Fluorescence Lifetime Analysis

Introduction

Fluorescence is widely used in life science and it is a gold standard for visualizing and analyzing biological molecules and processes. Thanks to the natural fluorescence of some proteins or small molecules, or to the labelling of specific or general macromolecules with extrinsic fluorophores, biological structures can be visualized both in vitro and in vivo using a microscope. Fluorescence can be described by 4 optical parameters: wavelength, intensity, polarization and lifetime. Wavelength and intensity are the most popular parameters used to study molecules and compounds through fluorescence microscopes. In fact, even though there are several models of fluorescence microscopes (such as widefield, confocal, lightsheet, multiphoton, etc.), most of them obtain or reconstruct the image based on intensity and wavelength.

However, during the last decades, fluorescence lifetime (FL) has drawn the attention of researches being a highly useful intrinsic property of fluorescence. FL is defined as the average time a fluorophore spends in an excited state prior to emit a photon and return to its relaxed ground state. This unique fluorescence parameter allows the access to a new level of information compared to standard imaging methods (Figure 1). In this application note, we will discuss FL and its applications, as well as its advantages and limits.

Figure 1: Fluorescence intensity-based image compared to fluorescence lifetime-based image of the same Convallaria majalis sample. Instead of detecting fluorescence intensity and wavelength only, fluorescence lifetime-based approaches provide a new level of information thanks to the quantification of the average time of photon emission. On the same section in fact, we can notice that molecules that appears similar in color and in intensity, show large differences in lifetime. Image from FLIM LABS. ?

Fluorescence Lifetime

Fluorescence lifetime is an intrinsic parameter of fluorescence and consists in the mean time elapsed between the activation of the fluorophore and the emission of a photon (Figure 2). When measured, FL provides information regarding direct or indirect changes of the fluorophore itself, as well as its surroundings. Every fluorescence molecule has a unique fluorescence lifetime, and the value of the lifetime is reflected by the chemical and physical characteristics of the microenvironment in which the fluorophore resides. Moreover, fluorescence lifetime is independent of fluorescence brightness and fluorophore concentration, thus, FL can be accurately detected even from dim fluorescence signals. With these unique characteristics, the lifetime of a fluorophore has become quite valuable in life sciences (1).

Fluorescence lifetime can be determined using different systems and depending on the used technology the approach is named differently. In general, there are 3 main strategies, called FLIM, FLIm and FLA. FLIM stands for Fluorescence Lifetime Imaging Microscopy, it is the most common approach and FL is quantified on an image with a fluorescence microscope. FLIm stands for Fluorescence Lifetime Imaging, where FL is detected on an image without a microscope (es. Fiber-based systems used during surgeries). Lastly, FLA stands for Fluorescence Lifetime Analysis and it is a general term referring to all the technologies that quantify FL without imaging (i.e. cuvette analysis or time-resolved flow cytometry).

Figure 2: A. When a fluorophore absorbs light with a sufficient energy, an electron is excited to a higher energy level for a short period. The electron will shortly return to its ground state (usually it ranges between 100ps and 15ns), releasing its energy into the form of fluorescence. Conventionally, fluorescence lifetime is defined as the average time a fluorophore remains in its excited state. B. Pulsed lasers are used to “hit” regularly the fluorophore and generate multiple photons; by quantifying the time of arrival of each photon fluorescence lifetime can be determined.

FLIM Applications

Even though FLIM is not as widely used as intensity-based fluorescence microscopy, numerous studies have used this approach in different fields. Hereafter we will discuss briefly some main examples, however, many more can be found in the comprehensive review from Datta and colleagues (1).

One of the greatest advantages of FLIM is the possibility to exploit molecule autofluorescence, thus to be a label-free approach. Many macromolecules are autofluorescent and some of the main ones are reported in the table in Figure 3. One of the first study in vivo exploiting endogenous autofluorescence was carried out by Koening and colleagues where they described changes in lifetime of skin cells within different layers of the skin (2). Furthermore, FLIM detected cells that had become diseased due to melanoma. Oncology is indeed a very important field of application for FLIM, where tumor cells are characterized by a different lifetime compared to healthy cells. In addition, tumor cell lifetime changes during the disease progression, consequently many studies used this parameter to analyze tumor stages and/or monitor treatment responses (1).

Figure 3: Table of the representative endogenous fluorophores responsible for autofluorescence in proteins, microorganisms, plants and animals. Adapted from Berezin et al. (3)

One of the main targets of FLIM analysis is NADH, an important metabolic macromolecule present in all cells and organisms. The balance between NADH and its phosphorylated form NAD(P)H, determines the metabolic state of cell. NADH and NAD(P)H are autofluorescent but cannot be distinguished using standard microscopes as they are spectrally identical. However, they show a different lifetime signature thus detectable using FLIM (4). For these reasons, many studies implement FLIM analysis to determine the metabolic index of cells, in both physiological and tumoral/disease contexts. Importantly, metabolic index evaluation and tumor treatment prediction can be performed in vitro on 3D cell cultures like organoids.? ?

Lastly, many examples of autofluorescent FLIM applications come from plant biology and entomology fields, where in both cases the samples are characterized by several endogenous fluorescence molecules and structures. ??

FLIM approach is sensitive to changes in the microenvironment, such as temperature, pH and viscosity. However, this limit can be exploited to quantify these changes, indeed, numerous optical probes have been developed for both?in vivo?and?in vitro?applications to determine changes in physical conditions including viscosity, temperature, acidity?and oxygenation. Other examples of probes generated to exploit FL advantages are probes for localizing molecular trafficking within a cell or to track ions within individual organelles.

FLIM LABS: a new life for lifetime!

Fluorescence lifetime-based technology has experienced a rapid growth during the last decade, becoming a unique tool for performing real-time, non-invasive analysis in a large variety of scientific fields, from life-sciences to medical surgery and agrifood. Its potential has aroused the interest of industry and a technological shift in the detection and estimation of fluorescence lifetime is around the corner. Nevertheless, devices capable of implementing state-of-the-art fluorescence lifetime analysis that are compact, customizable and affordable, are still incredibly hard to find on the market.

In the light of this, an Italian company decided to support this technological shift by developing instruments that are portable and extremely easy to use, either for beginners or experienced users. Its name is FLIM LABS and its mission is rooted in the intention of democratizing the use of fluorescence lifetime by creating cutting-edge, AI-driven instruments, with a focus on simplicity and modularity. FLIM LABS devices are portable, customizable and cheap, making FLIM and FLA accessible to every user and adaptable to every scientific challenge in both scientific and industrial environment. Lasers, detectors, acquisition cards, adapters, sample holders and software: FLIM LABS is an all-in-one solution that provides a user-friendly hardware and intuitive software for FLA and FLIM data analysis with phasor approach.

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It is important to underline that FLIM LABS devices not only are compatible with imaging systems enabling “classic” FLIM approach, but it can work as a stand-alone kit. In fact, FLIM LABS modules can be built in order to analyze cuvette samples or solid samples, thus providing a system for single-point FL analysis. FLIM LABS Kit is versatile and adaptable to every scientific challenge and its simple and intuitive software allow the user to manage FL data in a user-friendly manner both in FLIM and in FLA. In the next paragraph we will discuss some FLA applications.

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FLIM LABS Kit Applications

The compact design of FLIM LABS components led to the development of the FLIM LABS Kit, a portable device composed of laser, detector and data acquisition card that enables single-point FL analysis. This system for FLA opens new horizons in the scientific field thanks to its innovative real-time and imaging-free approach. By “pointing” the laser at a cuvette or directly at the sample, the FLIM LABS Kit can be used into numerous fields: quality control of compounds or solutions, water analysis for microplastics detection, waste sorting, plant biology, pigments identification in artworks, etc.. The list of FLA applications is very long, for brevity, we selected 2 innovative FLIM LABS projects.

The first project is called FLASH, which stands for Fluorescence Lifetime Analysis to the Screening of Healthcare nanoformulations. FLIM LABS in collaboration with NEST Laboratories of Scuola Normale Superiore di Pisa aim to develop a rapid and cost-effective screening of nanoformulations for commercial product analysis and new candidate formulation validation. Nanoformulations typically involve encapsulating active molecular drugs within specialized carriers to enhance stability and precise delivery to therapeutic targets. Despite the huge research efforts in this field, the number of nanoformulations making it to clinical use remains low with unpredictable outcomes. The main obstacle is represented by the limited understanding of their chemical and physical attributes, including potential variations among production batches. This experimental bottleneck motivated FLIM LABS and SNS to develop a new strategy to evaluate encapsulated luminescent molecules using FLIM LABS Kit. In this project, the researchers were able to discriminate the structural configuration of specific encapsulated nanoformulations in a non-invasive and label-free manner (Figure 4).

Figure 4: Schematic representation of the expected phasor-FLIM signature of water-dissolved drug analyzed in the FLASH project. This compound exists in 3 different configurations (water-dissolved, crystallized and associated with the capsule membrane) that exhibit 3 different lifetimes (respectively 1 ns, 0.2 ns and 4.5 ns). The position of the phasor-FLIM signature allows the identification of the drug configuration without imaging. Image from FLIM LABS. ?

Another innovative project coordinated by FLIM LABS is Laserblood, an EIC pathfinder project that aims to develop a new strategy for Pancreatic Dductal AdenoCarcinoma (PDAC) early diagnosis (Figure 5). Specifically, the goal of Laserblood is to use Fluorescence Lifetime Analysis (FLA) Technology to identify a PDAC marker in patient’s blood samples. This project will be coordinated by FLIM LABS and it involves many partners such as Sapienza Università di Roma, Istituti Fisioterapici Ospitalieri, Policlinico Universitario Campus Bio-Medico, Uniklinikum Erlangen and Crowdhelix. Together, they will develop a FLA-based strategy to predict the progression of pancreatic cancer through the implementation of the innovative FLIM LABS devices. In particular, liquid biopsies (blood samples from mice or patients) will be analyzed using FLIM LABS kit to determine the fluorescence lifetime of a PDAC markers. The goal is to determine the fluorescence lifetime fingerprint of this marker at every stage of the disease, in order to generate a non-invasive in vitro diagnosis test that will help to evaluate patient conditions and the real effectiveness of the treatments.

Figure 5: Laserblood logo. This research project was among the 53 Pathfinder projects selected by the European Innovation Council (EIC) out of almost 800 applications received. Image from FLIM LABS.

Conclusion

FLIM is a widely used tool for biomedical imaging and has advanced the field of microscopy in the past few decades. In this application note, we discussed the advantages of Fluorescence Lifetime-based approaches over classic fluorescence intensity-based imaging. Moreover, we described how FLA can be a valid alternative compared to standard imaging approaches. FLIM and FLA applications are various and can be applied in different fields, from life science to medical and industrial environments. Independently from the application, these strategies exploit an intrinsic fluorescence property and result in a highly sensitive, self-referenced and useful tool. Unfortunately, performing FL-based experiments and managing the data is challenging and requires expert users. For these reasons, emerging companies such as FLIM LABS are determined to provide a technology that is simple to implement and intuitive to use, by simplifying the hardware and improving the software for the data analysis.

References

(1) Datta R, Heaster TM, Sharick JT, Gillette AA, Skala MC. Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications. J Biomed Opt. 2020 May;25(7):1-43. doi: 10.1117/1.JBO.25.7.071203. PMID: 32406215; PMCID: PMC7219965.

(2) Konig K, Riemann I. High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution. J Biomed Opt. 2003 Jul;8(3):432-9. doi: 10.1117/1.1577349. PMID: 12880349.

(3) Berezin MY, Achilefu S. Fluorescence lifetime measurements and biological imaging. Chem Rev. 2010 May 12;110(5):2641-84. doi: 10.1021/cr900343z. PMID: 20356094; PMCID: PMC2924670.

(4) Blacker, T., Mann, Z., Gale, J. et al. Separating NADH and NADPH fluorescence in live cells and tissues using FLIM. Nat Commun 5, 3936 (2014). https://doi.org/10.1038/ncomms4936