Classroom | New tools for biomedical research: FLIM-FRET

Fluorescence lifetime imaging (FLIM) combined with Förster resonance energy transfer (FRET) has proven to be very beneficial for the study of various structural and cellular dynamics in biomedical research. Since the FRET signal is strongly dependent on the distance of the FRET ligand from the receptor, FRET allows monitoring of molecular interactions. This allows for the study of molecular interactions such as ligand-receptor complexes, protein-protein interactions, interactions of effector proteins with DNA, and the like. On the other hand, with the deep understanding of FRET, scientists have been able to design FRET probes to achieve controlled donor-receptor binding and release efficiency. For FRET probes, we can divide into two types of molecular interactions: FRET in the probe for the receptor and the interaction of the probe with the ligand. Today's small series will detail some of the application examples of FLIM and FRET, and the combination of FLIM-FRET.

“Currently, most bioprobes are based on fluorescence. The increase or decrease in the brightness of a fluorescent probe depends on its concentration. However, the fluorescence intensity is not only affected by the concentration of the subject, but also by the intensity of the illumination, photobleaching, and matrix absorption and shadowing. Effects, etc. In order to avoid these problems as much as possible, scientists tend to prefer ratio-dyes because they allow calibration of background interference. However, fluorescence-based measurements are not very reliable, because even with careful design The calibration method is not ideally reproducible. FLIM provides a better method because the fluorescence lifetime is independent of the dye concentration, the intensity of the illumination, and the absorption and scattering of the fluorescent signal in the sample, so there is no effect on the intermolecular interaction. On the other hand, the fluorescence lifetime is significantly affected by the environment, which allows FLIM to be used to measure the effects of environmental parameters: in some special molecular environments, there is a second dye that absorbs the energy emitted by the first dye— - FRET. This process provides a sensitive way for life measurement For this combination of techniques, more and more probes are developed, and for biomedical research:. FLIM-FRET biosensor "

What is FLIM?

The excitation of fluorescence occurs with the proper photon energy (excitation light). After absorbing photons, the atoms in the molecule release a small amount of heat). Therefore, the emitted light has a longer wavelength than the excitation light. In addition, energy release can also be triggered by interaction with low-energy photons, a condition known as stimulated emission, which is the basic principle of STED ultra-high resolution microscopy. Finally, energy can also be completely released without emitting photons, thereby reducing fluorescence efficiency. Fluorescent images are generally considered to be the two-dimensional intensity distribution of the emitted light. The measurable intensity is a function of the excitation wavelength or emission wavelength or both. The transmitted signal is measured by independent or simultaneous detection of a series of continuous or discontinuous spectra. The end result is used to construct color images, separate signal paths, resolve spatial and temporal interactions of targets, and more. However, the fluorescence process also provides an additional dimension of information: fluorescence lifetime independent of intensity. Fluorescence lifetime can be used to identify and separate fluorescent species under controlled conditions and to reveal details of the molecular environment. After fluorescence excitation, the fluorescent molecules will stay in the excited state for a period of time and then decay back to the ground state. Their time in the excited state is unpredictable because it is a stochastic process controlled by quantum mechanics. A well-known example of this process is the radioactive decay of unstable nuclei, and the half-life of each nuclide is very specific. For fluorescence, the time Ï„ in the excited state is the fluorescence lifetime. In addition to the intensity characteristics, the specificity of the fluorescence lifetime makes it an effective method for distinguishing fluorescent dyes. Therefore, the FLIM image does not represent the intensity of each pixel, but provides the lifetime information of the pixel site [1].

The classic FLIM measurement method (time-dependent single photon counting, TCSPC) does measure fluorescence lifetime events at a single site. The pixel information is the "average time of arrival", that is, the average of all life events measured in the pixel, or one or more feature times in the histogram for curve fitting extraction of the arrival time. To achieve significant results, approximately 400 TCSPCs should be measured per pixel. Therefore, we often hear complaints about fluorescence lifetime measurements: “Life analysis is too complicated!”.

What is FRET?

Förster resonance energy transfer (FRET) is a quenching phenomenon that affects fluorescence measurements. As described above, in addition to emitting photons, the molecules can release all of the excitation energy without generating radiation. If the quencher molecule is also a fluorescent dye, energy can be transferred to the acceptor (A) by resonance so that the donor (D) does not undergo photon radiation. Therefore, the released energy does not dissipate as heat, but is stored in the excited state of the acceptor fluorescent dye [2]. For the conditions under which FRET occurs, the excitation spectrum of the acceptor must overlap with the emission spectrum of the donor, and the two molecules must also be in close contact and arranged appropriately. In this case, "close contact" means that the distance should not exceed about 10 nanometers. The closer the molecule is, the higher the probability of FRET occurring. The relationship between the FRET model distance and the transfer rate kF: where R0 represents the "Förster radius", which is the distance between the acceptors with a transfer efficiency of 50%. The Förster radius is assumed to be a specific value for each donor-receptor pair. The characteristic lifetime of the donor excitation is represented by τD, where r is the actual distance between the two molecules. Since the rate of transfer is inversely proportional to r6, FRET occurs only when the donor and acceptor are in intimate contact. Since this relationship is unique, the initial FRET was only used to estimate the molecular distance ("molecular scale"). In addition, the absolute FRET efficiency is also dependent on the degree of overlap between donor and acceptor excitation and the energy transfer direction of the two fluorescent dyes. Throughout the process: donor D is excited by short-wavelength photons Ex, energy is delivered to receptor A in a non-radiative manner (FRET), and the acceptor emits long-wavelength photons Em.

The occurrence of the following two phenomena can distinguish the occurrence of FRET.

First, the sample (acceptor) emits a different color of fluorescence than the independent donor is excited to emit. For the example in Figure 3, the donor should not emit red light after blue excitation. The method by which this emission can be measured and compared to the donor emission is called "sensitized emission". Sensitized emission quantifies the occurrence of FRET. This measurement can be performed in a live sample, but if a measurement is made with fluorescence intensity, complex corrections are required and can therefore be used for various error calibrations. Again, life measurement is an alternative. The effect of FRET on lifetime will be described in the last section of this article.

Second, donor emission is reduced because some donor excitation will become receptor excitation. This phenomenon can be used for the measurement of "receptor photobleaching", which measures changes in donor emission when eradicating receptors by photobleaching. After the receptor is removed, the donor emission will increase. This method is only suitable for fixed samples. The biological methods of FLIM-FRET biosensors can be collectively referred to as “biosensors”, which can be proteins or peptides, DNA or RNA fragments, cells, etc., or even whole organisms can be used as biosensors, for example, to detect the toxicity of contaminated water. Rate screening freshwater fish in the experiment. In the larger concept, the biosensor may also include a composite sensor for measuring the analyte concentration containing the biological portion and optoelectronics. It is well known, for example, that glucose biosensors - a fast and simple device for controlling blood sugar in diabetic patients. The FLIM-FRET biosensor generally represents a fluorescence phenomenon in which fluorescence (life) is used as a signal and FRET occurs.

So far, we have learned how the FLIM-FRET biosensor works.

The first part is a probe: a protein or peptide that is usually interacting with an analyte (target molecule). Proteins and peptides can be conveniently introduced into the target by genetic engineering methods and are therefore preferred sensor molecules. The most famous example is calmodulin. Calmodulin binds to Ca2+ ions and undergoes a conformational change upon binding or unbinding.

The second part deals with fluorescence. We need a pair of molecules labeled with a fluorescent dye that can be used for energy transfer. The fluorescent dye molecules must be attached to the probe in a manner that they are far apart and cannot transfer energy in a conformational state, but in the alternative conformation state they are close together and arranged in an appropriate position and after triggering Can carry out energy transfer. FRET occurs or disappears when the probe binds or releases the analyte. We can measure the extent of binding or release by Sensitized emission. If a fluorescent protein is used as the fluorescent dye, the entire biosensor can be genetically expressed and can be introduced into any biological target, even a specific subcellular structure. Suitable receptor pairs are, for example, CFP (cyan fluorescent protein) as a donor and YFP (yellow fluorescent protein) as a receptor.

The third part is the life measurement. As mentioned above, Sensitized emission measurements by intensity are prone to significant errors and require cumbersome calibration and complex corrections (mainly because the intensity is affected by many other factors, not just those that have an effect on the FRET process). . Lifespan basically depends only on the type of fluorescent dye and the impact of the environment. In the case of FRET, the fluorescence lifetime will be shortened compared to pure fluorescence emission. In Figure 4, a reservoir (similar to the energy stored in the excited state) is used to explain the cause of life shortening. If the donor can only fluoresce (green), then it is equivalent to only one drain controlling the speed of emptying the reservoir (a). If the donor can transfer energy to the receptor, it would be like opening a second drain (red) in the reservoir, with the result that the water will be drained faster (b). a): In the absence of FRET, the excited state energy bank D of the donor molecule releases energy only by emitting photons. Therefore, the energy of the excited state is determined only by a drain (green droplet) that controls the lifetime of the excited state; b): if the acceptor molecule can absorb the excitation energy from the donor molecule, there is a second exhaust channel for the row Empty excited state energy library. Therefore, the depletion is faster, and therefore, in the case where FRET occurs, the life is shorter. This difference in lifetime is used to detect conformational changes in sensor molecules after binding of a particular analyte (intramolecular FRET). Of course, any interaction between two molecules in the receptor can be studied in the same manner with a suitable FRET. Therefore, if we measure the change in the life of the donor, we can monitor the conformational shift of the entire sensor and thereby monitor changes in analyte concentration. If the combined conformation results in the occurrence of lower FRET, the donor lifetime will increase relative to the analyte concentration and vice versa. In order to measure the fluorescence lifetime of dynamically changing in vivo with sufficient accuracy and speed, the instrument requires high sensitivity, a suitable method for fast frame rate and time-dependent single photon counting [3].

Application examples of FLIM-FRET To illustrate the role of FLIM-FRET in modern biomedical sciences, Xiaobian gives the following two examples. The FLIM-FRET application covers not only biosensors for analyte detection, but also various interactions of proteins, peptides, nucleic acids, and the like. The use of, for example, fluorescent proteins CFP and YFP or derivatives is typically employed.

One Cell Ca2+ Study Ca2+ ions have many important roles in cell physiology, for example, as a second messenger in cell signaling. It is an essential component of muscle contraction, releasing neurotransmitters and contributing to the stabilization of the membrane potential of living cells. It is also an important factor in many enzyme reactions and blood clotting. In order to study these functions, it is necessary to use probes that are rapidly and efficiently metabolized in living cells. One example of a standard FLIM-FRET biosensor is the Ca2+ indicator "cameleon" (please refer to the cited literature). There are a variety of cameleons whose names indicate sensitivity to Ca2+ and its color changes, such as chameleon [4]. The sensor in the Cameleon probe is calmodulin. It undergoes a conformational change upon binding to calcium. There are two fluorescent proteins, such as CFP and YFP, which form a FRET-biosensor. The Cameleon calcium sensor shows FRET when Ca2+ ions bind, ie the lifetime will be shortened in the presence of calcium.

The second well-known messenger in cell signaling of the two-cell cAMP study is cAMP, which was found to be a shape-regulating factor in the development of P. difficile fruiting bodies [5]. cAMP is a cellular intracellular messenger that does not pass through cell membranes and therefore plays a key role in carbohydrate cycling and lipid metabolism. It also involves the regulation of some ion channels. Similar to cameleon, biosensors can be constructed by fusing the cAMP-binding protein Epac with two fluorescent proteins [6] [7]. FLIM-FRET can also be used in a number of popular studies in recent years, such as non-interfering mapping of spatial and temporal changes in metabolic status in cell culture 3D culture [8], FRET-Biosensor (T2AMPKAR) for monitoring AMPK in tumor spheres ( Activity of 5' adenosine-phosphate activated protein kinase). In order to adequately image the 3D structure of the spheroid, the LEICA TCS SP8 DIVE multi-optical subsystem can be used for excitation. These examples are only a small part of the FLIM-FRET application in biomedical research. The interaction of proteins, donors and receptors, DNA and protein or DNA fragments with RNA can be studied by fluorescence lifetime combined with fluorescence excitation energy transfer. Therefore, the research results in the field of FLIM and FRET will increase significantly in the near future!

reference
1.Gerritsen et al: Fluorescence Lifetime Imaging in Scanning Microscopy. Pawley J (Ed) Handbook of Biological Confocal Microscopy, 3rd ed. Springer New York (2006)
2.Förster T: Energiewanderung und Fluoreszenz. Die Naturwissenschaften. 6, pp166-175 (1946) 3. Boringhaus RT: Lifetime - a Proper Alternative. Leica Science Lab (2018)
4. Miyawaki A et al: Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, pp882-887 (1997)
5.Konijn TM et al: The Acrasin Activity of Adenosine-3'-5'-cyclic Phosphate. PNAS 58, pp1152-1154 (1967)
6. Ponsioen B et al: Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator. EMBO reports 5/12, pp1176-1180 (2004)
7. Klarenbeek J et al: Fourth-Generation Epac-Based FRET Sensors for cAMP Feature Exceptional Brightness, Photostability and Dynamic Range: Characterization of Dedicated Sensors for FLIM, for Ratiometry and with High Affinity. PLoS One, 10/4 (2015)
8. Chennell G et al: Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM. Sensors (Basel), 16/8: 1312 (2016).