is typically performed using normal white light. However, certain tumors can be difficult to detect under white light since all structures are illuminated without specificity to malignant lesions. Fluorescence endoscopy is for this reason increasingly used in cancer diagnostics and surveillance to improve detection of cancerous and precancerous lesions. Special fluorescent dyes are normally administered to patient and preferentially accumulate into malignant lesions and this way increase contrast between malignant and heathy tissues when excited when specific wavelength of light.
Most common fluorescence endoscopy use cases include:
- Blue light cystoscopy with 5-ALA (bladder cancer)
- NIR endoscopy with ICG (indocyanine green)
- Autofluorescence endoscopy (bronchi and GI tract)
Fluorescence-guided surgery
Fluorescence-guided surgery is used in addition to standard white light surgery to improve intraoperative visualization of tumor margins and overall tumor visualization to achieve more complete tumor removal. Improved sensitivity of tumor resection generally correlates with longer patient survival. One important indication for fluorescence-guided surgery is glioblastoma. For this use, patients receive 5-aminolevulinic acid (5-ALA) which is converted into protoporphyrin-IX (PpIX) selectively in tumor cells to visualize them in red color by surgical microscope’s blue light. Several targeted fluorescence agents for many cancer types are currently being studied in clinical trials (see below table).
| Dye | Main indications | Fluorophore | Target | Targeting molety | Excitation (nm) | Manufacturer |
| EMI-137 | Colon, breast, esophagus |
Cy5-modified | c-MET | peptide | 650 | Edinburgh Molecular Imaging (UK) |
| LUM-015 | Colon, breast, brain | Cy5 | cathepsin (tumor microenvironment) |
protease-activated fluorophore |
650 | Lumicell Inc (USA) |
| SGM-101 | Colon, pancreas, brain | BM104 | carcino-embryonic antigen |
monoclonal antibody | 700 | SurgiMab (France) |
| AVB-620 | Breast | Cy5 & Cy7 | matrix metalloproteinases |
activatable peptide | 750 | Avelas Biosciences (USA) |
| OTL-38 | Ovarian, lung | ICG-type | folate receptor | folate (vitamin) | 775 | On Target Laboratories LLC (USA) |
Modulight’s solutions for fluorescence imaging
Fluorescence endoscopy
Modulight’s ML7710 next-generation light source for endoscopy delivers both white light and separate fluorescence wavelengths as customized for each use case, providing compact design for all light sources in one package. ML7710 can also include optional spectral fluorescence detection within the same unit. This is especially useful functionality in the development of novel dyes that are not yet supported by the optics of the commercial instruments to detect fluorescence. The system has been designed in close collaboration with end users, ensuring optimal clinical usability.
White light is produced with 3 individually controllable laser channels (RGB) that enable adjusting the white light balance according to the needs of the application and the used illumination mode. Additional lasers are specific to absorption spectra of the used fluorescent dyes. The example configuration below shows the 488 nm laser used for fluorescein together with the RGB channels. The system supports all clinically used fluorescent dyes like ICG (NIR endoscopy), 5-ALA (blue light cystoscopy), and many of the novel dyes under clinical investigation. Several laser lines can also be turned on simultaneously to support additional imaging modes like narrow-band imaging and autofluorescence endoscopy.
Modulight’s light source provides optimal beam profile for endoscopy. Speckle can be removed by a simple switch when very uniform illumination profile is needed.
Before speckle removal (Cs=16.09%)
After speckle removal (Cs=4.6%)
Modulight lasers match ideally the absorption peak of the dye, and as a result induce very strong fluorescence emission compared to gold standard Xenon lamps that can only target the lower part of the absorption peak. In contrast to Xenon lamps, lasers don’t require any filtering and all produced light can be used for efficient fluorescence excitation. This is especially useful for situations where fluorescence is faint, such as for autofluorescence endoscopy to create as bright image as possible without any additional heat production. Narrow laser wavelengths can also be more optimal than LEDs and Xenon lamps for image recognition algorithms that are being increasingly used in endoscopy as an assistance tool for the clinician.
Fluorescence-based treatment monitoring
Modulight’s ML7710 can also be used for monitoring light-based treatments with the help of monitoring changes in the fluorescence emission of the drug. For example drugs used in photodynamic therapy (PDT) typically have intrinsic fluorescence properties when excited with specific wavelength of light. Inducing fluorescence and monitoring photobleaching of the drug can help to verify that the drug is localized in the tissue and adjusting the light delivery time to be optimal for each individual patient, in accordance with the approach of personalized medicine. ML7710 is being studied for this purpose in a clinical trial, while the same approach can be suitable for other cancers as well, such as bladder, colorectal, and prostate cancer.
ML7710 is also being preclinically studied as a tool for future theranostic nanomedicine where a generic diagnosis, imaging, and treatment technology is being developed. Light-activated combination therapies can have multiple therapeutic and fluorescent components incorporated, which allow drug’s activation and monitoring with the same ML7710 system. Another possibility with the ML7710 is optical biopsies for cancer detection either utilizing the differences in autofluorescence between healthy and malignant tissues, or with the help of fluorescent dyes.
Fluorescence spectra of PpIX recorded with ML7710 at baseline (0 min) and 30 minutes after start of the illumination, indicating complete photobleaching and supporting to end the illumination. The spectra are recorded in real-time into Modulight Cloud for analytics.
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