Fluorescence intensity and lifetime distribution analysis: toward higher accuracy in fluorescence fluctuation spectroscopy. Solid thick lines show fits of a two-dimensional diffusion model to the CFs. From the amplitudes of ACFs and CCFs, rel.cc. To explore the full potential of SFSCS, we extended the approach to systems containing three spectrally overlapping fluorophores. 2021 Jul 7;22(14):7300. doi: 10.3390/ijms22147300. of ca. The https:// ensures that you are connecting to the Fluorescence fluctuation spectroscopy: a coming of age story. Germany. Fluorescence Fluctuation Spectroscopy. PMC Page 11 and 12: It would be interesting if the authors could determine whether the observed correlation amplitudes are consistent with the probability of the different FPs (mEGFP, mEYFP, mCherry2 with rel cc amplitudes ranging from 0.45-0.79). For both approaches, two excitation wavelengths are currently required for three- and four-species detection. 2015 Mar 16;16(3):6076-92. doi: 10.3390/ijms16036076. 2022 Jun 25;51(3):362-372. doi: 10.3724/zdxbyxb-2022-0017. Thus, FRET has a minor effect (<10%) on the cross-correlation and alone cannot explain the reduced rel.cc. Usually such studies are complex and done on specialized system, this work presents a method easily translatable to commercial system and compatible with standard fluorophores with a well-described analysis pipeline. 8600 Rockville Pike (C) Molecular brightness of LC3-mEYFP obtained from three-species scanning fluorescence spectral correlation spectroscopy (SFSCS) measurements shown in Figure 3 as a function of the ratio of LC3-mEYFP to M2-mCherry2 expression at the PM, in units of protein monomers. FPs linked by a short peptide displayed lower rel.cc., probably due to fluorescence resonance energy transfer (FRET), as previously reported (Foo et al., 2012). (D) Representative CFs (light red: ACF for mApple [A]; dark red: ACF for mCherry2 [Ch2]; gray: CCF calculated for both fluorophores) obtained from SFSCS measurements on the PM of HEK 293T cells co-expressing mp-mApple and mp-mCherry2. Several applications might aim to study interaction within proteins of different abundance, it is important to understand the relative concentration range where the method can be used and provide reliable results. Download it once and read it on your Kindle device, PC, phones or tablets. Pulsed interleaved excitation fluctuation imaging. for 1:1 stoichiometry). values were obtained for the polymerase samples, rel.3C. . For non-correlated data (e.g., G,Y and Y,Ch2 combinations), the second fit routine may converge to negative fit amplitudes, resulting in a distribution of rel.cc. The molecule then drops down to one of the various vibrational levels of the ground electronic state again, emitting a photon in the process. The expected experimental rel.3C. The obtained rel.cc. Aggregation distributions on cells determined by photobleaching image correlation spectroscopy. The number of cells measured is given in parentheses. Incomplete maturation or non-fluorescent photophysical states of FP tags will reduce the amount of detectable ternary complexes. Lower brightness and higher diffusion coefficients were obtained in cells where PA-mEYFP was present at much higher concentrations than PB1-mEGFP (Figure 6figure supplement 1B,C). Epub 2014 Oct 1. In these conditions, sc-FLCCS could not provide reliable results with sixfold (or higher) difference in relative protein abundance, even though the lower abundant protein was tagged with the brighter, longer lifetime FP (tefl et al., 2020). TRICS was performed using three-dimensional RSICS image stacks Iix,y,t detected for three species i. For (DF), the number of cells measured is given in parentheses. . For most UV, visible, and NIR measurements the use of precision quartz cuvettes is necessary. In contrast, we observed substantial CCFs when analyzing measurements on cells expressing mp-mEYFP-mEGFP heterodimers (Figure 1figure supplement 3A). The site is secure. Of note, in our experiments, rel.cc.values > 0.7 (relative to the positive control) were observed for all pair-wise interactions between PC subunits (detected average pair-wise rel.cc. Fluorescence fluctuation spectroscopy provides a powerful toolbox to quantify such interactions directly in living cells. The variance of each point of the ACF was calculated in the multiple tau algorithm (Wohland et al., 2001). 2.5 to 1.0, with 90% to 10% of the signal originating from mEGFP), the ACFs measured for mEYFP showed strong noise when mEGFP was present in much higher amount (i.e., SNR ranging from 2.5 to 0.2, with 90% to 10% of the signal originating from mEYFP). fluctuations within the 3 species of probes would influence the observable. We measured individual emission spectra (Figure 1figure supplement 1) for single-species samples to calculate three-species spectral filters (Figure 3figure supplement 1), which we then used to decompose the signal detected in cells expressing multiple FPs into the contribution of each species. The number of monomers was calculated by dividing the signal detected for PB1-mEGFP and PA-mEYFP in scanning fluorescence spectral correlation spectroscopy (SFSCS) measurements by the average molecular brightness detected for mEGFP and mEYFP fluorophores in the monomeric reference sample (cells co-expressing mp-mEGFP, mp-mEYFP, and mp-mCherry2). Annu Rev Biophys Biomol Struct. For SFSCS measurements, line scans of 256 1 pixels (pixel size 80 nm) was performed perpendicular to the PM with 403.20 s scan time. Some of these methodologies are classical protein aggregation analyses, while others are not yet widely used. ca. Kaliszewski MJ, Shi X, Hou Y, Lingerak R, Kim S, Mallory P, Smith AW. Similarly, molecular brightness and diffusion coefficients can be analyzed as a function of the abundance of each subunit (Figure 6figure supplement 1B,C). This value corresponds to a pf of ca. Biophys J. Firstly, photodecomposition may decrease the intensity of fluorescence over time. Several applications might aim to study interaction within proteins of different abundance, it is important to understand the relative concentration range where the method can be used and provide reliable results. On the other hand, rel.cc. Epub 2021 Jan 29. ACFs calculated for mEGFP and mEYFP were characterized by a higher SNR compared to those for the red FPs mApple and, in particular, mCherry2 (Figure 4AC). To confirm that absolute brightness values are not influenced by the spectral decomposition, we also determined the brightness of mEGFP in cells expressing mp-mEGFP alone (Figure 3G), resulting in values close to 1 (1.08 0.23, mean SD, n = 28 cells). Singapore Additionally, the brightness values determined for mEYFP in both two-species samples were similar, with a relative ratio of 1.07 0.18, as expected. Sample size is given in parentheses in each graph. eCollection 2021 Jun. An official website of the United States government. Normalized brightness of coat proteins. of 0.78 0.19 (mean SD, n = 18 cells) was obtained on cells expressing mp-mCherry2-mApple heterodimers (Figure 1E, Figure 1figure supplement 3B). Shi X, Foo YH, Sudhaharan T, Chong SW, Korzh V, Ahmed S, Wohland T. Determination of dissociation constants in living zebrafish embryos with single wavelength fluorescence cross-correlation spectroscopy. Originally developed for applications in physics and physical chemistry, fluorescence fluctuation spectroscopy is becoming widely used in cell biology. An official website of the United States government. values from the amplitude ratios of the ACFs and CCFs. Having demonstrated the capability of SFSCS to successfully quantify protein interactions and oligomerization, even in the case of three FPs with overlapping emission spectra, we applied this approach in a biologically relevant context. The pixels corresponding to the PM were defined as pixels within 2.5 SD of the peak. values were observed for all combinations: rel.cc.PB1-G,PA-Y = 0.93 0.18 (mean SD, n = 53 cells), rel.cc.PB1-G,PB2-Ch2 = 0.47 0.14, rel.cc.PA-Y,PB2-Ch2 = 0.39 0.14. In contrast, we detected a substantial rel.cc. See this image and copyright information in PMC. To evaluate this fit routine, we have reanalyzed a set of three-species SFSCS measurements on cells co-expressing mp-mCherry2-mEGFP hetero-dimers and mp-mEYFP. The obtained results (Author response image 1) are very similar and do not show significant differences in the average values or variation of parameter estimates. For allowing anisotropy measurements, the addition of two polarization filters is necessary: One after the excitation monochromator or filter, and one before the emission monochromator or filter. Petazzi RA, Aji AK, Chiantia S. Fluorescence microscopy methods for the study of protein oligomerization. Read reviews from worlds largest community for readers. We then performed three-species RSICS measurements in the cell nucleus, where all three proteins are enriched (Figure 6A). Epub 2015 Mar 11. Variance (f2, blue circles) and third central moment (f3, blue circles) were calculated with a moving average (window size F) for a set of 105 random numbers drawn from a Poissonian distribution with average 10. Correlation analysis resulted generally in noisier CFs (Figure 1D) compared to mEGFP and mEYFP. The mEGFP-tag is located at the C-terminus, with a 10aa linker. Signaling pathways in biological systems rely on specific interactions between multiple biomolecules. Molecular aggregation characterized by high order autocorrelation in fluorescence correlation spectroscopy. As these are membrane probes with the fluorescent proteins not expected to interact with the membrane, is there an explanation for the factor 2? We have examined both the day-to-day variability of the relative brightness values of the homo-dimer control and of the absolute brightness of the monomeric reference on the same day. Due to imperfect optical overlap, experimentally detectable rel.3C. The analysis of the intensity fluctuation of a fluorescence signal from a relatively small volume and from a few molecules contains information about the distribution of different species present in the solution and about kinetic parameters of the system. In each block, lines were summed up column-wise and across all spectral channels, and the lateral position with maximum fluorescence was determined. 2016;85:14. Unable to load your collection due to an error, Unable to load your delegates due to an error. J. Homodimers interacting with homodimers of the other species (i.e., AA-BB, AA-CC, BB-CC). . This will be a useful addition to live cell spectroscopy tools to study protein-protein interactions. values for ternary complexes of (i) 1:1:1 or (ii) 2:2:2 stoichiometry, under the assumption that each fluorescent protein can be detected with a probability Pf For simplicity, we assume the same Pf and molecular brightness for all three fluorophore species. Biochim Biophys Acta. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light; typically, but not necessarily, visible light. Int J Mol Sci. Another inner filter effect occurs because of high concentrations of absorbing molecules, including the fluorophore. Since it requires a single excitation line and is compatible with spectrally strongly overlapping FPs, it circumvents optical limitations such as imperfect overlap of the observation volumes. This is the case when measuring the quantum yield or when finding the wavelength with the highest emission intensity for instance. Therefore, we performed SFSCS measurements on HEK 293T cells co-expressing mp-mCherry2 and mp-mApple. 6:1 (yellow points) to ca. We think that this comment might arise from a misunderstanding. This work discusses different analysis methods used in fluctuation spectroscopy and evaluates their use for studying protein-protein interactions, and highlights some of the technical aspects. , : , 196006, -, , 22, 2, . M2 protein oligomerize but its interacting partner LC3 does not, does that mean each single LC3 associates with multiple M2 protein? 2 The authors applied SFSCS to study how the Influenza A virus matrix protein 2 interact with the autophagy protein LC3 and the tetraspanin CD9. Chemphyschem. Cellular homeostasis and aging. Error bars represent mean SD. To estimate the fraction of ternary complexes than can be detected, we compare experimental rel.cc. Biol Reprod. The proteostasis boundary in misfolding diseases of membrane traffic. [7] In fluorescence spectra, it is always seen at a constant wavenumber difference relative to the excitation wavenumber e.g. For the instrumentation utilized in the present work, the time resolution for SFSCS was limited to 0.5 ms. The increase in noise as a result of filtering may prevent detection of weak protein interactions due to the low SNR of CCFs in this case. In analytical chemistry, fluorescence detectors are used with HPLC. The presented approaches provide a powerful toolbox to investigate complex protein interaction networks in living cells and organisms. Here, we present scanning fluorescence spectral correlation spectroscopy (SFSCS), a versatile approach that can be implemented on commercial confocal microscopes, allowing the investigation of interactions between multiple protein species at the plasma membrane. Before Would you like email updates of new search results? I suggest the authors to provide further experiment and eventually simulation to characterize such dynamic range. FCS determines transport and chemical reaction rates from mea surements of spontaneous microscopic thermally driven molecular concentra tion fluctuations. Low rel.cc. Epub 2020 Feb 13. Jorba N, Area E, Ortn J. Oligomerization of the influenza virus polymerase complex in vivo. In the first sample, we observed an increased relative brightness of 1.67 0.38 (mean SD, n = 34 cells) for mEGFP, whereas values around 1 were obtained for mEYFP and mCherry2. We kindly thank Madlen Luckner for providing the plasmids for PA-mEYFP, PB1-mEGFP, and PB2-mCherry2 expression, Thorsten Wohland for providing the PMT-mApple plasmid, and Jelle Hendrix for fruitful discussion. Thank you for submitting your article "Multi-color fluorescence fluctuation spectroscopy in living cells via spectral detection" for consideration by eLife. Signal-to-noise ratio (SNR) (color coded) of autocorrelation functions (ACFs) for mEGFP (A), mEYFP (B), and mCherry2 (C) channels obtained from SFSCS measurements on HEK 293T cells co-expressing mp-mEGFP, mp-mEYFP, and mp-mCherry2 as a function of their signal relative to that of the other two fluorescent protein (FP) species. For a 2:2:2 stoichiometry, we obtained an estimated rel.3C. At each pH value, ca. lower pf corresponds to higher amplitude). We then calculate the maximum rel.cc. 2004 Feb;72(1):1-10. doi: 10.1111/j.1432-0436.2004.07201002.x. G3C(1,1) also includes in its averaged value the other seven correlation values corresponding, for example, to (1=0,1=1,2=1,2=0), (1=1,1=0,2=0,2=-1), etc. An ideal monochromator would only transmit light in the specified range and have a high wavelength-independent transmission. We can therefore assume that for the case of ternary complexes ca. Anna Akhmanova, Accessibility While CD9-mEGFP and LC3-mEYFP showed normalized brightness values close to 1 (BCD9-G = 0.89 0.25, BLC3-Y = 1.02 0.35), suggesting that both proteins are monomers, we observed significantly higher relative brightness values for M2-mCherry2 (BM2-Ch2 = 2.24 0.49). This observation volume This is a three dimensional surface data set: emission intensity as a function of excitation and emission wavelengths, and is typically depicted as a contour map. FRET artifacts can be minimized using appropriate linkers, for example, rigid linker peptides, as presented here. Fu Y, Liu Y, Wen T, Fang J, Chen Y, Zhou Z, Gu X, Wu H, Sheng J, Xu Z, Zou W, Chen B. J Cell Biol. This new volume of Methods in Enzymology continues the legacy of this premier serial by containing quality chapters authored by leaders in the field. The data shown on Page 19/20 were obtained by RSICS measurements in the cytoplasm, not on the plasma membrane. The authors use a range of protein constructs that include between one to four fluorescent proteins, which they use in different compositions to demonstrate that they can analyse all possible interactions of four probes in a single measurement. To calibrate the focal volume, point FCS measurements with Alexa Fluor 488 (Thermo Fisher Scientific) dissolved in water at 20 nM were performed at the same laser power. 2018;217:5163. Auer JMT, Stoddart JJ, Christodoulou I, Lima A, Skouloudaki K, Hall HN, Vukojevi V, Papadopoulos DK. Next, we proceeded with the analysis of PC oligomerization. For this reason and to keep the analysis comparable with our previously published work, we prefer not to modify the already proposed analysis. Since its inception, and As a reference for the absolute brightness, we also determined the relative molecular brightness of mEGFP in cells expressing mp-mEGFP alone, yielding a value of 1.03 0.21 (mean SD, n = 22 cells). CD9 belongs to the family of tetraspanins and is supposedly involved in virus entry and virion assembly (Florin and Lang, 2018; Hantak et al., 2019; Dahmane et al., 2019). Dunsing V, Mayer M, Liebsch F, Multhaup G, Chiantia S. Direct evidence of amyloid precursor-like protein 1 trans interactions in cell-cell adhesion platforms investigated via fluorescence fluctuation spectroscopy. Error bars represent mean SD. Firstly, the distortion arising from the instrument is discussed. Here, we review a suite of methods based on fluorescence fluctuation spectroscopy (FFS) and how they have been employed to interrogate chromatin Fluorescence correlation spectroscopy (FCS) is one of the widely applied techniques to determine diffusion dynamics in two and three dimensions. Four-dimensional image stacks Ix,y,t,k (time-lapse images acquired in k spectral channels) were imported in MATLAB (The MathWorks) from CZI image files using the Bioformats package (Linkert et al., 2010) and further analyzed using custom-written code (Dunsing and Chiantia, 2021). This in now stated in lines 1024-1029. The site is secure. For spectral imaging at different pH values, culture medium was exchanged with buffer containing 140 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, and 20 mM HEPES with pH ranging from 5.0 to 9.2. In contrast to the case of LC3, we did not detect significant binding of M2 with the tetraspanin CD9, a protein that was previously shown to be incorporated into IAV virions and supposedly plays a functional role during the infection process (Shaw et al., 2008; Hutchinson, 2014). The SNR is plotted as a color code in 2-d graphs, for each species as a function of the relative intensity to the other two species. The extension of FCS to four probes, the demonstration of triple correlations and the application to a biological context provides important progress in fluorescence fluctuation spectroscopy and will allow the measurements of complex interactions at the cellular membranes. Solid thick lines show fits of a two-dimensional diffusion model to the CFs. We then apply this scheme to simultaneously investigate the interaction of influenza A virus (IAV) matrix protein 2 (M2) with two cellular host factors, the tetraspanin CD9 and the autophagosome protein LC3, co-expressed in the same cell. RICS ACFs, CCFs, and the TRICS 3CF were calculated. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses. Acc Chem Res. The number of cells measured is given in parentheses. 1A) microscope, which enables selective imaging of the Erdinc Sezgin, The biology of proteostasis in aging and disease. between mEGFP and mEYFP for the polymerase subunits indicates higher order interactions, that is, higher stoichiometry than 1:1 (Kaliszewski et al., 2018). We discuss different analysis methods used in fluctuation spectroscopy and evaluate their use for studying protein-protein interactions. Light scattered by Rayleigh scattering has the same wavelength as the incident light, whereas in Raman scattering the scattered light changes wavelength usually to longer wavelengths. Accessibility Using a scan path perpendicular to the plasma membrane (PM), this technique provides enhanced stability and the ability to probe slow membrane dynamics (Ries and Schwille, 2006), protein interactions (Ries et al., 2009b; Dunsing et al., 2017), and oligomerization (Dunsing et al., 2018) at the PM of cells. Nederveen-Schippers LM, Pathak P, Keizer-Gunnink I, Westphal AH, van Haastert PJM, Borst JW, Kortholt A, Skakun V. Int J Mol Sci. Careers. at some distance from the binding region. G,Y,Ch2 and G,Ch2). We have evaluated the diffusion dynamics of M2 and LC3 (see Author response image 8). Assuming pf values of 75, 77, and 61% (as calculated from the determined relative brightness values of homodimers) for mEGFP, mEYFP, and mCherry2, respectively, pf corrected normalized brightness values of PB1-G = 2.1 0.7 (mean SD, n = 53 cells), PA-Y = 1.8 0.6, and PB2-Ch2 = 2.2 0.7 were obtained (see Materials and methods for details). 2. This is most evident for mCherry2 in four-species applications. Page 19/20: The diffusion coefficient for the hetero-tetramer is only about half of the one determined for hetero-dimers. 3. Finally, hetero-interactions of different molecular species can be detected by cross-correlation analysis of fluctuations emitted by spectrally separated fluorophores (Schwille et al., 1997). values will be lower than these values. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. They found that IAV preferentially interact LC3 but not with CD9 (Figure 3). Nevertheless, the previous analysis could only be performed between two of the three subunits at the same time. For all fluorophore species, ACFs with amplitudes significantly above zero were obtained. Generally, pair-wise cross-correlation analysis can only detect pair-wise interactions between fluorescently tagged protein species. Methods. R01 GM084364/GM/NIGMS NIH HHS/United States, R01 GM086217/GM/NIGMS NIH HHS/United States. FTIR spectroscopic imaging of protein aggregation in living cells. Therefore, unless otherwise noted, similar long rigid linkers were inserted in all constructs used in this study that contain multiple FPs (see Supplementary file 1a). Conventional two-color scanning FCCS has been previously applied to quantify receptor-ligand interactions in living zebrafish embryos (Ries et al., 2009b) and CRISPR/Cas9 edited cell lines to study such interactions at endogenous protein level (Eckert et al., 2020). To evaluate the performance of TRICS, we first analyzed simulated RICS data. Normalized brightness values were calculated by dividing the molecular brightness values detected in each SFSCS measurement by the average brightness obtained for mEGFP, mEYFP, and mCherry2 in cells co-expressing mp-mEGFP, mp-mEYFP, and mp-mCherry2. obtained for pairs of green/yellow and red FPs, e.g. The values obtained for cross-correlation of mEGFP and mEYFP signals (with a rigid linker) are indeed close to the expected value (e.g. values were compared to the values obtained on negative controls (i.e., cells co-expressing free mEGFP, mEYFP, and mCherry) and positive controls (i.e., cells expressing mEYFP-mCherry2-mEGFP heterotrimers) (Figure 6D). government site. Rep., 2018). The LSM 880 instrumentation was funded by the DFG grant INST 336/114-1 FUGG. value of 0.65 detected for mEGFP and mEYFP signal (see Figure 6D) was close to the expected value, hence, almost all complexes containing fluorescent mEGFP and mEYFP were detectable. values obtained for all FP combinations on a positive control (FP heterotrimers) in pair-wise cross-correlation analysis with the expected value of rel.cc. Surprisingly, we found that a high fluorescence background resulted from inefficient dimerization of fluorescent protein (FP)-labeled MS2 coat protein (MCP). Finally, we demonstrated that RSICS allows the quantification of the stoichiometry of higher order molecular complexes, based on molecular brightness analysis for each FP species. Finally, we analyzed the diffusion dynamics of PCs via RSICS (Figure 6F). 2012;12:26232640. Nevertheless, residual FRET may still occur. To this aim, a polygonal region of interest (ROI) was selected in the time- and channel-averaged image frame containing a homogeneous region in the cytoplasm (four-species measurements on FP constructs) or nucleus (three-species measurements on polymerase complex and related controls) of cells. The .gov means its official. about navigating our updated article layout. In the previous section, we showed that SFSCS enables cross-talk-free cross-correlation analysis of two fluorescent species excited with a single laser line, even in the case of strongly overlapping emission spectra. Utrecht University, Within each of these electronic states there are various vibrational states. The diffusion times obtained from mEGFP/mCherry2 ACFs and from the CCF are shown here. This site needs JavaScript to work properly. We agree with the reviewer that it is important to keep FRET into consideration when planning experiments and constructs, in particular because hetero-oligomers are commonly used as a positive control to normalize the rel.cc. Next, we tested whether the same approach can be used for FPs with overlapping emission in the red region of the visible spectrum, which generally suffer from reduced SNR in FFS applications (Dunsing et al., 2018; Foust et al., 2019). Copyright 2012 Biophysical Society. Cells were passaged every 35 days, no more than 15 times. That would give the readers a clear picture what can and cannot be done with this new technique. values (Figure 5G). The number of cells measured is given in parentheses. . Linkert M, Rueden CT, Allan C, Burel J-M, Moore W, Patterson A, Loranger B, Moore J, Neves C, Macdonald D, Tarkowska A, Sticco C, Hill E, Rossner M, Eliceiri KW, Swedlow JR. Metadata matters: Access to image data in the real world. Error bars represent mean SD. For true correlations, only positive values should be obtained. (DF) SFSCS cross-correlation functions (CCFs) (dark blue/ light blue/orange/yellow/red/dark red for CCFs calculated for mEGFP and mEYFP/mEGFP and mApple/mEGFP and mCherry2/mEYFP and mApple/mEYFP and mCherry2/mApple and mCherry2) from measurements described in (AC) (CCFs in (D)/(E)/(F)) corresponding to ACFs shown in (A)/(B)/(C). Ridgeway WK, Millar DP, Williamson JR. Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy. Resultingly, only a small percentage of the excitation light reaches the fluorophores that are visible for the detection system. 2021 Jun 1;120(11):2192-2204. doi: 10.1016/j.bpj.2021.03.035. As a consequence, the overlap of excitation volumes of the two laser lines might be limited, thus reducing the maximum achievable rel.cc., as previously discussed for standard FCCS (Foo et al., 2012). As a simple approximation, we assume therefore that each species, independently of its participation in hetero-complexes, is either (i) exclusively dimeric or (ii) present as a well-defined mixture of monomers and homotrimers. The https:// ensures that you are connecting to the This Arbitrary-region raster image correlation spectroscopy. The mApple_pcDNA3.1+ plasmid was generated by amplifying mApple from PMT-mApple and inserting it into pcDNA3.1+ vector by digestion with KpnI and BamHI. 2022 Jun 8;2(2):None. Cross-correlation analysis may be further affected by FRET between different FP species, potentially reducing experimental rel.cc. Ries J, Yu SR, Burkhardt M, Brand M, Schwille P. Modular scanning FCS quantifies receptor-ligand interactions in living multicellular organisms. 2015;84:435464. Dunsing V, Chiantia S. A fluorescence fluctuation spectroscopy assay of protein-protein interactions at cell-cell contacts. HHS Vulnerability Disclosure, Help To this aim, the variance and third central moment of a series of 105 random numbers, sampled from a Poissonian distribution (with mean f0=10), were calculated within windows with variable size F (Appendix 1figure 1). Fluorescence fluctuation spectroscopy (FFS) is an ensemble of microscopy tools that allow biomolecular dynamics, interactions, and structural changes Notably, rel.cc. , Employing two detectors to reduce autocorrelation curve distortion due to detector afterpulse. 6. Published by Elsevier Inc. All rights reserved. In the first part, we present two-species SFSCS using a single excitation wavelength and strongly overlapping fluorophores. From these amplitudes, the rel.3C. 2001 Sep;25(1):103-10. doi: 10.1006/meth.2001.1219. values are lower than expected (e.g. 2. Then, all aligned line scans were averaged over time and fitted with a Gaussian function. We obtain rel.3C. In particular, we present scanning fluorescence spectral correlation spectroscopy (SFSCS), combining SFCS and FSCS. Since all FPs (mEGFP, mEYFP, mCherry2) exhibit similar pf of 70%, the expected rel.cc. Bookshelf (solely based on pf, i.e. Epub 2021 Apr 5. The significant increase in diffusion time for mCherry2 (taudCh2/taudG=1.35) indicates a ca. To generate GPI-mApple and GPI-mCherry2, mApple and mCherry2 inserts were amplified from PMT-mApple and mCherry2-C1, respectively, and inserted into GPI-mEYFP using restriction by AgeI and BsrGI. Huet S, Avilov SV, Ferbitz L, Daigle N, Cusack S, Ellenberg J. In all samples, ACFs with amplitudes significantly above zero were obtained, with the highest noise level detected for mCherry2 (Figure 5A, C and E). doi: 10.1083/jcb.201709072. The .gov means its official. Based on the experimental q values, we can calculate the expected rel.cc. For each cell, 25 frames were acquired and pixels corresponding to the plasma membrane (PM) semi-manually segmented in the average image (manual selection followed by removal of pixels with intensities below 25% of the maximum pixel intensity in the selected region). This value was close (ca. PMC The mp-mApple plasmid was generated by amplifying mApple from PMT-mApple and inserting it into mp-mCherry2 by digestion with AgeI and BsrGI. In contrast, the addition of a surfactant to a protein which contains a tryptophan which is exposed to the aqueous solvent will cause a blue-shifted emission spectrum if the tryptophan is embedded in the surfactant vesicle or micelle. Fluorescence fluctuation spectroscopy determines the brightness, size, and concentration of fluorescent particles from the intensity bursts generated by individual particles passing through a small observation volume. Spectral scanning data were exported as TIFF files (one file per three spectral channels), imported, and analyzed in MATLAB (The MathWorks, Natick, MA) using custom-written code (Dunsing and Chiantia, 2021). The most versatile fluorimeters with dual monochromators and a continuous excitation light source can record both an excitation spectrum and a fluorescence spectrum. FFS studies are conventionally limited to the analysis of two spectrally distinguished species due to (i) broad emission spectra of fluorophores with consequent cross-talk artifacts and (ii) limited overlap of detection/excitation geometries for labels with large spectral separation. We furthermore apply raster spectral image correlation spectroscopy for the simultaneous analysis of up to four species and determine the stoichiometry of ternary IAV polymerase complexes in the cell nucleus. As we state in the discussion, the relative concentration range may depend on the specific sample, fluorophores and detection efficiency, and thus needs to be determined carefully. As a consequence, the approach provides reliable results only in a certain range of relative protein abundance. of 0.53 0.10 was also determined for mApple and mCherry2 signals, that is, from the CCFs exhibiting the lowest SNR. Progress in Molecular Biology and Translational Science. Can authors put their software in an open repository? values were obtained for all fluorophore combinations that were not expected to show interactions, for example, 0.05 0.08 (mean SD, n = 46 cells) between mEGFP and mEYFP signal in the first sample. More precisely, one would expect a slightly larger observation volume for mCherry2 than for mEGFP (due to the higher wavelength) and thus an amplitude ratio GCh2/GG smaller than 1. The https:// ensures that you are connecting to the In addition, tryptophan is a relatively rare amino acid; many proteins contain only one or a few tryptophan residues. This issue can be circumvented, if needed, by selecting adaptive initial values, for example, obtaining the initial amplitude value from averaging the first points of the CFs (see Figure 3figure supplement 2). Kimura S, Noda T, Yoshimori T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. The pf was calculated from the obtained molecular brightness Bi,2 of FP homodimers of species i: pf=Bi,2Bi,11 (Dunsing et al., 2018). 2008;1130:320-6. doi: 10.1196/annals.1430.040. Finally, we extend our calculations by considering incomplete maturation of FP tags based on the fluorescence probability Pf For simplicity, we assume the same Pf for each FP species, in agreement with the similar Pf values of ca. We establish fluorescence fluctuation spectroscopy (FFS) with nanoscale detection volumes generated by stimulated emission depletion. Since it is a new technique all the limitations of the technique can be discussed in a "limitations" subsection. The technique was independently developed by Watt Webb and Rudolf Rigler during the early 1970s. The obtained rel.cc. Here, we labeled all three subunits using FP fusion constructs and co-expressed PA-mEYFP, PB1-mEGFP, and PB2-mCherry2 in A549 cells. 6075% observed here for mEGFP, mEYFP, and mCherry2. ( a ) Jablonski diagram showing, MeSH GPI-mEGFP was cloned by amplifying mEGFP from an mEGFP-N1 vector and inserting it into GPI-EGFP using digestion with AgeI and BsrGI. 70% of the expected value (Figure 6D). We summarize in this section the main instrumental, conceptual, and sample-related limitations and requirements connected to the multicolor FFS approach employed in this work. Dis Model Mech. The molecular brightness of species i was calculated by dividing the average count rate in the ROI by the particle number determined from the fit to the ACF: Bi=IitNi. Disclaimer, National Library of Medicine The unique features of fluorescence fluctuation spectroscopy (FFS) make this technique attractive for cellular applications. There has also been a report of its use in differentiating malignant skin tumors from benign. In the case of interactions, the fit converges to positive values. values normalized to that of the positive control (i.e., the pair-wise rel.cc. intensities), which we show in Figure 1. . In the second sample (mp-Ch2-G + mp-Y + mp-A), similarly low rel.cc. For the second sample (Ch2-G + Y-A), values significantly above zero, that is, rel.cc.G,Ch2 = 0.46 0.09 (mean SD, n = 23 cells) and rel.cc.Y,A = 0.300.10, were only observed for two fluorophore pairs. Y1 - 2004/6 Reviewer #3 (Recommendations for the authors): This manuscript is a carefully conducted study of multi-color fluorescence fluctuation spectroscopy as applied to plasma membranes. rel.cc.=0.56 and 0.57 for mEGFP/mCherry2 and mEYFP/mCherry2 in heterotrimers). The single-channeled detector can only detect the intensity of one wavelength at a time, while the multichanneled one detects the intensity of all wavelengths simultaneously, making the emission monochromator or filter unnecessary. Would you like email updates of new search results? Finally, ACFs and pair-wise CCFs of fluorescence time series of species i and j were calculated as follows using a multiple tau algorithm: To avoid artifacts caused by long-term instabilities or single bright events, CFs were calculated segment-wise (1020 segments) and then averaged. (AC) Relative cross-correlation for PA-mEYFP and PB2-mCherry2 (A), normalized molecular brightness (B), and diffusion coefficient (C) detected for PA-mEYFP, obtained from three-species RSICS measurements on A549 cells co-expressing PA-mEYFP, PB1-mEGFP, and PB2-mCherry2. In addition, residual FRET (see point 3) may increase the ACF amplitude of the acceptor fluorophore, here mCherry2 (see Foo et al., BJ, 2012). 0.4 ms to 6 ms, see Author response image 1). Detected CCFs showed the expected pattern: all six CCFs were indistinguishable from noise for the first sample with four independent FPs (Figure 5B), whereas large CCF amplitudes were obtained for the pairs mEGFP and mCherry2, as well as mEYFP and mApple in the second sample (Ch2-G + Y-A) (Figure 5D). Epub 2019 Apr 22. Jameson DM, Ross JA, Albanesi JP. Fluorescence correlation spectroscopy (FCS) is a powerful technique for quantification of molecular dynamics, and it has been widely applied in diverse fields, e.g., biomedicine, biophysics, and chemistry. This volume covers fluorescence fluctuation spectroscopy and includes chapters on such topics as Frster resonance energy transfer (fret) with fluctuation algorithms, protein corona on nanoparticles by FCS, and FFS approaches to All solutions, buffers, and media used for cell culture were purchased from PAN-Biotech (Aidenbach, Germany). Only the amplitude ratio for mEGFP and mCherry2 ACFs is shown. Dunsing V, Luckner M, Zhlke B, Petazzi RA, Herrmann A, Chiantia S. Optimal fluorescent protein tags for quantifying protein oligomerization in living cells. PY - 2004/6. Furthermore, they showed the biological application of their methodology by investigating Influenza virus components. Briefly, plasmids were incubated for 20 min with 3 l Turbofect diluted in 50 l serum-free medium, or 15 min with 2 l P3000 and 2 l Lipofectamine3000 diluted in 100 l serum-free medium, and then added dropwise to the cells. The .gov means its official. For all three protein species (PA-mEYFP, PB1-mEGFP, PB2-mCherry2, referred here simply as A, B, and C), normalized brightness values close to the values of FP-homodimers were observed in this work. The average laser power used in two-photon excitation is much higher than in the single-photon excitation scheme. Clipboard, Search History, and several other advanced features are temporarily unavailable. Therein, we stress the point that for two-species measurements, the fact that only one laser line is needed provides a clear advantage of the spectral approach, resulting in higher rel.cc. Solid thick lines show fits of a two-dimensional diffusion model to the CFs. 0.50 for mp-mCherry2-mEGFP hetero-dimers, which is very close the experimental value of 0.45. Effect of bin time on the photon counting histogram for one-photon excitation. 35% difference in size and a slight shift of observation volumes (since the diffusion time obtained from the CCF does not fall in between the values obtained from ACFs for taudCh2 and taudG, see the discussion in Foo et al., 2012). This article is distributed under the terms of the, GUID:22105F3E-6D92-4F8B-951D-6DC572B6F6CF, GUID:EEDABAFC-36DA-4CE8-91E7-1CF6F22F3F00, GUID:231433D0-A16D-4237-ABFC-F63B141FE379, GUID:116C4987-25AA-4CB9-9694-1265F76CA8E3, GUID:075E40C9-5DFE-42CF-A098-C90143454C8F, GUID:CAD6AC5B-DD0E-4308-B62B-3FEC612AE21F, GUID:00FF800F-E30A-456E-8422-A7755BA60705, GUID:8B04CD14-80AD-4C68-A7B8-C94C34811362, GUID:6E7AE3F1-1109-4DED-BB84-87AE1F3BBB5E. This could also be compared with the actual correlation times measured. 8600 Rockville Pike 2015 Apr;92(4):100. doi: 10.1095/biolreprod.114.125781. Weidemann T, Mcksch J, Schwille P. Fluorescence fluctuation microscopy: a diversified arsenal of methods to investigate molecular dynamics inside cells. Overall, the differences obtained for the different fit procedures are small. = 4pf2/(pf + 1)2 0.68 (2:2:2 stoichiometry) for pf = 0.7. To correct this, a beam splitter can be applied after the excitation monochromator or filter to direct a portion of the light to a reference detector. Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy. From a linear interpolation of G3C to (0,0) (using the first point G3C(1,1) and the average of the four points G3C(1,2), G3C(2,1), G3C(2,2), G3C(3,0)) an approximate value of the 3CF amplitude was determined and corrected with the correction factor discussed in Section A1.1. Having demonstrated robust three-species cross-correlation analysis, we aimed to further explore the limits of SFSCS. The emission spectra of the FPs utilized in this study did not depend on cell lines or subcellular localization (Figure 5figure supplement 1) and showed no (mEGFP, mEYFP) or little (mApple, mCherry2) variation with pH over a range of 5.09.2 (Figure 5figure supplement 2). It would be interesting if the authors could comment on the day-to-day variability of their calibrations. (G) Relative cross-correlation values obtained from four-species SFSCS measurements described in (AC). 2004 May;58(5):122A-137A. The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. Using fluorescent proteins to study mRNA trafficking in living cells. 2008;85:273-92. doi: 10.1016/S0091-679X(08)85012-1. This results in a better signal-to-noise ratio, and lowers the detection limit by approximately a factor 10000,[3] when compared to the 180 geometry. Fluorescence spectroscopy is primarily concerned with electronic and vibrational states. Comparison of two types of linker peptides (short flexible or long rigid) between mEGFP and mEYFP showed that the linker length slightly affected rel.cc. The slight underestimation of the rel.3C. values normalized to the positive control were 0.71 for B-C, 0.97 for A-C, and 1.43 for A-B, see Figure 6D). 90% on average) to that obtained for this fluorophore combination in measurements on FP heterotrimers, suggesting very strong association of LC3-mEYFP with M2-mCherry2. values for all three pair combinations, indicating very low amounts of unbound PA, PB1, or PB2 and higher order interactions (see Appendix 1, Section A1.1 for additional details). 2022. They then apply the technique to the interaction of influenza A proteins. The lower rel.cc. In particular, we compared molecular brightness values obtained by SFSCS on HEK 293T cells co-expressing homodimeric mp-2x-mEGFP, mp-mEYFP, and mp-mCherry2 (mp-2x-G + mp-Y + mp-Ch2) to the values measured on cells co-expressing the three monomeric constructs mp-mEGFP, mp-mEYFP, and mp-mCherry2 (mp-G + mp-Y + mp-Ch2). 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