Quantitative Fluorescence Resonance Energy Transfer Research Articles

Heterodimerization of Human UDP-Glucuronosyltransferase 1A9 and UDP-Glucuronosyltransferase 2B7 Alters Their Glucuronidation Activities.

Human UDP-glucuronosyltransferases (UGTs) play a pivotal role as prominent phase II metabolic enzymes, mediating the glucuronidation of both endobiotics and xenobiotics. Dimerization greatly modulates the enzymatic activities of UGTs. In this study, we examined the influence of three mutations (H35A, H268Y, and N68A/N315A) and four truncations (signal peptide, single transmembrane helix, cytosolic tail, and di-lysine motif) in UGT2B7 on its heterodimerization with wild-type UGT1A9, using a Bac-to-Bac expression system. We employed quantitative fluorescence resonance energy transfer (FRET) techniques and co-immunoprecipitation assays to evaluate the formation of heterodimers between UGT1A9 and UGT2B7 allozymes. Furthermore, we evaluated the glucuronidation activities of the heterodimers using zidovudine and propofol as substrates for UGT2B7 and UGT1A9, respectively. Our findings revealed that the histidine residue at codon 35 was involved in the dimeric interaction, as evidenced by the FRET efficiencies and catalytic activities. Interestingly, the signal peptide and single transmembrane helix domain of UGT2B7 had no impact on the protein-protein interaction. These results provide valuable insights for a comprehensive understanding of UGT1A9/UGT2B7 heterodimer formation and its association with glucuronidation activity. SIGNIFICANCE STATEMENT: Our findings revealed that the H35A mutation in UGT2B7 affected the affinity of protein-protein interaction, leading to discernable variations in fluorescence resonance energy transfer efficiencies and catalytic activity. Furthermore, the signal peptide and single transmembrane helix domain of UGT2B7 did not influence heterodimer formation. These results provide valuable insights into the combined effects of polymorphisms and protein-protein interactions on the catalytic activity of UGT1A9 and UGT2B7, enhancing our understanding of UGT dimerization and its impact on metabolite formation.

Multiplexed imaging for probing RAS-RAF interactions in living cells

GTP-bound RAS interacts with its protein effectors in response to extracellular stimuli, leading to chemical inputs for downstream pathways. Significant progress has been made in measuring these reversible protein-protein interactions (PPIs) in various cell-free environments. Yet, acquiring high sensitivity in heterogeneous solutions remains challenging. Here, using an intermolecular fluorescence resonance energy transfer (FRET) biosensing approach, we develop a method to visualize and localize HRAS-CRAF interactions in living cells. We demonstrate that the EGFR activation and the HRAS-CRAF complex formation can be concurrently probed in a single cell. This biosensing strategy discriminates EGF-stimulated HRAS-CRAF interactions at the cell and organelle membranes. In addition, we provide quantitative FRET measurements for assessing these transient PPIs in a cell-free environment. Finally, we prove the utility of this approach by showing that an EGFR-binding compound is a potent inhibitor of HRAS-CRAF interactions. The outcomes of this work form a fundamental basis for further explorations of the spatiotemporal dynamics of various signaling networks.

Bad is essential for Bcl-xL-enhanced Bax shuttling between mitochondria and cytosol.

Although Bcl-xL has been shown to retrotranslocate Bax from mitochondria to cytosol, other studies have found that Bcl-xL also stabilizes the mitochondrial localization of Bax. It is still unclear what causes the difference in Bcl-xL-regulated Bax localization. Bad, a BH3-only protein with a high affinity for Bcl-xL, may play an important role in Bcl-xL-regulated Bax shuttling. Here, we found that Bcl-xL enhanced both translocalization and retrotranslocation of mitochondrial Bax, as evidenced by quantitative co-localization, western blots and fluorescence loss in photobleaching (FLIP) analyses. Notably, Bad knockdown prevented Bcl-xL-mediated Bax retrotranslocation, indicating Bad was essential for this process. Quantitative fluorescence resonance energy transfer (FRET) imaging in living cells and co-immunoprecipitation analyses showed that the interaction of Bcl-xL with Bad was stronger than that with Bax. The Bad mimetic ABT-737 dissociated Bax from Bcl-xL on isolated mitochondria, suggesting that mitochondrial Bax was directly liberated to cytosol due to Bad binding to Bcl-xL. In addition, MK-2206, an Akt inhibitor, decreased Bad phosphorylation while increasing cytosolic Bax proportion. Our data firmly demonstrate a notion that Bad binds to mitochondrial Bcl-xL to release Bax, resulting in retrotranslocation of Bax to cytosol, and that the amount of Bad involved is regulated by Akt signaling.

Dual-channel structured illumination super-resolution quantitative fluorescence resonance energy transfer imaging

The Structured illumination (SI)-based super resolution fluorescence resonance energy transfer (SR-FRET) imaging technique, known as SISR-FRET, enables the investigation of molecular structures and functions in cellular organelles by resolving sub-diffraction FRET signals within living cells. The FRET microscopy offers unique advantages for quantitatively detecting dynamic interactions and spatial distribution of biomolecules within living cells. The spatial resolution of conventional FRET microscopy is limited by the diffraction limit, and it can only capture the average behavior of these events within the resolution limits of conventional fluorescence microscopy. The SISR-FRET performs sequential linear reconstruction of the three-channel SIM images followed by FRET quantitative analysis by using a common localization mask-based filtering approach. This two-step process ensures the fidelity of the reconstructed SR-FRET signals while effectively removing false-positive FRET signals caused by SIM artifacts. However, the slow imaging speed resulting from the switching of excitation-emission channels in SISR-FRET imaging limits its application in fast imaging scenarios. To address this issue, this study proposes a dual-channel structured illumination super-resolution quantitative FRET imaging system and method. By incorporating an FRET dual-channel imaging and registration module into the imaging pathway, the spatial switching and channel multiplexing of the SISR-FRET excitation-emission channels are achieved. Combining the image reconstruction algorithm with channel sub-pixel registration correction, the dual-channel SISR-FRET technique enhances the temporal resolution by 3.5 times while preserving the quantitative super-resolution FRET analysis. Experimental results are obtained by using a multi-color SIM system to perform super-resolution imaging of living cells expressing mitochondria outer membrane FRET standard plasmids. These experiments validate the improved spatial and temporal resolution of dual-channel SISR-FRET and the fidelity of FRET quantification analysis. In summary, this research presents a novel dual-channel structured illumination super-resolution FRET imaging system and method. It overcomes the limitations of slow imaging speed in SISR-FRET by realizing the spatial switching and channel multiplexing of excitation-emission channels. The proposed technique enhances the temporal resolution while maintaining quantitative analysis of super-resolution FRET. Experimental validation demonstrates the increased spatial and temporal resolution of dual-channel SISR-FRET and the accuracy of FRET quantification analysis. This advancement contributes to the study of molecular structures and functions in cellular organelles, providing valuable insights into the intricate mechanisms of living cells.

Oligomerization of the HBV/HDV functional receptor NTCP expressed in Sf9 insect cell

BackgroundSodium taurocholate co-transportering polypeptide (NTCP, SLC10A1) is a vital bile acid transporter and the functional receptor of hepatitis B and D virus. The oligomerization of NTCP is important for the structural study of its interaction with HBV preS1 peptide. MethodsRecombinant NTCPs were expressed in Sf9 host cell using baculoviruses. Function of recombinant NTCP was verified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method. A quantitative fluorescence resonance energy transfer (FRET) method was established to analyze the interaction between NTCP wild type (WT) and mutants. Co-immunoprecipitation (Co-IP) was used to test the interaction between NTCP variants. ResultsSub-cellular location of recombinant NTCPs varies with the modification of NTCP. Bands of monomer, dimer and oligomers were shown in gel analysis of NTCP. Significant FRET was observed between cyan florescence protein (CFP) tagged NTCP and yellow florescence protein (YFP) tagged NTCP. FRET efficiency between CFP- and YFP-NTCP S267F mutants was lower than WT. Co-IP results showed that S267F interacts with WT NTCP when co-expressed in cell. ConclusionDimer is the predominant form of NTCP expressed in Sf9 when solubilized with detergent. FRET and Co-IP analysis support that NTCP forms oligomers in Sf9 cell. General significanceOur results showed that NTCP formed oligomers in Sf9 cell. Meanwhile the FRET analysis of NTCP variants further elucidated the molecular mechanism of NTCP oligomerization.

Measuring System Calibration Factors by Unmixing the Excitation–Emission Spectra of One Dish of Cells

Abstract Accurate predetermination of the quantum yield ratio (QA/QD) and the extinction coefficient ratio (KA/KD) between acceptor and donor is a prerequisite for quantitative fluorescence resonance energy transfer (FRET) imaging. We here propose a method to measure KA/KD and QA/QD by measuring the excitation–emission spectra (ExEm-spectra) of one dish of cells expressing m (≥3) kinds of FRET constructs. The ExEm-spectra images are unmixed to obtain the weight maps of donor (WD), acceptor (WA), and acceptor sensitization (WS). For each cell, the frequency distribution plots of the WS/WD and WS/WA images are fitted by using a single-Gaussian function to obtain the peak values of WS/WD (SD) and WS/WA (SA). The statistical frequency-SD/SA plots from all cells are fitted by using a multi-Gaussian function to obtain the peak values of both SD and SA, and then the ranges of WS/WD (RSD) and WS/WA (RSA) for each FRET construct are predetermined. Based on the predetermined RSD and RSA values of FRET constructs, our method is capable of automatically classifying cells expressing different FRET constructs. Finally, the WS/WD–WA/WD plot from different kinds of cells is linearly fitted to obtain KA/KD and QA/QD values.

Quantitative Fluorescence Resonance Energy Transfer Analysis on the Direct Interaction of Activation-2b with Histone H3/Switch-3B Protein in Arabidopsis Mesophyll Protoplasts

Interaction between the alteration/deficiency in activation-2b (ADA2b) and histone H3/switch-3B (SWI3B) proteins was evaluated in arabidopsis mesophyll protoplasts by quantitative fluorescence resonance energy transfer (FRET) analysis. Microscopic image showed that ADA2b, SWI3B and H3 proteins colocalized in nucleus, and quantitative FRET measurements showed 0.31 of FRET efficiency (E) for the protoplasts coexpressing ECFP-ADA2b and EYFP-SWI3B, and 0.285 of E for the protoplasts coexpressing ECFP-H3 and EYFP-ADA2b, demonstrating the direct interaction of ADA2b with SWI3B/H3 protein. Collectively, SWI3B and H3 proteins are the inherent components of the ADA2b complex in which ADA2b directly interacts with SWI3B/H3 protein.

Robustness Evaluation of Quantitative Fluorescence Resonance Energy Transfer Imaging Methods in Live Cells

Robustness Evaluation of Quantitative Fluorescence Resonance Energy Transfer Imaging Methods in Live Cells

Highly-efficient quantitative fluorescence resonance energy transfer measurements based on deep learning

Intensity-based quantitative fluorescence resonance energy transfer (FRET) is a technique to measure the distance of molecules in scale of a few nanometers which is far beyond optical diffraction limit. This widely used technique needs complicated experimental process and manual image analyses to obtain precise results, which take a long time and restrict the application of quantitative FRET especially in living cells. In this paper, a simplified and automatic quantitative FRET (saqFRET) method with high efficiency is presented. In saqFRET, photoactivatable acceptor PA-mCherry and optimized excitation wavelength of donor enhanced green fluorescent protein (EGFP) are used to simplify FRET crosstalk elimination. Traditional manual image analyses are time consuming when the dataset is large. The proposed automatic image analyses based on deep learning can analyze 100 samples within 30[Formula: see text]s and demonstrate the same precision as manual image analyses.

Interaction between Bax and Bcl-XL proteins confirmed by partial acceptor photobleaching-based FRET imaging

Exact interaction mechanism between Bax and Bcl-XL, two key Bcl-2 family proteins, is an interesting and controversial issue. Partial acceptor photobleaching-based quantitative fluorescence resonance energy transfer (FRET) measurement, PbFRET, is a widely used FRET quantification method in living cells. In this report, we implemented pixel-to-pixel PbFRET imaging on a wide-field microscope to map the FRET efficiency ([Formula: see text] images of single living HepG2 cells co-expressing CFP-Bax and YFP-Bcl-XL. The [Formula: see text] value between CFP-Bax and YFP-Bcl-XL was 4.59% in cytosol and 11.31% on mitochondria, conclusively indicating the direct interaction of the two proteins, and the interaction of the two proteins was strong on mitochondria and modest in cytosol.

Molecular Analysis of Liquid-Based Cytological Specimen Using Virtually Positive Sputum with Adenocarcinoma Cells.

Liquid-based cytology (LBC) analysis of sputum is a useful diagnostic and prognostic tool for detecting lung cancer. DNA and RNA derived from lung cancer cells can be used for this diagnosis. However, the quality of cytological material is not always adequate for molecular analysis due to the effect of formalin in the commercially available fixation kits. In this study, we examined DNA and RNA extraction methods for LBC analysis with formalin fixation, using lung carcinoma cell lines and sputum. The human non-small cell lung cancer cell lines were fixed with LBC fixation reagents, such as CytoRich red preservative. Quantification of thyroid transcription factor-1 (TTF-1) and actin mRNA, epidermal growth factor receptor (EGFR) DNA in HCC827, H1975, and H1299 cells, and mutation analysis of EGFR in HCC827 and H1975 cells were performed by quantitative PCR (qPCR) and fluorescence resonance energy transfer (FRET)-based preferential homoduplex formation assay (F-PHFA) method, respectively. mRNA and DNA extracted from cell lines using RNA and/or DNA extraction kits for formalin-fixed paraffin-embedded (FFPE) fixed with various LBC solutions were efficiently detected by qPCR. The detection limit of EGFR mutations was at a rate of 5% mutated positive cells in LBC. The detection limit of the EGFR exon 19 deletion in HCC827 was detected in more than 1.5% of the positive cells in sputum. In contrast, the detection limit of the T790M/L858R mutation in H1975 was detected in more than 13% of the positive cells. We also detected EGFR mutations using next generation sequencing (NGS). The detection limit of NGS for EGFR mutation was lower than that of the F-PHFA method. Furthermore, more than 0.1% of positive cells could be cytomorphologically detected. Our results demonstrate that LBC systems are powerful tools for cytopathological and genetic analyses. However, careful attention should be paid to the incidence of false negative results in the genetic analysis of EGFR mutations detected by LBC.

基于光谱分离的定量荧光共振能量转移检测

基于光谱分离的定量荧光共振能量转移检测

Mitofusin 2 but not mitofusin 1 mediates Bcl-XL-induced mitochondrial aggregation.

Bcl-2 family proteins, as central players of the apoptotic program, participate in regulation of the mitochondrial network. Here, a quantitative live-cell fluorescence resonance energy transfer (FRET) two-hybrid assay was used to confirm the homo-/hetero-oligomerization of mitofusins 2 and 1 (MFN2 and MFN1), and also demonstrate the binding of MFN2 to MFN1 with 1:1 stoichiometry. A FRET two-hybrid assay for living cells co-expressing CFP-labeled Bcl-XL (an anti-apoptotic Bcl-2 family protein encoded by BCL2L1) and YFP-labeled MFN2 or MFN1 demonstrated the binding of MFN2 or MFN1 to Bcl-XL with 1:1 stoichiometry. Neither MFN2 nor MFN1 bound with monomeric Bax in healthy cells, but both MFN2 and MFN1 bind to punctate Bax (pro-apoptotic Bcl-2 family protein) during apoptosis. Oligomerized Bak (also known as BAK1; a pro-apoptotic Bcl-2 family protein) only associated with MFN1 but not MFN2. Moreover, co-expression of Bcl-XL with MFN2 or MFN1 had the same anti-apoptotic effect as the expression of Bcl-XL alone to staurosporine-induced apoptosis, indicating the Bcl-XL has its full anti-apoptotic ability when complexed with MFN2 or MFN1. However, knockdown of MFN2 but not MFN1 reduced mitochondrial aggregation induced by overexpression of Bcl-XL, indicating that MFN2 but not MFN1 mediates Bcl-XL-induced mitochondrial aggregation.

Automated E-FRET microscope for dynamical live-cell FRET imaging.

Acceptor-sensitised 3-cube fluorescence resonance energy transfer (FRET) imaging (also termed as E-FRET imaging) is a popular fluorescence intensity-based FRET quantification method. Here, an automated E-FRET microscope with user-friendly interfaces was set up for dynamical online quantitative live-cell FRET imaging. This microscope reduces the time of a quantitative E-FRET imaging from 12 to 3 s. After locating cells, calibration of the microscope and E-FRET imaging of the cells can be performed automatically by clicking 'Capture' button on interfaces. E-FRET imaging was performed on the microscope for living cells expressing different FRET tandem constructs. Dynamical E-FRET imaging on the microscope for live cells coexpressing CFP-Bax and YFP-Bax treated by staurosporine (STS) revealed three Bax redistribution stages: Bax translocation from cytosol to mitochondria within 10 min, membrane insertion with conformational change on mitochondrial membrane within about 30 min, and subsequent oligomerisation within about 10 min. Because of excellent user-friendly interface and stability, the automated E-FRET microscope is a convenient tool for quantitative FRET imaging of living cell. LAY DESCRIPTION: Acceptor-sensitised 3-cube fluorescence resonance energy transfer (FRET) imaging (also termed as E-FRET) is a popular fluorescence intensity-based FRET quantification methods. E-FRET measurements are currently performed manually, and a complete FRET measurement takes about 12 s. E-FRET measurement necessitates not only a skilled operator and specialised equipment but also expertise in the interpretation of FRET signals, a considerable challenge in the application of FRET technology in living cells. Furthermore, manual E-FRET microscope is hard to perform dynamical quantitative FRET measurement, the ever-increasing applications in mapping the biochemical signal transduction within cells. Here, an automated E-FRET microscope with user-friendly interfaces was set up for dynamical online quantitative live-cell FRET imaging. This microscope reduces the time of a quantitative E-FRET imaging from 12 to 3 s. After locating cells, calibration of the microscope and E-FRET imaging of the cells can be performed automatically by clicking 'Capture' button on interfaces. Because of excellent user-friendly interface and stability, the automated E-FRET microscope is a convenient tool for quantitative FRET imaging of living cell.

Quantitative FRET measurement based on spectral unmixing of donor, acceptor and spontaneous excitation-emission spectra.

The spontaneous excitation-emission (ExEm) spectrum is introduced to the quantitative mExEm-spFRET methodology we recently developed as a spectral unmixing component for quantitative fluorescence resonance energy transfer measurement, named as SPEES-FRET method. The spectral fingerprints of both donor and acceptor were measured in HepG2 cells with low autofluorescence separately expressing donor and acceptor, and the spontaneous spectral fingerprint of HEK293 cells with strong autofluoresence was measured from blank cells. SPEES-FRET was performed on improved spectrometer-microscope system to measure the FRET efficiency (E) and concentration ratio (R C ) of acceptor to donor vales of FRET tandem plasmids in HEK293 cells, and obtained stable and consistent results with the expected values. Moreover, SPEES-FRET always obtained stable results for the bright and dim cells coexpressing Cerulean and Venus or Cyan Fluorescent Protein (CFP)-Bax and Yellow fluorescent protein (YFP)-Bax, and the E values between CFP-Bax and YFP-Bax were 0.02 for healthy cells and 0.14 for the staurosporine (STS)-treated apoptotic cells. Collectively, SPEES-FRET has very strong robustness against cellular autofluorescence, and thus is applicable to quantitative evaluation on the protein-protein interaction in living cells with strong autofluoresence.

Superior robustness of ExEm-spFRET to IIem-spFRET method in live-cell FRET measurement.

We recently developed two quantitative fluorescence resonance energy transfer (FRET) measurement methods based on spectral unmixing of emission spectra (IIem-spFRET) and excitation-emission spectra (ExEm-spFRET), respectively. We here evaluated robustness of the two methods by implementing them on a self-assembled quantitative FRET measurement system using the cells expressing different constructs. For the cells with larger signal-to-noise (S/N) ratio (>9), the two methods obtained consistent FRET efficiency (E) values and total concentration ratio (RC) values of acceptor to donor for all constructs; for the cells with 3 < S/N < 9, IIem-spFRET obtained bigger RC values than the expected value for VCV construct; for the cells with S/N < 3, although IIem-spFRET method obtained inaccurate E and RC values for VCV construct, the two methods also obtained consistent E and RC values for all other constructs. Collectively, both ExEm-spFRET and IIem-spFRET methods are very applicable for live-cell FRET measurement, and ExEm-spFRET has superior robustness especially for the cells with low S/N ratio. LAY DESCRIPTION: Fluorescent proteins (FPs)-based fluorescence resonance energy transfer (FRET) has been widely used as a powerful technique to study protein-protein interaction and stoichiometry of macromolecular complexes in living cells. There are two key issues for quantitative FRET measurement especially in living cells: donor emission crosstalk (donor fluorescence is collected in acceptor detection channel) and acceptor excitation crosstalk (acceptor is excited directly under donor excitation) due to the spectral overlap of FPs. Two-wavelengths excitation-based spectral linear unmixing of emission spectra can resolve donor emission crosstalk due to the obvious difference in emission spectra between donor and acceptor, but additional reference is necessary for the correction of acceptor excitation crosstalk. Spectral unmixing of excitation-emission spectra has inherent ability to resolve donor emission crosstalk and acceptor excitation crosstalk simultaneously without additional reference. We recently developed two quantitative FRET measurement methods based on spectral unmixing of emission spectra (IIem-spFRET) and excitation-emission spectra (ExEm-spFRET), respectively. We here evaluate robustness of the two methods by implementing them on a self-assembled quantitative FRET measurement system using the same cells expressing different constructs under different signal-to-noise (S/N) ratios. For the cells with S/N > 9, the two methods obtained consistent FRET efficiency (E) values and total concentration ratio (RC) values of acceptor to donor for all constructs; for the cells with 3 < S/N < 9, IIem-spFRET obtained bigger RC values than the expected value for VCV construct; for the cells with S/N < 3, although IIem-spFRET method obtained inaccurate E and RC values for VCV construct, the two methods also obtained consistent E and RC values for all other constructs. Collectively, our experimental results demonstrate that both ExEm-spFRET and IIem-spFRET methods are very applicable for live-cell FRET measurement, and ExEm-spFRET has superior robustness especially for the cells high E and low S/N ratio.

Molecular and biophysical analysis of apoptosis using a combined quantitative phase imaging and fluorescence resonance energy transfer microscope.

Apoptotic mechanisms are often dysregulated in cancerous phenotypes. Additionally, many anticancer treatments induce apoptosis and necrosis, and the monitoring of this apoptotic activity can allow researchers to identify therapeutic efficiency. Here, we introduce a microscope which combines quantitative phase imaging (QPI) with the ability to detect molecular events via fluorescence (or Förster) resonance energy transfer (FRET). The system was applied to study cells undergoing apoptosis to correlate the onset of apoptotic enzyme activity as observed using a FRET-based apoptosis sensor with whole cell morphological changes analyzed via QPI. The QPI data showed changes in cell disorder strength during the initiation of apoptotic enzymatic activity.

Simultaneous measurement of quantum yield ratio and absorption ratio between acceptor and donor by linearly unmixing excitation-emission spectra.

Quantum yield ratio (QA /QD ) and absorption ratio (KA /KD ) in all excitation wavelengths used between acceptor and donor are indispensable to quantitative fluorescence resonance energy transfer (FRET) measurement based on linearly unmixing excitation-emission spectra (ExEm-spFRET). We here describe an approach to simultaneously measure QA /QD and KA /KD values by linearly unmixing the excitation-emission spectra of at least two different donor-acceptor tandem constructs with unknown FRET efficiency. To measure the QA /QD and KA /KD values of Venus (V) to Cerulean (C), we used a wide-field fluorescence microscope to image living HepG2 cells separately expressing each of four different C-V tandem constructs at different emission wavelengths with 435 nm and 470 nm excitation respectively to obtain the corresponding excitation-emission spectrum (SDA ). Every SDA was linearly unmixed into the contributions (weights) of three excitation-emission spectra of donor (WD ) and acceptor (WA ) as well as donor-acceptor sensitisation (WS ). Plot of WS /WD versus WA /WD for the four C-V plasmids from at least 40 cells indicated a linear relationship with 1.865 of absolute intercept (QA /QD ) and 0.273 of the reciprocal of slope (KA /KD ), which was validated by quantitative FRET measurements adopting 1.865 of QA /QD and 0.273 of KA /KD for C32V, C5V, CVC and VCV constructs respectively in living HepG2 cells.

Magneto-Active Substrates for Local Mechanical Stimulation of Living Cells

Cells can feel and react to the mechanical properties of their environment such as stiffness or geometry by translating mechanical cues into biochemical ones, inducing biochemical and mechanical responses. This process, called mechanotransduction, drives critical functions such as cell differentiation, proliferation and migration. In order to assess to which extend cellular response depends on the temporal and spatial characteristics of the stimulation, it is essential to control temporally and spatially the mechanical cues. Methods have been proposed to apply global and continuous deformations, or local deformations though discrete substrates. Here we propose a novel method to apply mechanical stimuli in a local and dynamic way to cells plated on the continuous surface of deformable substrates. These substrates are made of a soft elastomer (PDMS) in which iron micro-pillars are embedded and actuated by two electromagnets. The amplitude of the surface deformation is controlled by the input current in the coils, and monitored by tracking fluorescent particles underneath the surface. Traction Force Microscopy (TFM) allows us to estimate the stress generated by the pillars, and the cellular mechanical response. Cells adhering to the magneto-active substrates can be stimulated both in traction and compression in the range of contractile cell stresses. Thanks to the compatibility with standard fluorescence techniques, we can furthermore observe the biochemical response of cells with fluorescence techniques such as quantitative Fluorescence Resonance Energy Transfer (FRET).

Improved spectrometer-microscope for quantitative fluorescence resonance energy transfer measurement based on simultaneous spectral unmixing of excitation and emission spectra.

Based on our recently developed quantitative fluorescence resonance energy transfer (FRET) measurement method using simultaneous spectral unmixing of excitation and emission spectra (ExEm-spFRET), we here set up an improved spectrometer-microscope (SM) for implementing modified ExEm-spFRET (mExEm-spFRET), in which a system correction factor (fsc) is introduced. Our SM system is very stable for at least six months. Implementation of mExEm-spFRET with four or two excitation wavelengths on SM for single living cells expressing different FRET constructs obtained consistent FRET efficiency (E) and acceptor-donor concentration ratio (Rc) values. We also performed mExEm-spFRET measurement for single living cells coexpressing cyan fluorescent protein (CFP)-Bax and yellow fluorescent protein (YFP)-Bax and found that the E values between CFP-Bax and YFP-Bax were very low (2.2%) and independent of Rc for control cells, indicating that Bax did not exist as homooligomer in healthy cells, but positively proportional to Rc in the case of Rc<1 and kept constant value (25%) when Rc>1 for staurosporine (STS)-treated cells, demonstrating that all Bax formed homooligomer after STS treatment for 6h.