FRAP, FLIM, and FRET: Detection and analysis of cellular dynamics on a molecular scale using fluorescence microscopy

Abstract

The combination of fluorescent-probe technology plus modern optical microscopes allows investigators to monitor dynamic events in living cells with exquisite temporal and spatial resolution. Fluorescence recovery after photobleaching (FRAP), for example, has long been used to monitor molecular dynamics both within cells and on cellular surfaces. Although bound by the diffraction limit imposed on all optical microscopes, the combination of digital cameras and the application of fluorescence intensity information on large-pixel arrays have allowed such dynamic information to be monitored and quantified. Fluorescence lifetime imaging microscopy (FLIM), on the other hand, utilizes the information from an ensemble of fluorophores to probe changes in the local environment. Using either fluorescence-intensity or lifetime approaches, fluorescence resonance energy transfer (FRET) microscopy provides information about molecular interactions, with Ångstrom resolution. In this review, we summarize the theoretical framework underlying these methods and illustrate their utility in addressing important problems in reproductive and developmental systems.

Figure 1. A simplified Jablonski diagram illustrating the fluorescence process

Photons that are absorbed by fluorochromes undergo a number of different processes including the emission of a photon (hv_F) of lower energy than the photon that is absorbed (hv_A. ). S₀ and S₁ are the ground and first-excited electronic states, respectively, and the horizontal lines represent different vibrational states of the fluorochrome. In condensed phases, following light absorption (hv_A), almost all molecules rapidly relax to the lowest vibrational state of the S₁ state, from which molecules return to the ground state via one of two decay processes: non-radiative or radiative decay. The radiative decay rate (Γ) depends on the electronic properties of an isolated fluorochrome. Molecular interactions, such as dynamic (or collisional) quenching and energy transfer, are treated in the non-radiative decay rate (k). Radiative decay is responsible for fluorescence emission (hv_F), providing detectable photons. k_q, bimolecular quenching constant; [Q], quencher concentration; k_T, energy transfer rate constant; k_j, rate constant for non-radiative processes other than dynamic quenching and FRET (Chang et al., 2007).

Figure 2. Principles of FRAP

FRAP can be broken down into three steps: Pre-bleach, bleach, and post-bleach. In the pre-bleach step, the entire sample is illuminated using a low intensity light source in order obtain a background reference signal. In the bleach step, the molecules within the region of interest are photobleached using a high-intensity laser for a brief period of time (typically ≤ 20 msec). During the post-bleach step, the sample is illuminated with a low-intensity light source, and the movement of bleached molecules out of the original bleach spot and the movement of fluorescent molecules into the original bleach spot are monitored. The post-bleach step provides information about the fractional recovery (three possibilities are shown), and the diffusion coefficient of the mobile fraction (often taken from the half-time for recovery).

Figure 3. Use of FRAP to document the assembly of a fluorescent acrosomal matrix during spermiogenesis

The acrosomal matrix of sperm from the water strider, Aquarius remigis, is formed in post-meiotic spermatogenic cells. Pre-bleach and post-bleach images of round spermatids (RS) and mature sperm bundles (MS) are shown; the red rectangles represent the areas that were photobleached. The recovery curves document that flavin adenine dinucleotide (FAD)-conjugated monomers are freely diffusing in round spermatids, but become immobilized into a rigid helical matrix in mature sperm. Reprinted with permission from Miyata et al., (2011).

Fig. 4. FLIM instrumentation design

Time-domain (A) and frequency-domain (B) lifetime imaging instrumentation and concepts. The variable r indicates parameters that are spatially varying. PD, photodiode; CCD, charge-coupled device (camera); RF synth, RF synthesizer; mod, intensity modulator; thick solid lines, light path; thin solid line, electronic path; dashed line, RF signal path. Reprinted with permission from Urayama and Mycek (2003).

Figure 5. In vivo multi-photon FLIM detection of globally elevated levels of resting calcium in astrocytic networks

A: Multi-photon laser illumination simultaneously excited methoxy-XO4 (blue, Aβ), OGB (green, neurons and astrocytes), and SR-101 (red, astrocytes) through a cranial window. The resulting fluorescence emission was sent to either (1) a three-channel intensity-based photomultiplier tube (PMT) module or (2) a 16-channel multispectral FLIM detector. A single-photon counter (TCSPC) recorded fluorescence lifetime data. B to E: fluorescence decay curves were fit with a calcium-bound lifetime (2359 ps) and an unbound calcium lifetime (569 ps) for each pixel. The pixel data were averaged to obtain single-cell calcium levels, depicted with a calibrated color bar (in C and E). Figure adapted and reprinted with permission from Kuchibhotla et al. (2009).

Figure 6. FRET occurs over short molecular distances

Energy transfer efficiency falls off inversely as the sixth power of the separation distance (R). The distance at which the energy transfer efficiency is 50% is defined by R_o, which is a characteristic of the spectroscopic properties of the donor and acceptor fluorochromes. In most cases, a separation of more than 50 Ångstroms will yield very low energy transfer efficiencies whereas separations on the order of 30 Ångstroms or less can be detected by either steady-state mechanisms (quenching of the donor fluorescence and sensitized emission of the acceptor fluorescence) or a decrease in fluorescence lifetime of the donor.

Figure 7. Detection of FRET by FLIM reveals GRB2 dimerization in the cell and on the plasma membrane

A: GFP-tagged GRB2 (GFP-Grb2, as FRET donor) and RFP-tagged GRB2 (RFP-Grb2, as FRET acceptor) were cotransfected into HEK293T cells, and the GFP lifetime was measured. Lifetime measurements revealed that the cells expressing both donor and acceptor GRB2 show a reduction in GFP lifetime due to FRET. Cells expressing only donor GRB2 show longer lifetimes in the absence of FRET, and act as an internal control for the FLIM experiments. The lifetime histogram reveals two average lifetime peaks corresponding to the shorter (GFP-GRB2/RFP-GRB2 dimer) lifetime, centered at 1.9 ns, and the GFP-GRB2-alone lifetime, centered at 2.2 ns (see arrows). This clearly demonstrates GRB2 can dimerize under the appropriate cellular context. Fluorescence decays were fitted with a single exponential decay model shown at the bottom. B: GRB2 dimers localize to the plasma membrane in the presence of FGFR2. In order to investigate if the dimeric GRB2 could localize to the plasma membrane in the presence of fibroblast growth factor receptor 2 (FGFR2), GFP-GRB2 and RFP-GRB2 were cotransfected together with the FGFR2 in HEK293T cells. FLIM measurement revealed that colocalized GRB2 forms dimers on the plasma membrane (see FLIM image). This confirms that GRB2 dimers are recruited to the cell membrane in the presence of FGFR2. Reprinted with permission from Lin et al. (2012).

Figure 8. Phasor analysis of frequency-domain FLIM for FRET detection of a single chain RhoA biosensor, with normal sub-cellular localization

A: Intensity images of COS7 cell transfected with (RBD-Citrine)-1L-(ECFP-RhoA) before and after LPA stimulation (donor channel). B: Phasor plot of (RBD-Citrine)-1L-(ECFP-RhoA) experiment. C: FRET efficiency calculator of (RBD-Citrine)-1L-(ECFP-RhoA) experiment. D: Painted FLIM image of this (RBD-Citrine)-1L-(ECFP-RhoA) experiment. E: Painted generalized polarization images of an (RBD-Citrine)-1L-(ECFP-RhoA) experiment. Reprinted with permission from Hinde et al. (2012).

Figure 9. FRET studies in transgenic zebrafish hearts

Transgenic zebrafish expressing (A) YFP- CBD1- CFP or (B) a YFP-CBD1-CFP mutant (D447V/D498I) with decreased Ca²⁺ binding affinity. Representative FRET (YFP/CFP) images of transgenic zebrafish hearts (ventricle, shown in pseudocolor) and the corresponding changes in the YFP-to-CFP ratio (black, top traces), YFP (black), and CFP (gray) emissions (bottom traces) during spontaneous contractions are shown. The decrease in the ratio of YFP/CFP emissions correlates with cardiac contraction (not shown). Pseudocolor scale bars are shown to the left of the images. Reprinted with permission from Xie et al. (2008).