## Dual-Color Fluorescence Cross-Correlation Spectroscopy on a Single Plane Illumination Microscope (SPIM-FCCS) |

Optics Express, Vol. 22, Issue 3, pp. 2358-2375 (2014)

http://dx.doi.org/10.1364/OE.22.002358

Acrobat PDF (2786 KB)

### Abstract

Single plane illumination microscopy based fluorescence correlation spectroscopy (SPIM-FCS) is a new method for imaging FCS in 3D samples, providing diffusion coefficients, flow velocities and concentrations in an imaging mode. Here we extend this technique to two-color fluorescence cross-correlation spectroscopy (SPIM-FCCS), which allows to measure molecular interactions in an imaging mode. We present a theoretical framework for SPIM-FCCS fitting models, which is subsequently used to evaluate several test measurements of *in-vitro* (labeled microspheres, several DNAs and small unilamellar vesicles) and *in-vivo* samples (dimeric and monomeric dual-color fluorescent proteins, as well as membrane bound proteins). Our method yields the same quantitative results as the well-established confocal FCCS, but in addition provides unmatched statistics and true imaging capabilities.

© 2014 Optical Society of America

## 1. Introduction

1. D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy I: Conceptual basis and theory,” Biopolymers **13**, 1–27 (1974). [CrossRef]

2. D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. an experimental realization.” Biopolymers **13**, 29–61 (1974). [CrossRef] [PubMed]

3. K. M. Berland, P. T. So, and E. Gratton, “Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment,” Biophys. J. **68**, 694–701 (1995). [CrossRef] [PubMed]

^{−15}l = 1

*μ*m

^{3}). FCS was extended to fluorescence cross-correlation spectroscopy (FCCS) to evaluate the cross-correlation between two or more separate color channels [4

4. P. Schwille, F. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multi-component diffusional analysis in solution,” Biophys. J. **72**, 1878–1886 (1997). [CrossRef] [PubMed]

9. D. M. Shcherbakova, M. A. Hink, L. Joosen, T. W. J. Gadella, and V. V. Verkhusha, “An orange fluorescent protein with a large stokes shift for single-excitation multicolor FCCS and FRET imaging,” J. Am. Chem. Soc. **134**, 7913–7923 (2012). [CrossRef]

10. O. Krichevsky and G. Bonnet, “Fluorescence correlation spectroscopy: the technique and its applications,” Rep. Prog. Phys. **65**, 251–297 (2002). [CrossRef]

12. E. Haustein and P. Schwille, “Fluorescence correlation spectroscopy: novel variations of an established technique,” Annu. Rev. Biophys. Biomol. Struct. **36**, 151–169 (2007). [CrossRef] [PubMed]

13. N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, and J. Langowski, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE **4**, e5041 (2009). [CrossRef]

14. J. Ries, S. Chiantia, and P. Schwille, “Accurate determination of membrane dynamics with line-scan FCS,” Biophys. J. **96**, 1999–2008 (2009). [CrossRef] [PubMed]

15. Q. Ruan, M. A. Cheng, M. Levi, E. Gratton, and W. W. Mantulin, “Spatial-temporal studies of membrane dynamics: Scanning fluorescence correlation spectroscopy (SFCS),” Biophys. J. **87**, 1260–1267 (2004). [CrossRef] [PubMed]

8. F. Bestvater, Z. Seghiri, M. S. Kang, N. Gröner, J. Y. Lee, I. Kang-Bin, and M. Wachsmuth, “EMCCD-based spectrally resolved fluorescence correlation spectroscopy,” Opt. Express **18**, 23818–23828 (2010). [CrossRef]

16. G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. **38**, 813–828 (2009). [CrossRef] [PubMed]

17. D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: Highly parallel fluorescence correlation spectroscopy,” Biophys. J. **96**, 5050–5059 (2009). [CrossRef] [PubMed]

18. R. A. Colyer, G. Scalia, I. Rech, A. Gulinatti, M. Ghioni, S. Cova, S. Weiss, and X. Michalet, “High-throughput FCS using an LCOS spatial light modulator and an 8×1 spad array,” Biomed. Opt. Express **1**, 1408–1431 (2010). [CrossRef]

19. M. Kloster-Landsberg, D. Tyndall, I. Wang, R. Walker, J. Richardson, R. Henderson, and A. Delon, “Note: Multi-confocal fluorescence correlation spectroscopy in living cells using a complementary metal oxide semiconductor-single photon avalanche diode array,” Rev. Sci. Instrum. **84**, 076105 (2013). [CrossRef] [PubMed]

20. B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. **79**, 4463–4470 (2007). [CrossRef] [PubMed]

21. T. Wohland, X. Shi, J. Sankaran, and E. H. K. Stelzer, “Single plane illumination fluorescence correlation spectroscopy (SPIM-FCS) probes inhomogeneous three-dimensional environments,” Opt. Express **10**, 10627–10641 (2010). [CrossRef]

22. J. Capoulade, M. Wachsmuth, L. Hufnagel, and M. Knop, “Quantitative fluorescence imaging of protein diffusion and interaction in living cells,” Nat. Biotechnol. **29**, 835—839 (2011). [CrossRef] [PubMed]

*supplementary information*(SI [23

23. SI: The supplementary notes are available at http://www.dkfz.de/Macromol/publications/files/spimfccs2013_supplement.pdf.

24. M. Gösch, A. Magnusson, S. Hård, H. Blom, S. Anderegg, K. Korn, P. Thyberg, M. Wells, T. Lasser, and R. Rigler, “Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements,” J. Biomed. Opt. **10**, 054008 (2005). [CrossRef] [PubMed]

25. J. Ries, Z. Petrášek, A. J. García-Sáez, and P. Schwille, “A comprehensive framework for fluorescence cross-correlation spectroscopy,” New J. Phys. **12**, 113009 (2010). [CrossRef]

26. V. Betaneli, E. P. Petrov, and P. Schwille, “The role of lipids in VDAC oligomerization,” Biophys. J. **102**, 523–531 (2012). [CrossRef] [PubMed]

28. T. Toplak, E. Pandzic, L. Chen, M. Vicente-Manzanares, A. R. Horwitz, and P. W. Wiseman, “STICCS reveals matrix-dependent adhesion slipping and gripping in migrating cells,” Biophys. J. **103**, 1672–1682 (2012). [CrossRef] [PubMed]

29. M. A. Digman, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Detecting protein complexes in living cells from laser scanning confocal image sequences by the cross correlation raster image spectroscopy method,” Biophys. J. **96**, 707–716 (2009). [CrossRef] [PubMed]

*z*-sectioning) limited by the used microscopy techniques. To overcome these limitations, we extend FCCS into a fast and true imaging mode by using single plane illumination microscopy (SPIM-FCCS) with a high-speed electron-multiplying charge coupled device (EMCCD) camera, giving moderate to high temporal resolution (0.3 – 1.0 ms) and good

*z*-sectioning.

*μ*m) in a sample. The difference in SPIM-FCCS is that two light sheets of different wavelength are used simultaneously to excite different fluorophores for the cross-correlation analysis. For detection we use an image splitter optics to separate the full spectral range into two distinct color channels which are imaged onto the same EMCCD camera. This sensor has been chosen due to its relatively high temporal resolution and high detection efficiency (quantum efficiency > 95%). The properties of several other possible detectors have recently been studied and compared in [30

30. A. P. Singh, J. W. Krieger, J. Buchholz, E. Charbon, J. Langowski, and T. Wohland, “The performance of 2D array detectors for light sheet based fluorescence correlation spectroscopy,” Opt. Express **21**, 8652–8668 (2013). [CrossRef] [PubMed]

## 2. SPIM-FCCS: The method

### 2.1. Introduction to SPIM-FCCS

*r⃗*of a sample is split spectrally into a green color channel signal

*I*

_{g}(

*t; r⃗*) and red signal

*I*

_{r}(

*t; r⃗*). Then the normalized correlation functions (with

*γρ*∈ {gg, rr, gr, rg}) are calculated. Here 〈·〉 denotes a temporal average and

*G*(

_{γρ}*τ*) = 〈

*δI*(

_{γ}*t*) ·

*δI*(

_{ρ}*t*+

*τ*)〉is the non-normalized correlation function between the fluorescence fluctuations

*δI*(

_{γ}*t*) =

*I*(

_{γ}*t*) − 〈

*I*〉 in channels

_{γ}*γ*and

*ρ*respectively. The autocorrelations

*g*

_{gg}(

*τ*;

*r⃗*) and

*g*

_{rr}(

*τ*;

*r⃗*) mainly contain information about the diffusion coefficient and concentration of the different labeled particles, while the amplitude of the cross-correlation

*g*

_{gr}(

*τ*;

*r⃗*) reports on their interaction. As we will not use spatial cross-correlations between different points in the sample, we will omit the parameter

*r⃗*from this point on. If theoretical models are known for the measured correlation functions, a parameter fit can yield parameters describing the mobility and interaction of the observed species.

23. SI: The supplementary notes are available at http://www.dkfz.de/Macromol/publications/files/spimfccs2013_supplement.pdf.

### 2.2. SPIM-FCCS models

21. T. Wohland, X. Shi, J. Sankaran, and E. H. K. Stelzer, “Single plane illumination fluorescence correlation spectroscopy (SPIM-FCS) probes inhomogeneous three-dimensional environments,” Opt. Express **10**, 10627–10641 (2010). [CrossRef]

30. A. P. Singh, J. W. Krieger, J. Buchholz, E. Charbon, J. Langowski, and T. Wohland, “The performance of 2D array detectors for light sheet based fluorescence correlation spectroscopy,” Opt. Express **21**, 8652–8668 (2013). [CrossRef] [PubMed]

31. J. Sankaran, X. Shi, L. Ho, E. Stelzer, and T. Wohland, “ImFCS: A software for imaging FCS data analysis and visualization,” Opt. Express **18**, 25468–25481 (2010). Available at http://staff.science.nus.edu.sg/~chmwt/resources/imfcs_software.html. [CrossRef]

*D*

_{A},

*D*

_{B}and

*D*

_{AB}, as well as the concentrations

*c*

_{A},

*c*

_{B}and

*c*

_{AB}of three molecular species A, B and AB present in this process. In a typical experiment, A and B are the green and red labeled monomers, whereas AB represents the double-labeled dimer formed from A and B.

25. J. Ries, Z. Petrášek, A. J. García-Sáez, and P. Schwille, “A comprehensive framework for fluorescence cross-correlation spectroscopy,” New J. Phys. **12**, 113009 (2010). [CrossRef]

32. L. C. Hwang and T. Wohland, “Single wavelength excitation fluorescence cross-correlation spectroscopy with spectrally similar fluorophores: Resolution for binding studies,” J. Chem. Phys. **122**, 114708 (2005). [CrossRef] [PubMed]

33. P. Liu, T. Sudhaharan, R. M. Koh, L. C. Hwang, S. Ahmed, I. N. Maruyama, and T. Wohland, “Investigation of the dimerization of proteins from the epidermal growth factor receptor family by single wavelength fluorescence cross-correlation spectroscopy,” Biophys. J. **93**, 684–698 (2007). [CrossRef] [PubMed]

*I*(

_{g}*t*),

*δI*(

_{g}*t*) in the green and

*I*(

_{r}*t*),

*δI*(

_{r}*t*) in the red color channel. These can be written for any color channel

*γ*and the set of molecular species 𝕊 = {A, B, AB,...} as: Here

*χ*in channel

*γ*. The symbol

*c*(

_{χ}*t*,

*r⃗*) denotes the local particle concentration of species

*χ*at time

*t*and position

*r⃗*and MDE

*(*

_{γ}*r⃗*) is the molecular detection efficiency of channel

*γ*for an emitting particle at position

*r⃗*. The fluorescence fluctuations

*δI*(

_{γ}*t*) are calculated form the concentration fluctuations

*δc*(

_{χ}*t*,

*r⃗*).

21. T. Wohland, X. Shi, J. Sankaran, and E. H. K. Stelzer, “Single plane illumination fluorescence correlation spectroscopy (SPIM-FCS) probes inhomogeneous three-dimensional environments,” Opt. Express **10**, 10627–10641 (2010). [CrossRef]

30. A. P. Singh, J. W. Krieger, J. Buchholz, E. Charbon, J. Langowski, and T. Wohland, “The performance of 2D array detectors for light sheet based fluorescence correlation spectroscopy,” Opt. Express **21**, 8652–8668 (2013). [CrossRef] [PubMed]

31. J. Sankaran, X. Shi, L. Ho, E. Stelzer, and T. Wohland, “ImFCS: A software for imaging FCS data analysis and visualization,” Opt. Express **18**, 25468–25481 (2010). Available at http://staff.science.nus.edu.sg/~chmwt/resources/imfcs_software.html. [CrossRef]

*e*

^{2}-halfwidth

*w*

_{LS}for illumination, a pixel detector with square pixels (side length

*a*) and a point spread function (PSF), that describes the imaging properties of the detection optics. The overall MDE is then calculated by an integral over the volume sampled by each point on the pixels (

*r⃗*= (

*x*,

*y*,

*z*)

^{T}): where

*𝒩*

^{−1}is a normalization constant, set to fulfill

*χ*between channels

*γ*and

*ρ*(Note: we defined

^{6}): To describe the diffusive motion of species

*χ*, we use a Green’s function of the following form: The integrals in Eq. (5) can be separated into three directional components

*δx*=

*δy*=

*δz*= 0), which was well achieved during the measurements for this paper, as shown in sections 3.2 and 3.3. The full correlation function including a non-zero shift and a possible directed flow, is provided in the SI [23

23. SI: The supplementary notes are available at http://www.dkfz.de/Macromol/publications/files/spimfccs2013_supplement.pdf.

*xz*-plane, we separated the integrals in Eq. (5) only into two factors

*χ*in channel

*γ*can be written in terms of the molar channel brightness

*η*

_{g}of fluorophore A in the green channel and

*η*

_{r}of the fluorophore B in the red channel: Here

*κ*

_{gr}is the crosstalk factor of the green into the red channel and we assume that there is no crosstalk of the red into the green channel, which is well justified for our dual-wavelength FCCS setup. The factors

*η*

_{g}and

*η*

_{r}can be estimated from the measured and background corrected average fluorescence intensities 〈

*I*

_{g}(

*t*)〉 and 〈

*I*

_{r}(

*t*)〉, as where the crosstalk coefficient

*κ*

_{gr}is determined in an A-only (green-only) sample as

*κ*

_{gr}= 〈

*I*

_{r}(

*t*)〉/〈

*I*

_{g}(

*t*)〉. Note that we only used molar brightnesses

*η*,

_{γ}*V*

_{eff}:=(∫MDE(

*r⃗*) d

*V*)

^{2}/∫ MDE

^{2}(

*r⃗*) d

*V*.

*c*

_{A},

*c*

_{B}and

*c*

_{AB}, the amount of binding (as relative dimer concentration) can be calculated: More general, the amount of cross-correlation (also a measure of binding) can also be defined from the correlation function amplitudes without assuming any concentrations: where

*g*(

_{γρ}*τ*) are either directly measured correlation functions (then

*τ*

_{min}is the minimum lag time) or fitted model functions (then

*τ*

_{min}= 0).

### 2.3. Data evaluation

**10**, 10627–10641 (2010). [CrossRef]

22. J. Capoulade, M. Wachsmuth, L. Hufnagel, and M. Knop, “Quantitative fluorescence imaging of protein diffusion and interaction in living cells,” Nat. Biotechnol. **29**, 835—839 (2011). [CrossRef] [PubMed]

**21**, 8652–8668 (2013). [CrossRef] [PubMed]

31. J. Sankaran, X. Shi, L. Ho, E. Stelzer, and T. Wohland, “ImFCS: A software for imaging FCS data analysis and visualization,” Opt. Express **18**, 25468–25481 (2010). Available at http://staff.science.nus.edu.sg/~chmwt/resources/imfcs_software.html. [CrossRef]

34. J. Buchholz, J. W. Krieger, G. Mocsár, B. Kreith, E. Charbon, G. Vámosi, U. Kebschull, and J. Langowski, “Fpga implementation of a 32×32 autocorrelator array for analysis of fast image series,” Opt. Express **20**, 17767–17782 (2012). [CrossRef] [PubMed]

*Ĩ*(

*t; r⃗*) of the sample with 5 · 10

^{4}– 5 · 10

^{5}frames at a frame rate above 1000 frames per second (fps) using both lasers for illumination. In addition a short (∼ 2000 frames) background series

*B*(

*t; r⃗*) without light sheet illumination is recorded with the same camera settings. Then the data is preprocessed by subtracting the averaged background (

*Ĩ*(

*t; r⃗*) − 〈

*B*(

*t; r⃗*)〉), splitting the images series into two color channels and computing the auto- and cross-correlation functions using multi-

*τ*-algorithm. If necessary a bleach correction is performed independently for every pixel, by fitting e. g. an exponential decay (or a more complex function) to the time-series in each pixel and then detrending the initial time-series, while preserving its ideal average and variance, as described in [14

14. J. Ries, S. Chiantia, and P. Schwille, “Accurate determination of membrane dynamics with line-scan FCS,” Biophys. J. **96**, 1999–2008 (2009). [CrossRef] [PubMed]

*g*(

_{γρ}*τ*;

*c*

_{A},

*c*

_{AB},

*D*

_{A},

*D*

_{AB},...), derived in the last section are fitted to the experimental curves (

*τ*,

_{i}*ĝ*

_{gg,}

*,*

_{i}*ĝ*

_{gr,}

*,...), measured at discrete lag times*

_{i}*τ*. We choose a global fitting method that simultaneously minimizes the least-squares deviations of the fit functions from the measurements. The optimal parameter vector

_{i}*β⃗*is then the solution of this least-squares optimization problem: where

*π⃗*(

_{γρ}*β⃗*) maps the “global” parameter vector

*β⃗*to the “local” vector containing only the parameters used by the model function

*g*(·;·). To solve this optimization problem we used either a version of the Levenberg-Marquardt algorithm [35

_{γρ}35. D. W. Marquardt, “An algorithm for least-squares estimation of nonlinear parameters,” J. Soc. Ind. Appl. Math. **11**, 431–441 (1963). [CrossRef]

37. J. Wuttke, “lmfit 3.2 – a c/c++ routine for levenberg-marquardt minimization with wrapper for least-squares curve fitting, based on work by B.S. Garbow, K.E. Hillstrom, J.J. Moré, and S. Moshier, available at http://apps.jcns.fz-juelich.de/doku/sc/lmfit,” (2010).

38. A. Corana, M. Marchesi, C. Martini, and S. Ridella, “Minimizing multimodal functions of continuous variables with the ’simulated annealing’ algorithm corrigenda for this article is available here,” ACM T. Math. Software **13**, 262–280 (1987). [CrossRef]

*c*

_{A},

*c*

_{B}and

*c*

_{AB}are always linked over these three correlation curves. In simple cases, where only one diffusing component is visible in the correlation functions, also the diffusion coefficients

*D*

_{A},

*D*

_{B}and

*D*

_{AB}are linked in the same manner. In more complex cases, where two diffusing fractions per species are required to describe the data, we found that the models do no longer converge reliably and with physically meaningful fit parameter values. So we simplified Eq. (11)–Eq. (13), by assuming two diffusing components per channel which were no longer assigned to any specific species. where the correlation functions are defined using Eq. (7) and Eq. (8) as

33. P. Liu, T. Sudhaharan, R. M. Koh, L. C. Hwang, S. Ahmed, I. N. Maruyama, and T. Wohland, “Investigation of the dimerization of proteins from the epidermal growth factor receptor family by single wavelength fluorescence cross-correlation spectroscopy,” Biophys. J. **93**, 684–698 (2007). [CrossRef] [PubMed]

39. Y. H. Foo, N. Naredi-Rainer, D. C. Lamb, S. Ahmed, and T. Wohland, “Factors affecting the quantification of biomolecular interactions by fluorescence cross-correlation spectroscopy,” Biophys. J. **102**, 1174–1183 (2012). [CrossRef] [PubMed]

40. QuickFit 3.0 can be downloaded free of charge from http://www.dkfz.de/Macromol/quickfit/.

**18**, 25468–25481 (2010). Available at http://staff.science.nus.edu.sg/~chmwt/resources/imfcs_software.html. [CrossRef]

## 3. Experimental setup

### 3.1. A light sheet microscope with dual color excitation and detection

41. K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum. **78**, 023705 (2007). [CrossRef] [PubMed]

*μ*m thick (1/

*e*

^{2}-halfwidth) and the field of view is about 50 × 50

*μ*m

^{2}at a pixels size of 400 nm. The detection volume defined by each pixel of the camera is about

*V*

_{eff}= 2.5 − 3

*μ*m

^{3}, as compared to

*V*

_{eff}= 0.5 – 1

*μ*m

^{3}in a confocal microscope [30

**21**, 8652–8668 (2013). [CrossRef] [PubMed]

41. K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum. **78**, 023705 (2007). [CrossRef] [PubMed]

^{2}during all measurements, which is a factor of 10 – 30 below typical intensities used for confocal FCS in live cells (∼ 1500 W/cm

^{2}).

*μ*s for all measurements. Binning was always performed during the data processing step and not on the camera. As different filter sets were used for the measurements in this paper, the crosstalk

*κ*

_{gr}is given in the text where necessary. Generally the crosstalk for eGFP was between

*κ*

_{gr}= 3.5% and 9%, and for Alexa-488 between

*κ*

_{gr}= 5.4% and 11.8% (both depending on the used filter set). Note that all model fits in this paper already incorporate a correction for the respective crosstalk.

40. QuickFit 3.0 can be downloaded free of charge from http://www.dkfz.de/Macromol/quickfit/.

### 3.2. Alignment procedure

**21**, 8652–8668 (2013). [CrossRef] [PubMed]

*δz*< 100 nm (at a typical light sheet width of ∼ 1.3

*μ*m).

*μ*m or 12.7

*μ*m respectively; Latech Scientific Supply Pte. Ltd, Singapore) in transmission illumination mode. The overlap of the two color channels was optimized using a special live-view of the difference between the two half images in QuickFit 3.0 and then maximizing the image cross-correlation coefficient between them. It is also possible to use the open source software

*μ*Manager [42] with its plugins “Split View” and “Co-localization” [43

43. E. M. M. Manders, F. J. Verbeek, and J. A. Aten, “Measurement of co-localization of objects in dual-colour confocal images,” J. Microsc. **169**, 375–382 (1993). [CrossRef]

*z*-scan of TetraSpec fluorescent micro-spheres (100 nm diameter, T7279 Microspheres, Life Technologies GmbH, Darmstadt, Germany) embedded in a gel cylinder (0.5% Phytagel, P8169, Sigma-Aldrich Chemie Gmbh, Munich, Germany) supplemented with 0.1% MgSO

_{4}. A Matlab script (Matlab 2012a, Math-Works, Ismaning, Germany) was developed and used to fit a 3D Gaussian model function to each bead in both channels. This allowed us to measure the displacement of the MDEs in all directions (available online: [44

44. The matlab scripts for the bead scan evaluation is freely available at: http://www.dkfz.de/Macromol/quickfit/beadscan.html.

*δx*,

*δy*and

*δz*of better than 100 nm. In section 3.3 we show FCCS simulations which confirm that this displacement leads to a negligible (< 5%) reduction of the measured relative concentrations. Exemplary results and detailed protocols are shown in the SI [23

**21**, 8652–8668 (2013). [CrossRef] [PubMed]

45. N. Bag, J. Sankaran, A. Paul, R. S. Kraut, and T. Wohland, “Calibration and limits of camera-based fluorescence correlation spectroscopy: A supported lipid bilayer study,” ChemPhysChem **13**, 2784–2794 (2012). [CrossRef] [PubMed]

*w*

_{g}and

*w*

_{r}(

*z*

_{g}and

*z*

_{r}were taken from the bead or light sheet scan), which also stayed constant with a relative standard deviation of ≤ 10% over a period of 9 months (data shown in SI [23

### 3.3. Simulations on the error due to misalignment

*δx*,

*δy*and

*δz*between the green and red detection volume. We used FCCS simulations to determine a threshold for these, which allows to do measurements with a small to negligible error. We extended the simulation program described in Refs. [30

**21**, 8652–8668 (2013). [CrossRef] [PubMed]

34. J. Buchholz, J. W. Krieger, G. Mocsár, B. Kreith, E. Charbon, G. Vámosi, U. Kebschull, and J. Langowski, “Fpga implementation of a 32×32 autocorrelator array for analysis of fast image series,” Opt. Express **20**, 17767–17782 (2012). [CrossRef] [PubMed]

46. T. Wocjan, J. Krieger, O. Krichevsky, and J. Langowski, “Dynamics of a fluorophore attached to superhelical DNA: Fcs experiments simulated by brownian dynamics,” Phys. Chem. Chem. Phys. **11**, 10671–10681 (2009). [CrossRef]

*c*

_{all}≈ 1 nM). The particles carry green, red or both fluorophores in different proportions

*c*

_{DL}/

*c*

_{all}ranging from single-label only (

*c*

_{DL}/

*c*

_{all}= 0) to double-label (DL) only (

*c*

_{DL}/

*c*

_{all}= 1). A blue and a green light sheet illuminate the walkers, and two detection volumes for the green and red color channels are set up. The focal parameters approximately match those of the actual SPIM-FCCS setups used for this paper. The distance between the volumes is varied in eleven steps between

*δx*= 0 nm and

*δx*= 2000 nm. Detailed simulation parameters are given in the SI [23

*c*

_{DL}/

*c*

_{all}, shift

*δx*and one of two realistic crosstalk coefficients

*κ*

_{gr}= 3.5% and

*κ*

_{gr}= 11.2%, the two autocorrelations and the cross-correlation curves were simulated and evaluated with a global fit to the SPIM-FCCS models described before. As in all measurements in this paper, we assumed

*δx*=

*δy*=

*δz*= 0 for the fit. For each combination the fit yields a focus shift dependent relative concentration from which we estimated the relative error, when assuming

*δx*= 0, instead of the actual

*δx*> 0:

*δx*) < 5%, we can draw the conclusion that the instrument has to be aligned to

*δx*,

*δy*,

*δz*<

*δ*

_{5%}= 200 nm. As shown in the last section 3.2, this is easily possible with the methods described there.

### 4. *In-vitro* samples

47. C.-H. Huang, “Phosphatidylcholine vesicles. formation and physical characteristics,” Biochemistry **8**, 344–352 (1969). PMID: [PubMed] . [CrossRef]

48. L. A. Maguire, H. Zhang, and P. A. Shamlou, “Preparation of small unilamellar vesicles (SUV) and biophysical characterization of their complexes with poly-l-lysine-condensed plasmid DNA,” Biotechnol Appl. Biochem. **37**, 73–81 (2003). [CrossRef] [PubMed]

*μ*l and 50

*μ*l of each solution into plastic sample bags, as described elsewhere [30

**21**, 8652–8668 (2013). [CrossRef] [PubMed]

**21**, 8652–8668 (2013). [CrossRef] [PubMed]

51. J. R. Unruh and E. Gratton, “Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied ccd camera,” Biophys. J. **95**, 5385–5398 (2008). [CrossRef] [PubMed]

*K*

_{D}=

*c*

_{A}·

*c*

_{B}/

*c*

_{AB}can estimated using SPIM-FCCS.

## 5. *In-vivo* measurements

### 5.1. Sample preparation

^{2}) of No. 3 cover slips (0.28 mm − 0.32 mm thick, No. 16301, Neolab, Heidelberg, Germany) put into the culture dishes before seeding the cells. Before use, the glass pieces were thoroughly washed with acetone or 70% ethanol and deionized water, then sterilized. For the measurement, a glass cover slip was clamped into self-closing tweezers. Then it was mounted from above in the SPIM sample chamber, which was filled with Hanks’ balanced salts solution (PAN-Biotech, Aidenbach, Germany). To avoid any direct reflection of the laser light to the sensor the glass cover slip was positioned under an angle below 45° with respect to the light sheet.

### 5.2. Proteins in the cyto- and nucleoplasm

52. N. Baudendistel, G. Müller, W. Waldeck, P. Angel, and J. Langowski, “Two-hybrid fluorescence cross-correlation spectroscopy detects protein–protein interactions in vivo,” ChemPhysChem **6**, 984–990 (2005). [CrossRef] [PubMed]

49. J. Sankaran, N. Bag, R. S. Kraut, and T. Wohland, “Accuracy and precision in camera-based fluorescence correlation spectroscopy measurements,” Anal. Chem. **85**, 3948–3954 (2013). [CrossRef] [PubMed]

*f*(

*t*) =

*A*· exp[−

*t*/

*τ*]) or modified mono-exponential model

_{B}*f*(

*t*) =

*A*· exp[−(

*t*+

*f*

_{2}

*t*

^{2})/

*τ*] was used.

_{B}*D*

_{A}=

*D*

_{B}=

*D*

_{AB}. Fig. 6 displays example results of these measurements, showing a shift of the relative dimer concentration from

*p*

_{AB}= (0.025 ± 0.023) for the monomers to (0.18 ± 0.06) for eGFP-mRFP1-transfected cells. The models already contain an implicit crosstalk-correction. The non-corrected relative correlation amplitude averaged over several cells was

*q*= (0.46 ± 0.07) for the dimers and (0.25 ± 0.04) for the monomers. These values are comparable to typical results from confocal FCCS experiments [52

52. N. Baudendistel, G. Müller, W. Waldeck, P. Angel, and J. Langowski, “Two-hybrid fluorescence cross-correlation spectroscopy detects protein–protein interactions in vivo,” ChemPhysChem **6**, 984–990 (2005). [CrossRef] [PubMed]

53. G. Vámosi, N. Baudendistel, C.-W. von der Lieth, N. Szalóki, G. Mocsár, G. Müller, P. Brázda, W. Waldeck, S. Damjanovich, J. Langowski, and K. Tóth, “Conformation of the c-Fos/c-Jun complex in vivo: A combined FRET, FCCS, and MD-modeling study,” Biophys. J. **94**, 2859–2868 (2008). [CrossRef]

54. B. K. Müller, E. Zaychikov, C. Bräuchle, and D. C. Lamb, “Pulsed interleaved excitation,” Biophys. J. **89**, 3508–3522 (2005). [CrossRef] [PubMed]

*D*

_{monomer}= (34 ± 5)

*μ*m

^{2}/s for the monomers to

*D*

_{dimer}= (25 ± 5)

*μ*m

^{2}/s ≈ 0.74 ·

*D*

_{monomer}for the dimer, which is comparable to the values for eGFP monomers and dimers reported for confocal FCS/FCCS measurements in [13

13. N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, and J. Langowski, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE **4**, e5041 (2009). [CrossRef]

52. N. Baudendistel, G. Müller, W. Waldeck, P. Angel, and J. Langowski, “Two-hybrid fluorescence cross-correlation spectroscopy detects protein–protein interactions in vivo,” ChemPhysChem **6**, 984–990 (2005). [CrossRef] [PubMed]

### 5.3. Membrane-bound proteins

33. P. Liu, T. Sudhaharan, R. M. Koh, L. C. Hwang, S. Ahmed, I. N. Maruyama, and T. Wohland, “Investigation of the dimerization of proteins from the epidermal growth factor receptor family by single wavelength fluorescence cross-correlation spectroscopy,” Biophys. J. **93**, 684–698 (2007). [CrossRef] [PubMed]

*g*(

_{γρ}*τ*) decayed to 0 for large lag times

*τ*. We used a strong variant of the bleach correction, which fits a modified exponential function

*f*(

*t*) =

*A*· exp[−(

*t*+

*f*

_{2}

*t*

^{2}+

*f*

_{3}

*t*

^{3})/

*τ*] to the data. Again a 2 × 2 pixel binning was used to improved CF statistics. This also reduces artifacts due to membrane motion, as the relation between motion and focus size is improved. Finally a 2-component SPIM-FCCS model for 2D diffusion in the

_{B}*xz*-plane was fitted to the data. Only the concentrations were linked over all channels, whereas the diffusion coefficients and diffusing species fractions were specific to each channel. Fig. 7 shows exemplary results. From a larger set of measurements, distributed over several weeks, we get an average

*p*

_{AB}= (0.36 ± 0.20) for the PMT-cells and

*p*

_{AB}= (0.93 ± 0.62) for the EGFR samples. These numbers are average and standard deviation over 42 (PMT) and 33 (EGFR) single-cell average values (see SI [23

*p*

_{AB}for the negative control (PMT-cells) can be explained by a non-perfect crosstalk correction and especially by remaining and non-corrected membrane motion. The results compare well to the curves and values reported in Ref. [33

**93**, 684–698 (2007). [CrossRef] [PubMed]

**93**, 684–698 (2007). [CrossRef] [PubMed]

## 6. Conclusion

*in-vitro*and

*in-vivo*measurements. We used custom-built light sheet microscopes, which were equipped with a dual-color excitation. An image splitter was used to separately image the detected fluorescence into two separate color channels on a single EMCCD camera. Based on our previous work [21

**10**, 10627–10641 (2010). [CrossRef]

**21**, 8652–8668 (2013). [CrossRef] [PubMed]

**18**, 25468–25481 (2010). Available at http://staff.science.nus.edu.sg/~chmwt/resources/imfcs_software.html. [CrossRef]

**93**, 684–698 (2007). [CrossRef] [PubMed]

**6**, 984–990 (2005). [CrossRef] [PubMed]

*in-vitro*and in living cells. The technique can easily be extended with fast, frame-based alternating of the excitation lasers, comparable to “pulsed interleaved excitation” [54

54. B. K. Müller, E. Zaychikov, C. Bräuchle, and D. C. Lamb, “Pulsed interleaved excitation,” Biophys. J. **89**, 3508–3522 (2005). [CrossRef] [PubMed]

55. A. N. Kapanidis, T. A. Laurence, N. K. Lee, E. Margeat, X. Kong, and S. Weiss, “Alternating-laser excitation of single molecules,” Acc. Chem. Res. **38**, 523–533 (2005). [CrossRef] [PubMed]

56. J. Roszik, D. Lisboa, J. Szöllősi, and G. Vereb, “Evaluation of intensity-based ratiometric FRET in image cytometry—approaches and a software solution,” Cytometry A **75A**, 761–767 (2009). [CrossRef]

57. S. Talwar, A. Kumar, M. Rao, G. I. Menon, and G. V. Shivashankar, “Correlated spatio-temporal fluctuations in chromatin compaction states characterize stem cells.” Biophys. J. **104**, 553–564 (2013). [CrossRef] [PubMed]

**21**, 8652–8668 (2013). [CrossRef] [PubMed]

34. J. Buchholz, J. W. Krieger, G. Mocsár, B. Kreith, E. Charbon, G. Vámosi, U. Kebschull, and J. Langowski, “Fpga implementation of a 32×32 autocorrelator array for analysis of fast image series,” Opt. Express **20**, 17767–17782 (2012). [CrossRef] [PubMed]

58. S. Burri, D. Stucki, Y. Maruyama, C. Bruschini, E. Charbon, and F. Regazzoni, “Jailbreak imagers: Transforming a single-photon image sensor into a true random number generator,” in “International Image Sensor Workshop,” (2013). Snowbird, June 2013, available online http://www.imagesensors.org/PastWorkshops/2013Workshop/2013Papers/07-20_099_regazzoni_paper_revised.pdf.

## Acknowledgments

## References and links

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2. | D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. an experimental realization.” Biopolymers |

3. | K. M. Berland, P. T. So, and E. Gratton, “Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment,” Biophys. J. |

4. | P. Schwille, F. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multi-component diffusional analysis in solution,” Biophys. J. |

5. | U. Kettling, A. Koltermann, P. Schwille, and M. Eigen, “Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy,” Proc. Natl. Acad. Sci. |

6. | K. G. Heinze, M. Jahnz, and P. Schwille, “Triple-color coincidence analysis: One step further in following higher order molecular complex formation,” Biophys. J. |

7. | L. C. Hwang, M. Gösch, T. Lasser, and T. Wohland, “Simultaneous multicolor fluorescence cross-correlation spectroscopy to detect higher order molecular interactions using single wavelength laser excitation,” Biophys. J. |

8. | F. Bestvater, Z. Seghiri, M. S. Kang, N. Gröner, J. Y. Lee, I. Kang-Bin, and M. Wachsmuth, “EMCCD-based spectrally resolved fluorescence correlation spectroscopy,” Opt. Express |

9. | D. M. Shcherbakova, M. A. Hink, L. Joosen, T. W. J. Gadella, and V. V. Verkhusha, “An orange fluorescent protein with a large stokes shift for single-excitation multicolor FCCS and FRET imaging,” J. Am. Chem. Soc. |

10. | O. Krichevsky and G. Bonnet, “Fluorescence correlation spectroscopy: the technique and its applications,” Rep. Prog. Phys. |

11. | K. Bacia, S. A. Kim, and P. Schwille, “Fluorescence cross-correlation spectroscopy in living cells,” Nat. Methods |

12. | E. Haustein and P. Schwille, “Fluorescence correlation spectroscopy: novel variations of an established technique,” Annu. Rev. Biophys. Biomol. Struct. |

13. | N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, and J. Langowski, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE |

14. | J. Ries, S. Chiantia, and P. Schwille, “Accurate determination of membrane dynamics with line-scan FCS,” Biophys. J. |

15. | Q. Ruan, M. A. Cheng, M. Levi, E. Gratton, and W. W. Mantulin, “Spatial-temporal studies of membrane dynamics: Scanning fluorescence correlation spectroscopy (SFCS),” Biophys. J. |

16. | G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. |

17. | D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: Highly parallel fluorescence correlation spectroscopy,” Biophys. J. |

18. | R. A. Colyer, G. Scalia, I. Rech, A. Gulinatti, M. Ghioni, S. Cova, S. Weiss, and X. Michalet, “High-throughput FCS using an LCOS spatial light modulator and an 8×1 spad array,” Biomed. Opt. Express |

19. | M. Kloster-Landsberg, D. Tyndall, I. Wang, R. Walker, J. Richardson, R. Henderson, and A. Delon, “Note: Multi-confocal fluorescence correlation spectroscopy in living cells using a complementary metal oxide semiconductor-single photon avalanche diode array,” Rev. Sci. Instrum. |

20. | B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. |

21. | T. Wohland, X. Shi, J. Sankaran, and E. H. K. Stelzer, “Single plane illumination fluorescence correlation spectroscopy (SPIM-FCS) probes inhomogeneous three-dimensional environments,” Opt. Express |

22. | J. Capoulade, M. Wachsmuth, L. Hufnagel, and M. Knop, “Quantitative fluorescence imaging of protein diffusion and interaction in living cells,” Nat. Biotechnol. |

23. | SI: The supplementary notes are available at http://www.dkfz.de/Macromol/publications/files/spimfccs2013_supplement.pdf. |

24. | M. Gösch, A. Magnusson, S. Hård, H. Blom, S. Anderegg, K. Korn, P. Thyberg, M. Wells, T. Lasser, and R. Rigler, “Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements,” J. Biomed. Opt. |

25. | J. Ries, Z. Petrášek, A. J. García-Sáez, and P. Schwille, “A comprehensive framework for fluorescence cross-correlation spectroscopy,” New J. Phys. |

26. | V. Betaneli, E. P. Petrov, and P. Schwille, “The role of lipids in VDAC oligomerization,” Biophys. J. |

27. | P. W. Wiseman, J. A. Squier, and K. R. Wilson, “Dynamic image correlation spectroscopy (ICS) and two-color image cross-correlation spectroscopy (ICCS): concepts and application,” in “BiOS 2000 Int. Symp. Biomed. Opt.”, (Int. Soc. Opt. Photon., 2000), pp. 14–20. |

28. | T. Toplak, E. Pandzic, L. Chen, M. Vicente-Manzanares, A. R. Horwitz, and P. W. Wiseman, “STICCS reveals matrix-dependent adhesion slipping and gripping in migrating cells,” Biophys. J. |

29. | M. A. Digman, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Detecting protein complexes in living cells from laser scanning confocal image sequences by the cross correlation raster image spectroscopy method,” Biophys. J. |

30. | A. P. Singh, J. W. Krieger, J. Buchholz, E. Charbon, J. Langowski, and T. Wohland, “The performance of 2D array detectors for light sheet based fluorescence correlation spectroscopy,” Opt. Express |

31. | J. Sankaran, X. Shi, L. Ho, E. Stelzer, and T. Wohland, “ImFCS: A software for imaging FCS data analysis and visualization,” Opt. Express |

32. | L. C. Hwang and T. Wohland, “Single wavelength excitation fluorescence cross-correlation spectroscopy with spectrally similar fluorophores: Resolution for binding studies,” J. Chem. Phys. |

33. | P. Liu, T. Sudhaharan, R. M. Koh, L. C. Hwang, S. Ahmed, I. N. Maruyama, and T. Wohland, “Investigation of the dimerization of proteins from the epidermal growth factor receptor family by single wavelength fluorescence cross-correlation spectroscopy,” Biophys. J. |

34. | J. Buchholz, J. W. Krieger, G. Mocsár, B. Kreith, E. Charbon, G. Vámosi, U. Kebschull, and J. Langowski, “Fpga implementation of a 32×32 autocorrelator array for analysis of fast image series,” Opt. Express |

35. | D. W. Marquardt, “An algorithm for least-squares estimation of nonlinear parameters,” J. Soc. Ind. Appl. Math. |

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37. | J. Wuttke, “lmfit 3.2 – a c/c++ routine for levenberg-marquardt minimization with wrapper for least-squares curve fitting, based on work by B.S. Garbow, K.E. Hillstrom, J.J. Moré, and S. Moshier, available at http://apps.jcns.fz-juelich.de/doku/sc/lmfit,” (2010). |

38. | A. Corana, M. Marchesi, C. Martini, and S. Ridella, “Minimizing multimodal functions of continuous variables with the ’simulated annealing’ algorithm corrigenda for this article is available here,” ACM T. Math. Software |

39. | Y. H. Foo, N. Naredi-Rainer, D. C. Lamb, S. Ahmed, and T. Wohland, “Factors affecting the quantification of biomolecular interactions by fluorescence cross-correlation spectroscopy,” Biophys. J. |

40. | QuickFit 3.0 can be downloaded free of charge from http://www.dkfz.de/Macromol/quickfit/. |

41. | K. Greger, J. Swoger, and E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum. |

42. | A. Edelstein, N. Amodaj, K. Hoover, R. Vale, and N. Stuurman, “Computer control of microscopes using μManager,” Curr. Protoc. Mol. Biol. |

43. | E. M. M. Manders, F. J. Verbeek, and J. A. Aten, “Measurement of co-localization of objects in dual-colour confocal images,” J. Microsc. |

44. | The matlab scripts for the bead scan evaluation is freely available at: http://www.dkfz.de/Macromol/quickfit/beadscan.html. |

45. | N. Bag, J. Sankaran, A. Paul, R. S. Kraut, and T. Wohland, “Calibration and limits of camera-based fluorescence correlation spectroscopy: A supported lipid bilayer study,” ChemPhysChem |

46. | T. Wocjan, J. Krieger, O. Krichevsky, and J. Langowski, “Dynamics of a fluorophore attached to superhelical DNA: Fcs experiments simulated by brownian dynamics,” Phys. Chem. Chem. Phys. |

47. | C.-H. Huang, “Phosphatidylcholine vesicles. formation and physical characteristics,” Biochemistry |

48. | L. A. Maguire, H. Zhang, and P. A. Shamlou, “Preparation of small unilamellar vesicles (SUV) and biophysical characterization of their complexes with poly-l-lysine-condensed plasmid DNA,” Biotechnol Appl. Biochem. |

49. | J. Sankaran, N. Bag, R. S. Kraut, and T. Wohland, “Accuracy and precision in camera-based fluorescence correlation spectroscopy measurements,” Anal. Chem. |

50. | M. Wachsmuth, “Fluoreszenzfluktuationsmikroskopie: Entwicklung eines prototyps, theorie und messung der beweglichkeit von biomolekülen im zellkern,” Ph.D. thesis, Ruprecht-Karls-Universität, Heidelberg. (2001). |

51. | J. R. Unruh and E. Gratton, “Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied ccd camera,” Biophys. J. |

52. | N. Baudendistel, G. Müller, W. Waldeck, P. Angel, and J. Langowski, “Two-hybrid fluorescence cross-correlation spectroscopy detects protein–protein interactions in vivo,” ChemPhysChem |

53. | G. Vámosi, N. Baudendistel, C.-W. von der Lieth, N. Szalóki, G. Mocsár, G. Müller, P. Brázda, W. Waldeck, S. Damjanovich, J. Langowski, and K. Tóth, “Conformation of the c-Fos/c-Jun complex in vivo: A combined FRET, FCCS, and MD-modeling study,” Biophys. J. |

54. | B. K. Müller, E. Zaychikov, C. Bräuchle, and D. C. Lamb, “Pulsed interleaved excitation,” Biophys. J. |

55. | A. N. Kapanidis, T. A. Laurence, N. K. Lee, E. Margeat, X. Kong, and S. Weiss, “Alternating-laser excitation of single molecules,” Acc. Chem. Res. |

56. | J. Roszik, D. Lisboa, J. Szöllősi, and G. Vereb, “Evaluation of intensity-based ratiometric FRET in image cytometry—approaches and a software solution,” Cytometry A |

57. | S. Talwar, A. Kumar, M. Rao, G. I. Menon, and G. V. Shivashankar, “Correlated spatio-temporal fluctuations in chromatin compaction states characterize stem cells.” Biophys. J. |

58. | S. Burri, D. Stucki, Y. Maruyama, C. Bruschini, E. Charbon, and F. Regazzoni, “Jailbreak imagers: Transforming a single-photon image sensor into a true random number generator,” in “International Image Sensor Workshop,” (2013). Snowbird, June 2013, available online http://www.imagesensors.org/PastWorkshops/2013Workshop/2013Papers/07-20_099_regazzoni_paper_revised.pdf. |

**OCIS Codes**

(040.1490) Detectors : Cameras

(180.2520) Microscopy : Fluorescence microscopy

(180.6900) Microscopy : Three-dimensional microscopy

(300.6280) Spectroscopy : Spectroscopy, fluorescence and luminescence

**ToC Category:**

Microscopy

**History**

Original Manuscript: October 29, 2013

Revised Manuscript: December 8, 2013

Manuscript Accepted: December 8, 2013

Published: January 28, 2014

**Virtual Issues**

Vol. 9, Iss. 4 *Virtual Journal for Biomedical Optics*

**Citation**

Jan Wolfgang Krieger, Anand Pratap Singh, Christoph S. Garbe, Thorsten Wohland, and Jörg Langowski, "Dual-Color Fluorescence Cross-Correlation Spectroscopy on a Single Plane Illumination Microscope (SPIM-FCCS)," Opt. Express **22**, 2358-2375 (2014)

http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-22-3-2358

Sort: Year | Journal | Reset

### References

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- D. Magde, E. L. Elson, W. W. Webb, “Fluorescence correlation spectroscopy. II. an experimental realization.” Biopolymers 13, 29–61 (1974). [CrossRef] [PubMed]
- K. M. Berland, P. T. So, E. Gratton, “Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment,” Biophys. J. 68, 694–701 (1995). [CrossRef] [PubMed]
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- U. Kettling, A. Koltermann, P. Schwille, M. Eigen, “Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy,” Proc. Natl. Acad. Sci. 95, 1416–1420 (1998). [CrossRef] [PubMed]
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- D. M. Shcherbakova, M. A. Hink, L. Joosen, T. W. J. Gadella, V. V. Verkhusha, “An orange fluorescent protein with a large stokes shift for single-excitation multicolor FCCS and FRET imaging,” J. Am. Chem. Soc. 134, 7913–7923 (2012). [CrossRef]
- O. Krichevsky, G. Bonnet, “Fluorescence correlation spectroscopy: the technique and its applications,” Rep. Prog. Phys. 65, 251–297 (2002). [CrossRef]
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- N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, J. Langowski, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE 4, e5041 (2009). [CrossRef]
- J. Ries, S. Chiantia, P. Schwille, “Accurate determination of membrane dynamics with line-scan FCS,” Biophys. J. 96, 1999–2008 (2009). [CrossRef] [PubMed]
- Q. Ruan, M. A. Cheng, M. Levi, E. Gratton, W. W. Mantulin, “Spatial-temporal studies of membrane dynamics: Scanning fluorescence correlation spectroscopy (SFCS),” Biophys. J. 87, 1260–1267 (2004). [CrossRef] [PubMed]
- G. Heuvelman, F. Erdel, M. Wachsmuth, K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38, 813–828 (2009). [CrossRef] [PubMed]
- D. J. Needleman, Y. Xu, T. J. Mitchison, “Pin-hole array correlation imaging: Highly parallel fluorescence correlation spectroscopy,” Biophys. J. 96, 5050–5059 (2009). [CrossRef] [PubMed]
- R. A. Colyer, G. Scalia, I. Rech, A. Gulinatti, M. Ghioni, S. Cova, S. Weiss, X. Michalet, “High-throughput FCS using an LCOS spatial light modulator and an 8×1 spad array,” Biomed. Opt. Express 1, 1408–1431 (2010). [CrossRef]
- M. Kloster-Landsberg, D. Tyndall, I. Wang, R. Walker, J. Richardson, R. Henderson, A. Delon, “Note: Multi-confocal fluorescence correlation spectroscopy in living cells using a complementary metal oxide semiconductor-single photon avalanche diode array,” Rev. Sci. Instrum. 84, 076105 (2013). [CrossRef] [PubMed]
- B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79, 4463–4470 (2007). [CrossRef] [PubMed]
- T. Wohland, X. Shi, J. Sankaran, E. H. K. Stelzer, “Single plane illumination fluorescence correlation spectroscopy (SPIM-FCS) probes inhomogeneous three-dimensional environments,” Opt. Express 10, 10627–10641 (2010). [CrossRef]
- J. Capoulade, M. Wachsmuth, L. Hufnagel, M. Knop, “Quantitative fluorescence imaging of protein diffusion and interaction in living cells,” Nat. Biotechnol. 29, 835—839 (2011). [CrossRef] [PubMed]
- SI: The supplementary notes are available at http://www.dkfz.de/Macromol/publications/files/spimfccs2013_supplement.pdf .
- M. Gösch, A. Magnusson, S. Hård, H. Blom, S. Anderegg, K. Korn, P. Thyberg, M. Wells, T. Lasser, R. Rigler, “Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements,” J. Biomed. Opt. 10, 054008 (2005). [CrossRef] [PubMed]
- J. Ries, Z. Petrášek, A. J. García-Sáez, P. Schwille, “A comprehensive framework for fluorescence cross-correlation spectroscopy,” New J. Phys. 12, 113009 (2010). [CrossRef]
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