Ratiometric Fluorescence Resonance Energy Transfer Research Articles

Comparison of intensity versus lifetime-based approaches for quantitative in vivo FRET imaging.

Fluorescence Resonance Energy Transfer (FRET) enables measurement of the distance separating two light-sensitive molecules. The method has been employed in countless biological applications to monitor inter and intra-molecular interactions and conformations in the 1-10 nm range. Importantly, FRET measurements can be performed with both ratiometric intensity FRET (ratiometric FRET) and fluorescence lifetime imaging-based FRET (FLI-FRET) approaches. Although FRET has long been used for in vitro microscopy, using either the intensity- or lifetime-based approaches, it has recently been extended to in vivo application, for instance to quantify drug-target engagement in animal models of cancer. The in vivo method had thus far only been demonstrated using macroscopic FLI-FRET (MFLI-FRET). Herein, we present a comparison of FRET quantification in vivo using either ratiometric FRET (3-cube, IVIS system) and MFLI-FRET (time-resolved ICCD system) in order to demonstrate the superiority of the latter for in vivo applications.

Water-stable Perovskite Quantum Dots-based FRET Nanosensor for the Detection of Rhodamine 6G in Water, Food, and Biological Samples

The practical application of perovskite quantum dots (QDs) for sensing in the aqueous phase has been restricted by their poor resistance to moisture and oxygen due to their highly ionic characteristic. In this work, we employed silica and phospholipid co-encapsulated water-stable all-inorganic CsPbBr3 QDs as a ratiometric fluorescence resonance energy transfer (FRET)-based fluorescence nanosensor for the detection of Rhodamine 6G (R6G) in food, water, and biological samples. The nanosensor on its own exhibits a strong green emission signal at 518 nm. However, in the presence of R6G, the original fluorescence signal at 518 nm decreases while a new emission peak at 565 nm increases, accordingly, indicating a typical ratiometric fluorescence relationship. The fluorescence intensity ratio (I565/I518) was found to be linearly correlated to the concentration of R6G present. The proposed R6G nanosensor has a linear operating range of 0 – 10 μg/mL and a detection limit of 0.01 μg/mL. In addition, the proposed nanosensor displayed good selectivity towards R6G when tested with other color additives and was also able to detect R6G in tap water, food, and biological samples that contain complex interfering background species. Overall, this work opens a new avenue for water-stable perovskite quantum dots for aqueous-phase sensing applications.

Ratiometric fluorescence resonance energy transfer for reliable and sensitive detection of intracellular telomerase RNA via strand displacement reaction amplification

Human telomerase RNA (hTR) is one essential component of telomerase and is overexpressed in tumor cells. Therefore, the reliable and sensitive detection of hTR is essential for the early cancer diagnosis. Herein, to avoid the false positive signals caused by co-existing components in the cell, a ratiometric fluorescence resonance energy transfer (FRET) strategy was developed to achieve reliable detection of intracellular hTR. Manganese dioxide nanosheets (MnO2NS) with good biocompatibility carry two fluorophore-labelled hairpin DNA probes into the cancer cell and then release the probes via decomposition of MnO2NS by intracellular L-glutathione reduced (GSH). Then, hTR triggered the cyclic strand displacement reaction (SDR) between two hairpin DNA probes to continuously form DNA duplexes, which made two fluorophores close to each other and led to an effective FRET. Fluorescence imaging demonstrated a higher expression level of hTR in HeLa cells than that in normal HL-7702 cells. The high specificity of hairpin DNA probes and SDR make it easy to discriminate the single-base mutation. Therefore, it provides a highly sensitive, simple and reliable method for the extracellular and intracellular detection of hTR.

Ratiometric FRET Encoded Hierarchical ZrMOF @ Au Cluster for Ultrasensitive Quantifying MicroRNA In Vivo.

Here, Zirconium metal-organic frameworks @ gold (ZrMOF @ Au) cluster architectures have been fabricated and then functionalized with two fluorescent dyes (Quasar [QS] and Cyanine5.5 [Cy5.5]) through deoxyribonucleic acid hybridization, to form a fluorescence resonance energy transfer (FRET) encoded ZrMOF @ Au-QS/Cy5.5 complex. In the presence of the target intracellular microRNA (miR)-21, the fluorescence of Cy5.5 at 705nm (F705 ) decreases and the fluorescence of QS at 665nm (F665 ) increases when Cy5.5 is released from the surface of ZrMOF @ Au-QS/Cy5.5. The change in the fluorescence ratio (F705 /F665 ) shows an outstanding linear range of 0.006-67.9 amol/ngRNA , and the limit of detection is 4.51 zmol/ngRNA in living cells. The high ratio loading of nucleic acid on surface of ZrMOF @ Au cluster and two fluorescence encoded signal enables better sensitivity and reliability. Zeptomolar sensitivity and good linearity against target affords distinct imaging-based monitoring of the cancer marker miR-21 both in living cells and in vivo. At the same time, the architecture displays remarkable photothermal conversion efficiency (53.7%) and gives rise to outstanding therapy ability in vivo. This strategy offers new avenues for the intelligent quantification of miRNAs for simultaneous diagnoses and treatments of early-stage cancers.

Graphdiyne/graphene quantum dots for development of FRET ratiometric fluorescent assay toward sensitive detection of miRNA in human serum and bioimaging of living cancer cells

An effective ratiometric fluorescence resonance energy transfer (FRET) assay developed for the sensitive and specific measurement of microRNAs-21 (miRNA-21) using graphdiyne quantum dots (GDQDs) as the new fluorescent probe. GDQDs with uniform size, narrow defined tunable emission peak and good crystallization synthesized via a classical solvothermal approach. In the sensing strategy, GDQDs modified with DNA probe exhibited strong fluorescence (FL) signal at 405 nm, while in the presence of graphene quantum dots (GQDs) duo to Förster-resonance-energy transfer process between GDQDs and GQDs, the fluorescence of GDQDs was efficiently quenched, meanwhile, a new fluorescence signal at 505 nm for GQDs provided. In the presence of miRNA-21, with the hybridization between miRNA-21 and GDQDs modified with DNA probe, the distance of GDQDs (donor) and GQD (acceptor) is increased, and the intensity fluorescent signal of GDQD recovered, which confirmed by decreasing of the average life times for GDQDs-DNA probe from 1.99 to 0.61ns after addition of GQDs, while the average life time increased again to 1.26 ns in the presence of micRNA-21 target. The ratio of the FL intensity (F405/F505) behaved precise and sensitive analytical efficiency for miRNA-21 detection with a good linear range from 5 pM to 200 nM and a detection limit of 0.5 pM (S/N = 3). More importantly, this RNA sensor was feasible to detect target microRNA-21 in the human serum sample and MCF-7 cell lines with a detection limit of 4 cells/10 μL. Furthermore, due to low cytotoxicity of GDQDs multicolour imaging of MDA-MB231 living cancer cells also utilized.

Dual dye-labeled G-quadruplex aptasensor for detection of thallium(I) using ratiometric fluorescence resonance energy transfer

A fluorescent probe was developed for ratiometric detection of thallium ions in mineral water samples by modifying a G-rich aptamer (PS2.M − 7) with a fluorescence donor (Cyanine-3, Cy3) and a quencher (Cyanine-5, Cy5). The probe had a random coil structure that changed into a G-quadruplex structure upon binding with Tl+. This change in structure decreased the distance between the donor and acceptor moieties, which resulted in fluorescence resonance energy transfer between Cy3 and Cy5. Under optimized conditions, the limit of detection and linear concentration range for Tl+ were 30.1 μM (3σ) and 10 μM–10 mM (R2 = 0.9981), respectively. This simple and cost-effective fluorescence sensor provided satisfactory results for detection of thallium ions in spiked mineral water samples.

Carbon Nanodots as a Multifunctional Fluorescent Sensing Platform for Ratiometric Determination of Vitamin B2 and “Turn-Off” Detection of pH

In this work, we synthesized carbon nanodots (CNDs) by a one-pot hydrothermal method to carbonize precursors of dry carnation petals and polyethylenimine. The obtained CNDs possess favorable photostability, good biocompatibility, and excellent water solubility, which can serve as a dual-responsive nanosensor for the determination of vitamin B2 (VB2) and pH. A unique ratiometric fluorescence resonance energy transfer probe was developed through a strong interaction between VB2 and surface moieties of CNDs. CNDs emitted at 470 nm; however, in the presence of VB2, an enhanced emission peak was clearly observed at 532 nm. The value of I532/I470 exhibits a stable response to the VB2 concentration from 0.35 to 35.9 μM with a detection limit of 37.2 nM, which has been used for VB2 detection in food and medicine samples and ratiometric imaging of VB2 in living cells with satisfying performance. In addition, the proposed CNDs also displayed pH-sensitive behavior and can be a turn-off fluorescent sensor to monitor pH. The fluorescent intensity at 470 nm is a good linear response against pH values from 3.6 to 8, affording the capability as a single-emissive nanoprobe for intracellular pH sensing.

Controlling dopamine binding by the new aptamer for a FRET-based biosensor

Dopamine is one of the most important neurotransmitters. A high-quality DNA aptamer for dopamine was reported in 2018. However, fundamental understanding of its binding and folding is lacking, which is critical for related biosensor design. Herein, we performed careful assays using a label-free technique called isothermal titration calorimetry (ITC) to study its secondary structure. We divided this aptamer into four regions and individually examined each of them. We confirmed two stems, but the third stem is believed to be part of a loop. The aptamer was then truncated. The original aptamer had a Kd of 2.2 ± 0.3 μM at 25 °C. Shortening the structure by one or two base pairs increased the Kd to 6.9 and 44.4 μM, respectively. Dopamine binding was promoted by both increasing the Mg2+ concentration and decreasing the temperature. At 5 °C, a Kd of 0.4 μM was achieved. Based on this understanding, we designed two fluorescence resonance energy transfer (FRET) quenching biosensors that differ only by a base pair. The shorter sensor had 3-fold higher sensitivity and a detection limit of 0.9 μM. In 1% fetal bovine serum, the sensor retained a similar limit of detection of 1.14 μM. A two-fluorophore ratiometric FRET sensor was also demonstrated with a low detection limit of 0.12 μM. This work indicated the feasibility of designing folding-based sensors for dopamine, and this design can be extended to other sensing modalities such as electrochemistry and colorimetry.

Ratiometric fluorescence resonance energy transfer aptasensor for highly sensitive and selective detection of Acinetobacter baumannii bacteria in urine sample using carbon dots as optical nanoprobes

Development of sensitive and selective analytical method for accurate diagnosis of Acinetobacter baumannii (Ab) bacteria in biological samples is a challenge. Herein, we developed an ingenious ratiometric fluorescent aptasensor for sensitive and selective detection of (Ab) bacteria based on fluorescence resonance energy transfer (FRET) between ortho-phenylenediamines carbon dot (o-CD), nitrogen-doped carbon nanodots (NCND) as donor's species and graphene oxide (GO) as acceptor. NCND that assembled onto the edge of graphene oxide (GO) exhibited quenched photoluminescence emission, and with the absorption of the modified o-CD with aptamer (o-CD-ssDNA) onto the graphene oxide surface the fluorescence of o-CD was efficiently quenched. The aptamer (ssDNA) as a biorecognition element is bound with A. baumannii specifically which releases the o-CD-ssDNA from GO and the recovery of the fluorescence signal of o-CD, while the fluorescence intensity of NCND only slightly altered and acted as the reference signal in ratiometric fluorescence assay. The fluorescence intensity ratio (I550 nm/I440nm) varied from 2.0 to 10.0 with the concentration of bacteria changing from 2.0 × 103 to 4.5 × 107 cfu/mL and the low detection limit of 3.0 × 102 cfu/mL (S/N = 3). The feasibility of the developed aptasensor for selective detection of A. baumannii in urine sample with satisfactory results was also demonstrated.

Sensitive detection of intracellular telomerase activity via double signal amplification and ratiometric fluorescence resonance energy transfer.

As an important and universal tumor marker, the reliable and in situ detection of intracellular telomerase activity is crucial for cancer diagnosis. Herein, a ratiometric fluorescence resonance energy transfer (FRET) method was developed for detecting intracellular telomerase activity. It takes full advantage of manganese dioxide nanosheets (MnO2NS) that can carry DNA probes with different conformations into cells and then completely release the DNA probes via decomposition of MnO2NS by intracellular reduced glutathione (GSH). In the presence of telomerase, a telomere substrate (TS) could be extended to form long telomerase extension products (TEPs), which trigger the cycling strand displacement reaction (SDR) between two fluorophore-labeled hairpin DNA probes to form lots of DNA duplexes. The close contact of two fluorophores led to an effective ratiometric FRET for reliable detection of telomerase activity. Fluorescence confocal imaging demonstrated that the activity of telomerase in tumor cells was reliably detected. The inhibition of telomerase activity by an inhibitor resulted in a decrease in FRET signal. For extracellular detection, the FRET ratio (FA/FD) shows a good linear relationship with the number of HeLa cells in the range of 20-1000 cells. Therefore, it offers a more facile method for reliable and sensitive detection of intracellular telomerase activity.

Activity-based ratiometric FRET probe reveals oncogene-driven changes in labile copper pools induced by altered glutathione metabolism

Copper is essential for life, and beyond its well-established ability to serve as a tightly bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low-molecular-weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest-activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and rhodamine dyes through a Tris[(2-pyridyl)methyl]amine bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and reduced/oxidized glutathione (GSH/GSSG) ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader cross-talk between metal and redox pathways in health and disease with activity-based probes.

Ratiometric enhanced fluorometric determination and imaging of intracellular microRNA-155 by using carbon dots, gold nanoparticles and rhodamine B for signal amplification

An ultrasensitive and highly reliable ratiometric assay is described for the determination of microRNA-155. It works at the attomolar concentration level and hashigh selectivity which warrants its potential application in cancer biomarker tracking. The excellent performance of this method results from (a) the use of a hybrid conjugate prepared from Rhodamine B (RhB), carbon dots (CDs) and probe-microRNA, and (b) from the measurement of fluorescence resonance energy transfer (FRET) that is observed in the AuNP/target-microRNA system as a result of RNA hybridization. The dye RhB (emission peak at 580nm) serves as an internal reference. The sensitivity of this assay is increased by about 30% because of the broad emissions of CDs (489nm and 665nm) through a sequential FRET phenomenon. RhB-CDs were covalently bio-conjugated to probe microRNA. In the presence of AuNPs, the fluorescence of the CDs is quenched, while in the presence of microRNA-155, the ratio of fluorescences at 489 and 665nm (I489/I665) is enhanced again. A linear relationship exists between the ratio of fluorescence and the concentration of microRNA-155 in the range from 1 aM to 0.1μM, and the detection limit is 0.3 aM. The assay was applied to quantitative studies of target microRNA-155 in multiple pathways associated with cancer progression in biological fluids include human serum samples and cancer cells. The nanoprobe also deliver clear signal to microRNA target in fixed and lived MDA-MB-231 cells. Graphical abstract A ratiometric FRET sensing method used for microRNA-155 detection at aM concentration level using CDs and AuNPs as donor-acceptor respectively and Rhodamine B as amplification reagent. The application of assay for imaging of microRNA-155 in fixed and live MDA-MB-231 cells is demonstrated.

G-quadruplex specific dye-based ratiometric FRET aptasensor for robust and ultrafast detection of toxin

G-quadruplex specific dyes are powerful tools for probing nucleic acid structures. Among nucleic acids, aptamers are of great interest, and widely exploited to construct versatile bioassays. Herein, based on G-quadruplex selective dye, thioflavin T (ThT), for probing the intrinsic structure of aptamers, we proposed a ratiometric fluorescence resonance energy transfer (FRET) aptasensor enabling robust and ultrafast detection of toxin. The binding of target ochratoxin A (OTA) would destruct the G-quadruplex structure of aptamer. It would lead to the detachment of ThT dye from aptamer which diminished the FRET effect between ThT and terminal-labeled dye, thus allowing quantification of OTA via FRET signals. The FRET aptasensor would confer an enhancement of 76.9% of signal to background ratio compared to the ThT-based non-FRET aptasensor. Remarkably, the FRET mechanism would eliminate the signal fluctuation resulted from varied probe concentration, thus benefiting the robustness of the assay. The aptasensor could achieve a detection of limit of 0.38 ng/mL for OTA detection. And the detection of OTA could be finished within 30 s. Besides, the assay was successful in analyzing OTA in coffee and oat samples with recoveries rate of 93.93%–107.59%. Therefore, G-quadruplex specific dye-based probing and FRET method would be a compelling design strategy for aptasensor, and may facilitate their practical application in food safety and environmental screening.

Universal Ti3C2 MXenes Based Self-Standard Ratiometric Fluorescence Resonance Energy Transfer Platform for Highly Sensitive Detection of Exosomes.

Exosomes, as novel noninvasive biomarkers for disease prediction and diagnosis, have shown fascinating prospects in monitoring cancer-linked public health issues. Herein, a unique Cy3 labeled CD63 aptamer (Cy3-CD63 aptamer)/Ti3C2 MXenes nanocomplex was constructed as a self-standard ratiometric fluorescence resonance energy transfer (FRET) nanoprobe for quantitative detection of exosomes. The Cy3-CD63 aptamer can be selectively adsorbed onto the Ti3C2 MXene nanosheets by hydrogen bond and metal chelate interaction between the aptamer and MXenes, and the fluorescence signal from Cy3-CD63 aptamer was quenched quickly owing to the FRET between the Cy3 and MXenes. The fluorescence of Cy3 greatly recovered after the addition of the exosomes which can specifically combine with the aptamer and release from the surface of Ti3C2 MXenes due to the high affinity between the aptamer and CD63 protein on exosome surface. Meanwhile, the self-fluorescence signal of MXenes in the whole process showed little change, which can be used as a standard reference. Based on the self-standard turn-on FRET biosensing platform the detection limit of exosome was determined as 1.4 × 103 particles mL-1, which was over 1000× lower than that of conventional ELISA method. This fluorescence sensor can also be used for the identification of multiple biomarkers on the exosome surface and different kinds of exosomes, combining with the fluorescent confocal scanning microscope image. The proposed strategy not only provides a universal nanoplatform for exosomes, but also can be extensively expanded to multiple biomarkers detection, which may promise the prospect of MXenes as robust candidates in biological fields.

Role of quantum dot in designing FRET based sensors

Nowadays Fluorescence Resonance Energy Transfer (FRET), which involves the nonradiative transfer of excitation energy from an excited state donor to a proximal ground-state acceptor, may be regarded as a useful tool in the design of ratiometric sensors. Recently, quantum dots (QDs), a kind of semiconductor nanoparticles, have been employed to modulate FRET. Compared with conventional FRET, quantum dot mediated FRET (QD-FRET) is advantageous because of their unique photophysical properties such as size tunable, narrow emission spectra but broad excitation spectra, strong signal intensity, high photostability, and good biocompatibility. Owing to these superior properties QDs have obtained huge development in many fields, such as drug testing, cell imaging, virus detection, molecular biology, medical diagnosis, electron transfer detection and so on. In view of above-mentioned achievements, QDs is a kind of promising material which can definitely be used as donors or acceptors in the FRET system. This review highlights the modern development of sensitive and selective ratiometric FRET based sensors where QDs has been used as fluorophores.

Ratiometric fluorescent pH nanoprobes based on in situ assembling of fluorescence resonance energy transfer between fluorescent proteins.

pH-dependent protein adsorption on mesoporous silica nanoparticle (MSN) was examined as a unique means for pH monitoring. Assuming that the degree of protein adsorption determines the distance separating protein molecules, we examined the feasibility of nanoscale pH probes based on fluorescence resonance energy transfer (FRET) between two fluorescent proteins (mTurquoise2 and mNeonGreen, as donor and acceptor, respectively). Since protein adsorption on MSN is pH-sensitive, both fluorescent proteins were modified to make their isoelectric points (pIs) identical, thus achieving comparable adsorption between the proteins and enhancing FRET signals. The adsorption behaviors of such modified fluorescent proteins were examined along with ratiometric FRET signal generation. Results demonstrated that the pH probes could be manipulated to show feasible sensitivity and selectivity for pH changes in hosting solutions, with a good linearity observed in the pH range of 5.5-8.0. In a demonstration test, the pH probes were successfully applied to monitor progress of enzymatic reactions. Such an "in situ-assembling" pH sensor demonstrates a promising strategy in developing nanoscale fluorescent protein probes. Graphical abstract Working principle of the developed pH sensor TNS; and FRET Ratio (I528/I460) as a function of pH under different protein feed ratios (mNeonGreen to mTurquoise2).

Silver enhanced ratiometric nanosensor based on two adjustable Fluorescence Resonance Energy Transfer modes for quantitative protein sensing

We developed a silver decahedral nanoparticles (Ag10NPs)-enhanced ratiometric Fluorescence Resonance Energy Transfer (FRET) nanosensor based on two adjustable FRET modes. Alexa Fluor 488 (Alexa) and Cyanine3 (Cy3)-aptamer-Black hole quencher-2 (BHQ-2) were bound with Ag10NPs to form the ratiometric FRET nanosensor (Ag-Alexa/Cy3/BHQ-2). Alexa act as donor and Cy3 as acceptor in the FRET mode 1 while Cy3 was donor and BHQ-2 was acceptor in the FRET mode 2. In the absence of platelet-derived growth factor (PDGF-BB), the fluorescence intensity of Alexa was lowest while that of Cy3 was highest. Upon the addition of PDGF-BB, Cy3-aptamer-BHQ-2 binds with PDGF-BB resulting in the change of structure of aptamer. The fluorescence intensity of Alexa increased while that of Cy3 decreased. In addition, the fluorescence intensity ratio of Alexa to Cy3 increased remarkably with PDGF-BB concentration in the range of 0.4–400ng/mL. A good linear response was obtained when the PDGF-BB concentrations were in the range of 3.1–200ng/mL, with the limit of detection at 0.4ng/mL. When compared to sensors without Ag10NPs (Alexa/Cy3/BHQ-2) and one without BHQ-2 (Ag-Alexa/Cy3), the new nanosensor Ag-Alexa/Cy3/BHQ-2 showed remarkable increase in sensitivity.

Fast, Ratiometric FRET from Quantum Dot Conjugated Stabilized Single Chain Variable Fragments for Quantitative Botulinum Neurotoxin Sensing.

Botulinum neurotoxin (BoNT) presents a significant hazard under numerous realistic scenarios. The standard detection scheme for this fast-acting toxin is a lab-based mouse lethality assay that is sensitive and specific, but slow (∼2 days) and requires expert administration. As such, numerous efforts have aimed to decrease analysis time and reduce complexity. Here, we describe a sensitive ratiometric fluorescence resonance energy transfer scheme that utilizes highly photostable semiconductor quantum dot (QD) energy donors and chromophore conjugation to compact, single chain variable antibody fragments (scFvs) to yield a fast, fieldable sensor for BoNT with a 20-40 pM detection limit, toxin quantification, adjustable dynamic range, sensitivity in the presence of interferents, and sensing times as fast as 5 min. Through a combination of mutations, we achieve stabilized scFv denaturation temperatures of more than 60 °C, which bolsters fieldability. We also describe adaptation of the assay into a microarray format that offers persistent monitoring, reuse, and multiplexing.

FRET Imaging by Laser Scanning Cytometry on Large Populations of Adherent Cells.

The application of FRET (fluorescence resonance energy transfer) sensors for monitoring protein-protein interactions under vital conditions is attracting increasing attention in molecular and cell biology. Laser-scanning cytometry (LSC), a slide-based sister procedure to flow cytometry, provides an opportunity to analyze large populations of adherent cells or 2-D solid tissues in their undisturbed physiological settings. Here we provide an LSC-based three-laser protocol for high-throughput ratiometric FRET measurements utilizing cyan and yellow fluorescent proteins as a FRET pair. Membrane labeling with Cy5 dye is used for cell identification and contouring. Pixel-by-pixel and single-cell FRET efficiencies are calculated to estimate the extent of the molecular interactions and their distribution in the cell populations examined. We also present a non-high-throughput donor photobleaching FRET application, for obtaining the required instrument parameters for ratiometric FRET. In the biological model presented, HeLa cells are transfected with the ECFP- or EYFP-tagged Fos and Jun nuclear proteins, which heterodimerize to form active AP1 transcription factor.

High throughput FRET analysis of protein–protein interactions by slide‐based imaging laser scanning cytometry

Laser scanning cytometry (LSC) is a slide-based technique combining advantages of flow and image cytometry: automated, high-throughput detection of optical signals with subcellular resolution. Fluorescence resonance energy transfer (FRET) is a spectroscopic method often used for studying molecular interactions and molecular distances. FRET has been measured by various microscopic and flow cytometric techniques. We have developed a protocol for a commercial LSC instrument to measure FRET on a cell-by-cell or pixel-by-pixel basis on large cell populations, which adds a new modality to the use of LSC. As a reference sample for FRET, we used a fusion protein of a single donor and acceptor (ECFP-EYFP connected by a seven-amino acid linker) expressed in HeLa cells. The FRET efficiency of this sample was determined via acceptor photobleaching and used as a reference value for ratiometric FRET measurements. Using this standard allowed the precise determination of an important parameter (the alpha factor, characterizing the relative signal strengths from a single donor and acceptor molecule), which is indispensable for quantitative FRET calculations in real samples expressing donor and acceptor molecules at variable ratios. We worked out a protocol for the identification of adherent, healthy, double-positive cells based on light-loss and fluorescence parameters, and applied ratiometric FRET equations to calculate FRET efficiencies in a semi-automated fashion. To test our protocol, we measured the FRET efficiency between Fos-ECFP and Jun-EYFP transcription factors by LSC, as well as by confocal microscopy and flow cytometry, all yielding nearly identical results. Our procedure allows for accurate FRET measurements and can be applied to the fast screening of protein interactions. A pipeline exemplifying the gating and FRET analysis procedure using the CellProfiler software has been made accessible at our web site.