Written by Federico Citterich
Conceived and reviewed by Alessandro Rossetta
Laser-induced fluorescence reveals hidden details within tissues by making invisible structures visible through light emission. In this article, we explore the principles behind laser-induced fluorescence and its applications in cancer detection and surgery. By combining theory with clinical practice, we show how this spectroscopic method is transforming oncology.
A surgeon leans over an operating table, pointing a laser at a patient’s brain. On the screen, the tumor tissue glows with an eerie light, while the healthy tissue remains dark. This fluorescence indicates exactly where to cut – and where to stop.
Many biological tissues auto-fluoresce – they naturally emit light when excited with certain wavelengths. This is due to the so-called fluorophores, naturally fluorescent molecules present in cells and tissues. Examples include collagen, elastin, NADH, and FAD.
FLUORESCENCE CAN HELP PHYSICIANS TO DETECT TUMOR SURGICAL MARGINS IN THE OR
But what happens when tumor cells are present? As we explained in one of our previous articles, tumors often have altered metabolism, collagen structure, and cell density, all of which change their autofluorescence signature. Hence, detecting these changes in autofluorescence can help researchers and physicians to differentiate between healthy and tumorous tissue, even intraoperatively: first, blue or UV light is shone onto the surgical site by a laser. Normal tissue would remain dark, while tumor tissue would shine more intensely because disrupted collagen architecture and metabolic shifts enhance the accumulation of fluorescent molecules such as porphyrins1. The transition from bright (tumor tissue) to dimmer autofluorescence (healthy tissue) signals where the tumor ends and healthy tissue begins.
Laser-induced fluorescence can be one of the most precise and versatile tools in modern biology. It’s non-invasive, non-destructive, and extremely sensitive and, sometimes, specific; it’s no coincidence that it’s widely used in medicine and considered one of the most promising tools for the future of the field.
FLUORESCENCE MEASUREMENT SYSTEMS: SOME EXAMPLES
Several commercial systems for autofluorescence measurements are nowadays available in the market. Endoscopic systems LIFE, D-light, SAFE-100, and DAFE are designed to analyze autofluorescence contrast with a built-in fluorescence excitation-collection module, to diagnose tumors in the bronchi and gastrointestinal tract. Or again, VELscope is used to detect oral carcinoma via direct visualization of the oral cavity’s autofluorescence.
VELscope results before a tongue biopsy. Image taken from Ciccù et al. (2019).
These systems demonstrate high sensitivity in detecting specific stages of a disease, but they specificity is limited. This means they are really good at identifying people who have the disease, but they also often give false alarms, wrongly flagging healthy people as sick (the so-called false positives).
This is because measuring autofluorescence in vivo is not at all straightforward. Tissue movement, variation in tissue surface profile, presence and change in concentration of endogenous absorbers (e.g., hemoglobin), and other factors can in fact change the light excitation-collection geometries, hence affecting the measurements.
CANCER DETECTION USING FLUORESCENT PROBES: PASSIVE TARGETING
One way to overcome this problem is using fluorescent probes. Fluorescent probes are special molecules or molecular tags that emit light when excited by a certain wavelength. By naturally accumulating in tumor tissue, they generate cancer-targeted signals that stand out from the background autofluorescence, thereby reducing false positives and improving diagnostic accuracy.
This process relies on something known as enhanced permeability and retention (EPR) effect. The EPR effect is a hallmark of tumors, and derives from two main biological changes present in cancers. First, blood vessels feeding tumors are often “leaky”, with larger gaps than normal vessels – hence permeability is enhanced. Second, tumors usually have poor lymphatic drainage2, so once something enters the tumor tissue, it tends to stay there longer than in healthy tissue – hence retention is enhanced.
Because of EPR, fluorescence tracers that circulate in the bloodstream naturally tend to collect more in tumor tissue than in healthy tissue. This process is known as passive targeting.
Examples of passive fluorescent indicators include methylene blue, indocyanine green, 5-Aminolevulinic Acid, and fluorescein.
CANCER DETECTION USING FLUORESCENT PROBES: ACTIVE TARGETING
But the EPR effect is not completely unique to tumors. Inflammation, wounds, or other highly vascularized tissues can also have leaky vessels and poor drainage, meaning that a passive fluorescence probe might accumulate in those areas too and potentially leading to false positives.
To further reduce false positives, researchers and physicians use active targeting. Active targeting refers to the process where fluorescent probes bind selectively to cancer biomarkers, hence accumulating in the tumor tissue “actively”.
A common approach relies on using antibodies, proteins that naturally bind very specifically to some molecules. The process is simple: an antibody designed to target a tumor-associated marker is tagged with a fluorescent dye. Then, the antibody is injected, and accumulates only in tumor tissue. Under excitation light, the dye on the antibody fluoresces strongly. Since the antibody binds only where the cancer is, the cancerous region lights up much more clearly than the surrounding normal tissue, creating a contrast between healthy and diseases tissue that is far sharper than with autofluorescence alone.
This can also be used in the OR to detect tumor surgical margins in a more efficient way than how described above: with fluorescent probes, the tumor literally glows under special light, guiding the surgeon’s scalpel.
Visualization of a glioblastoma tumor under white light (left) and under fluorescence microscope (right). Image taken from Alfonso-García et al. (2022).
CANCER DETECTION USING FLUORESCENT PROBES: ACTIVABLE TARGETING
However, because active probes remain fluorescent all the time, background signal from circulation and nonspecific uptake3 can persist, potentially leading to some false positives. This challenge has led to the development of activable probes, which remain dark until triggered by disease-specific processes, thereby adding an extra layer of specificity and greatly reducing false positives.
In other words, activable probes are “off” (non-fluorescent) until they encounter a specific biological trigger in the target tissue. Once triggered, they become “on” (fluorescent), so their light is only emitted exactly where the disease is active.
Activable probes can be triggered in several ways. For example, a probe may have a quencher4 attached that blocks fluorescence, and could be chemically designed so that a tumor-specific process separates the fluorophore and the quencher. Once the quencher is removed, fluorescence “switches on”.
Other examples include pH-activated probes, redox-activated probes, photo-activated probes, dual-activation probes, etc.
LASER-INDUCED FLUORESCENCE AND LIQUID BIOPSY
But laser-induced fluorescence can also be used outside the OR to diagnose or monitor cancer without surgery – ideally catching it early or tracking response to treatment.
This is where liquid biopsy comes in. Liquid biopsy is the sampling and analysis of non-solid biological tissues, primarily blood, and can be used to detect tumor-specific markers in a sensitive and non-invasive way.
The idea is similar to what was described above with fluorescent probes: a laser excites fluorescently labeled antibodies bound to tumor-specific markers. These markers light up as they pass through a microfluidic channel5, enabling identification and counting.
Liquid biopsy can also directly analyze fluorescent biomarkers without the need for labeling. For instance, cancer-related porphyrins or other endogenous fluorophores can be directly excited with a laser.
Importantly, this process can achieve single-cell sensitivity and can detect extremely low concentrations of tumor markers, crucial for early detection.
Schematic of liquid biopsy analysis. A blood sample flows through microchannels inside a microfluidic chip. A laser excites fluorescently labeled antibodies bound to tumor-specific markers, which emit a detectable glow that enables sensitive identification and enumeration.
While laser-induced fluorescence has already proven invaluable in highlighting diseased tissues, researchers are now looking beyond brightness alone. Properties such as fluorescence lifetime and emission spectra carry an additional layer of diagnostic information – a topic we’ll explore in the next articles.
GLOSSARY
- Porphyrins are a group of organic compounds that accumulate abnormally in tumors and fluoresce strongly under certain laser light.
- Lymphatic drainage is the process by which the lymphatic system removes excess fluid, proteins, and waste from tissues and returns them to the bloodstream.
- Nonspecific uptake is when a molecule accumulates in tissues other than its intended target.
- A quencher is a molecular group that suppresses (quenches) fluorescence from a nearby fluorophore by absorbing the fluorophore’s emitted energy or by directly dissipating the energy as heat.
- A microfluidic channel is a tiny passage, often narrower than a millimeter, that guides and controls the flow of liquids at the microscale. In cancer diagnostics, it is used to align and transport cells or molecules in a controlled stream for precise analysis or detection.
REFERENCES
Alfonso-García, A., Zhou, X., Bec, J., Anbunesan, S. N., Fereidouni, F., Jin, L. W., … & Marcu, L. (2022). First in patient assessment of brain tumor infiltrative margins using simultaneous time-resolved measurements of 5-ALA-induced PpIX fluorescence and tissue autofluorescence. Journal of Biomedical Optics, 27(2), 020501-020501.
https://pubmed.ncbi.nlm.nih.gov/35112514/
Alfonso-Garcia, A., Anbunesan, S. N., Bec, J., Lee, H. S., Jin, L. W., Bloch, O., & Marcu, L. (2023). In vivo characterization of the human glioblastoma infiltrative edge with label-free intraoperative fluorescence lifetime imaging. Biomedical Optics Express, 14(5), 2196-2208.
https://pmc.ncbi.nlm.nih.gov/articles/PMC10191664/
Cicciù, M., Cervino, G., Fiorillo, L., D’Amico, C., Oteri, G., Troiano, G., … & Laino, L. (2019). Early diagnosis on oral and potentially oral malignant lesions: a systematic review on the VELscope® fluorescence method. Dentistry journal, 7(3), 93.
https://www.mdpi.com/2304-6767/7/3/93
Gabrecht, T., Lovisa, B., Borle, F., & Wagnieres, G. (2007). Design of an endoscopic optical reference to be used for autofluorescence bronchoscopy with a commercially available diagnostic autofluorescence endoscopy (DAFE) system. Physics in Medicine & Biology, 52(8), N163.
https://pubmed.ncbi.nlm.nih.gov/17404451/
Kravchenko, Y., Sikora, K., Wireko, A. A., & Lyndin, M. (2024). Fluorescence visualization for cancer DETECTION: EXPERIENCE and perspectives. Heliyon, 10(2).
https://www.sciencedirect.com/science/article/pii/S2405844024004213
Kwaśny, M., & Bombalska, A. (2022). Applications of laser-induced fluorescence in medicine. Sensors, 22(8), 2956.
https://www.mdpi.com/1424-8220/22/8/2956
Lam, S., MacAulay, C., Hung, J., LeRiche, J., Profio, A. E., & Palcic, B. (1993). Detection of dysplasia and carcinoma in situ with a lung imaging fluorescence endoscope device. The Journal of thoracic and cardiovascular surgery, 105(6), 1035-1040.
https://pubmed.ncbi.nlm.nih.gov/8501931/
Marcu, L. (2012). Fluorescence lifetime techniques in medical applications. Annals of biomedical engineering, 40(2), 304-331.
https://pubmed.ncbi.nlm.nih.gov/22273730/
Marsden, M., Fukazawa, T., Deng, Y. C., Weyers, B. W., Bec, J., Gregory Farwell, D., & Marcu, L. (2020). FLImBrush: dynamic visualization of intraoperative free-hand fiber-based fluorescence lifetime imaging. Biomedical Optics Express, 11(9), 5166-5180.
https://pubmed.ncbi.nlm.nih.gov/33014606/
McNamara, K. K., Martin, B. D., Evans, E. W., & Kalmar, J. R. (2012). The role of direct visual fluorescent examination (VELscope) in routine screening for potentially malignant oral mucosal lesions. Oral surgery, oral medicine, oral pathology and oral radiology, 114(5), 636-643.
https://pubmed.ncbi.nlm.nih.gov/23083477/
Molpeceres, C., Ramos-Medina, R., Marquez, A., Romero, P., Gomez-Fontela, M., Candorcio-Simon, R., … & Martin, M. (2023). Laser transfer for circulating tumor cell isolation in liquid biopsy. International Journal of Bioprinting, 9(4), 720.
https://europepmc.org/article/MED/37323505
Sieroń, A., Sieroń-Stołtny, K., Kawczyk-Krupka, A., Latos, W., Kwiatek, S., Straszak, D., & Bugaj, A. M. (2013). The role of fluorescence diagnosis in clinical practice. OncoTargets and therapy, 977-982.