Abstract

Fluorescence microspectroscopy (FMS) with environmentally sensitive dyes provides information about local molecular surroundings at microscopic spatial resolution. Until recently, only probes exhibiting large spectral shifts due to local changes have been used. For filter-based experimental systems, where signal at different wavelengths is acquired sequentially, photostability has been required in addition. Herein, we systematically analyzed our spectral fitting models and bleaching correction algorithms which mitigate both limitations. We showed that careful analysis of data acquired by stochastic wavelength sampling enables nanometer spectral peak position resolution even for highly photosensitive fluorophores. To demonstrate how small spectral shifts and changes in bleaching rates can be exploited, we analyzed vesicles in different lipid phases. Our findings suggest that a wide range of dyes, commonly used in bulk spectrofluorimetry but largely avoided in microspectroscopy due to the above-mentioned restrictions, can be efficiently applied also in FMS.

© 2013 Optical Society of America

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2013 (1)

I. Urbančič, A. Ljubetič, Z. Arsov, and J. Štrancar, “Coexistence of probe conformations in lipid phases-a polarized fluorescence microspectroscopy study,” Biophys. J.105(4), 919–927 (2013).
[CrossRef] [PubMed]

2012 (1)

2011 (5)

Z. Arsov, I. Urbančič, M. Garvas, D. Biglino, A. Ljubetič, T. Koklič, and J. Štrancar, “Fluorescence microspectroscopy as a tool to study mechanism of nanoparticles delivery into living cancer cells,” Biomed. Opt. Express2(8), 2083–2095 (2011).
[CrossRef] [PubMed]

L. Opilik, T. Bauer, T. Schmid, J. Stadler, and R. Zenobi, “Nanoscale chemical imaging of segregated lipid domains using tip-enhanced Raman spectroscopy,” Phys. Chem. Chem. Phys.13(21), 9978–9981 (2011).
[CrossRef] [PubMed]

I. Kusters, N. Mukherjee, M. R. de Jong, S. Tans, A. Koçer, and A. J. M. Driessen, “Taming membranes: functional immobilization of biological membranes in hydrogels,” PLoS ONE6(5), e20435 (2011).
[CrossRef] [PubMed]

A. M. Valm, J. L. Mark Welch, C. W. Rieken, Y. Hasegawa, M. L. Sogin, R. Oldenbourg, F. E. Dewhirst, and G. G. Borisy, “Systems-level analysis of microbial community organization through combinatorial labeling and spectral imaging,” Proc. Natl. Acad. Sci. U.S.A.108(10), 4152–4157 (2011).
[CrossRef] [PubMed]

S. Pajk, M. Garvas, J. Štrancar, and S. Pečar, “Nitroxide-fluorophore double probes: a potential tool for studying membrane heterogeneity by ESR and fluorescence,” Org. Biomol. Chem.9(11), 4150–4159 (2011).
[CrossRef] [PubMed]

2010 (1)

D. Wüstner, A. Landt Larsen, N. J. Faergeman, J. R. Brewer, and D. Sage, “Selective visualization of fluorescent sterols in Caenorhabditis elegans by bleach-rate-based image segmentation,” Traffic11(4), 440–454 (2010).
[CrossRef] [PubMed]

2009 (4)

A. S. Klymchenko, S. Oncul, P. Didier, E. Schaub, L. Bagatolli, G. Duportail, and Y. Mély, “Visualization of lipid domains in giant unilamellar vesicles using an environment-sensitive membrane probe based on 3-hydroxyflavone,” Biochim. Biophys. Acta1788(2), 495–499 (2009).
[CrossRef] [PubMed]

A. P. Demchenko, Y. Mély, G. Duportail, and A. S. Klymchenko, “Monitoring biophysical properties of lipid membranes by environment-sensitive fluorescent probes,” Biophys. J.96(9), 3461–3470 (2009).
[CrossRef] [PubMed]

R. A. Neher, M. Mitkovski, F. Kirchhoff, E. Neher, F. J. Theis, and A. Zeug, “Blind source separation techniques for the decomposition of multiply labeled fluorescence images,” Biophys. J.96(9), 3791–3800 (2009).
[CrossRef] [PubMed]

H. Xu and B. W. Rice, “In-vivo fluorescence imaging with a multivariate curve resolution spectral unmixing technique,” J. Biomed. Opt.14(6), 064011 (2009).
[CrossRef] [PubMed]

2007 (2)

Z. Arsov and L. Quaroni, “Direct interaction between cholesterol and phosphatidylcholines in hydrated membranes revealed by ATR-FTIR spectroscopy,” Chem. Phys. Lipids150(1), 35–48 (2007).
[CrossRef] [PubMed]

A. Kalauzi, D. Mutavdzić, D. Djikanović, K. Radotić, and M. Jeremić, “Application of asymmetric model in analysis of fluorescence spectra of biologically important molecules,” J. Fluoresc.17(3), 319–329 (2007).
[CrossRef] [PubMed]

2005 (1)

L. Li, H. Wang, and J.-X. Cheng, “Quantitative coherent anti-Stokes Raman scattering imaging of lipid distribution in coexisting domains,” Biophys. J.89(5), 3480–3490 (2005).
[CrossRef] [PubMed]

2004 (3)

R. Neher and E. Neher, “Optimizing imaging parameters for the separation of multiple labels in a fluorescence image,” J. Microsc.213(1), 46–62 (2004).
[CrossRef] [PubMed]

S. Duhr, S. Arduini, and D. Braun, “Thermophoresis of DNA determined by microfluidic fluorescence,” Eur. Phys,” J. E Soft Matter15(3), 277–286 (2004).
[CrossRef]

T. Zal and N. R. J. Gascoigne, “Photobleaching-corrected FRET efficiency imaging of live cells,” Biophys. J.86(6), 3923–3939 (2004).
[CrossRef] [PubMed]

2003 (1)

T. Zimmermann, J. Rietdorf, and R. Pepperkok, “Spectral imaging and its applications in live cell microscopy,” FEBS Lett.546(1), 87–92 (2003).
[CrossRef] [PubMed]

2001 (2)

R. Lansford, G. Bearman, and S. E. Fraser, “Resolution of multiple green fluorescent protein color variants and dyes using two-photon microscopy and imaging spectroscopy,” J. Biomed. Opt.6(3), 311–318 (2001).
[CrossRef] [PubMed]

J. Markham and J.-A. Conchello, “Artefacts in restored images due to intensity loss in three-dimensional fluorescence microscopy,” J. Microsc.204(2), 93–98 (2001).
[CrossRef] [PubMed]

2000 (1)

L. A. Bagatolli and E. Gratton, “Two photon fluorescence microscopy of coexisting lipid domains in giant unilamellar vesicles of binary phospholipid mixtures,” Biophys. J.78(1), 290–305 (2000).
[CrossRef] [PubMed]

1999 (2)

J. Löbau, M. Sass, W. Pohle, C. Selle, M. H. J. Koch, and K. Wolfrum, “Chain fluidity and phase behaviour of phospholipids as revealed by FTIR and sum-frequency spectroscopy,” J. Mol. Struct.480–481, 407–411 (1999).
[CrossRef]

A. Bullen and P. Saggau, “High-speed, random-access fluorescence microscopy: II. Fast quantitative measurements with voltage-sensitive dyes,” Biophys. J.76(4), 2272–2287 (1999).
[CrossRef] [PubMed]

1998 (2)

R. Koynova and M. Caffrey, “Phases and phase transitions of the phosphatidylcholines,” Biochim. Biophys. Acta1376(1), 91–145 (1998).
[CrossRef] [PubMed]

F. Iachello and M. Ibrahim, “Analytic and algebraic evaluation of Franck−Condon overlap integrals,” J. Phys. Chem. A102(47), 9427–9432 (1998).
[CrossRef]

1996 (3)

T. Schmidt, G. J. Schütz, W. Baumgartner, H. J. Gruber, and H. Schindler, “Imaging of single molecule diffusion,” Proc. Natl. Acad. Sci. U.S.A.93(7), 2926–2929 (1996).
[CrossRef] [PubMed]

E. A. Burstein and V. I. Emelyanenko, “Log-normal description of fluorescence spectra of organic fluorophores,” Photochem. Photobiol.64(2), 316–320 (1996).
[CrossRef]

K. Akashi, H. Miyata, H. Itoh, and K. Kinosita., “Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope,” Biophys. J.71(6), 3242–3250 (1996).
[CrossRef] [PubMed]

1994 (3)

G. J. Brakenhoff, K. Visscher, and E. J. Gijsbers, “Fluorescence bleach rate imaging,” J. Microsc.175(2), 154–161 (1994).
[CrossRef]

W. K. Subczynski, A. Wisniewska, J.-J. Yin, J. S. Hyde, and A. Kusumi, “Hydrophobic barriers of lipid bilayer membranes formed by reduction of water penetration by alkyl chain unsaturation and cholesterol,” Biochemistry33(24), 7670–7681 (1994).
[CrossRef] [PubMed]

T. Parasassi, M. Di Stefano, M. Loiero, G. Ravagnan, and E. Gratton, “Influence of cholesterol on phospholipid bilayers phase domains as detected by Laurdan fluorescence,” Biophys. J.66(1), 120–132 (1994).
[CrossRef] [PubMed]

1993 (1)

S. Fery-Forgues, J.-P. Fayet, and A. Lopez, “Drastic changes in the fluorescence properties of NBD probes with the polarity of the medium: involvement of a TICT state?” J. Photochem. Photobiol. Chem.70(3), 229–243 (1993).
[CrossRef]

1991 (1)

J. P. Rigaut and J. Vassy, “High-resolution three-dimensional images from confocal scanning laser microscopy. Quantitative study and mathematical correction of the effects from bleaching and fluorescence attenuation in depth,” Anal. Quant. Cytol. Histol.13(4), 223–232 (1991).
[PubMed]

1986 (1)

N. Bobroff, “Position measurement with a resolution and noise‐limited instrument,” Rev. Sci. Instrum.57(6), 1152–1157 (1986).
[CrossRef]

1985 (1)

D. M. Benson, J. Bryan, A. L. Plant, A. M. Gotto, and L. C. Smith, “Digital imaging fluorescence microscopy: spatial heterogeneity of photobleaching rate constants in individual cells,” J. Cell Biol.100(4), 1309–1323 (1985).
[CrossRef] [PubMed]

1982 (1)

S. Waldenstrøm and K. R. Naqvi, “The overlap integrals of two harmonic-oscillator wavefunctions: some remarks on originals and reproductions,” Chem. Phys. Lett.85(5-6), 581–584 (1982).
[CrossRef]

1976 (2)

1969 (1)

D. B. Siano and D. E. Metzler, “Band shapes of the electronic spectra of complex molecules,” J. Chem. Phys.51(5), 1856–1861 (1969).
[CrossRef]

Akashi, K.

K. Akashi, H. Miyata, H. Itoh, and K. Kinosita., “Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope,” Biophys. J.71(6), 3242–3250 (1996).
[CrossRef] [PubMed]

Arduini, S.

S. Duhr, S. Arduini, and D. Braun, “Thermophoresis of DNA determined by microfluidic fluorescence,” Eur. Phys,” J. E Soft Matter15(3), 277–286 (2004).
[CrossRef]

Arsov, Z.

I. Urbančič, A. Ljubetič, Z. Arsov, and J. Štrancar, “Coexistence of probe conformations in lipid phases-a polarized fluorescence microspectroscopy study,” Biophys. J.105(4), 919–927 (2013).
[CrossRef] [PubMed]

Z. Arsov, I. Urbančič, M. Garvas, D. Biglino, A. Ljubetič, T. Koklič, and J. Štrancar, “Fluorescence microspectroscopy as a tool to study mechanism of nanoparticles delivery into living cancer cells,” Biomed. Opt. Express2(8), 2083–2095 (2011).
[CrossRef] [PubMed]

Z. Arsov and L. Quaroni, “Direct interaction between cholesterol and phosphatidylcholines in hydrated membranes revealed by ATR-FTIR spectroscopy,” Chem. Phys. Lipids150(1), 35–48 (2007).
[CrossRef] [PubMed]

Bader, A. N.

Bagatolli, L.

A. S. Klymchenko, S. Oncul, P. Didier, E. Schaub, L. Bagatolli, G. Duportail, and Y. Mély, “Visualization of lipid domains in giant unilamellar vesicles using an environment-sensitive membrane probe based on 3-hydroxyflavone,” Biochim. Biophys. Acta1788(2), 495–499 (2009).
[CrossRef] [PubMed]

Bagatolli, L. A.

L. A. Bagatolli and E. Gratton, “Two photon fluorescence microscopy of coexisting lipid domains in giant unilamellar vesicles of binary phospholipid mixtures,” Biophys. J.78(1), 290–305 (2000).
[CrossRef] [PubMed]

Bauer, T.

L. Opilik, T. Bauer, T. Schmid, J. Stadler, and R. Zenobi, “Nanoscale chemical imaging of segregated lipid domains using tip-enhanced Raman spectroscopy,” Phys. Chem. Chem. Phys.13(21), 9978–9981 (2011).
[CrossRef] [PubMed]

Baumgartner, W.

T. Schmidt, G. J. Schütz, W. Baumgartner, H. J. Gruber, and H. Schindler, “Imaging of single molecule diffusion,” Proc. Natl. Acad. Sci. U.S.A.93(7), 2926–2929 (1996).
[CrossRef] [PubMed]

Bearman, G.

R. Lansford, G. Bearman, and S. E. Fraser, “Resolution of multiple green fluorescent protein color variants and dyes using two-photon microscopy and imaging spectroscopy,” J. Biomed. Opt.6(3), 311–318 (2001).
[CrossRef] [PubMed]

Benson, D. M.

D. M. Benson, J. Bryan, A. L. Plant, A. M. Gotto, and L. C. Smith, “Digital imaging fluorescence microscopy: spatial heterogeneity of photobleaching rate constants in individual cells,” J. Cell Biol.100(4), 1309–1323 (1985).
[CrossRef] [PubMed]

Biglino, D.

Bobroff, N.

N. Bobroff, “Position measurement with a resolution and noise‐limited instrument,” Rev. Sci. Instrum.57(6), 1152–1157 (1986).
[CrossRef]

Borisy, G. G.

A. M. Valm, J. L. Mark Welch, C. W. Rieken, Y. Hasegawa, M. L. Sogin, R. Oldenbourg, F. E. Dewhirst, and G. G. Borisy, “Systems-level analysis of microbial community organization through combinatorial labeling and spectral imaging,” Proc. Natl. Acad. Sci. U.S.A.108(10), 4152–4157 (2011).
[CrossRef] [PubMed]

Bowyer, S.

M. Lampton, B. Margon, and S. Bowyer, “Parameter estimation in X-ray astronomy,” Astrophys. J.208, 177 (1976).
[CrossRef]

Brakenhoff, G. J.

G. J. Brakenhoff, K. Visscher, and E. J. Gijsbers, “Fluorescence bleach rate imaging,” J. Microsc.175(2), 154–161 (1994).
[CrossRef]

Braun, D.

S. Duhr, S. Arduini, and D. Braun, “Thermophoresis of DNA determined by microfluidic fluorescence,” Eur. Phys,” J. E Soft Matter15(3), 277–286 (2004).
[CrossRef]

Brewer, J. R.

D. Wüstner, A. Landt Larsen, N. J. Faergeman, J. R. Brewer, and D. Sage, “Selective visualization of fluorescent sterols in Caenorhabditis elegans by bleach-rate-based image segmentation,” Traffic11(4), 440–454 (2010).
[CrossRef] [PubMed]

Bryan, J.

D. M. Benson, J. Bryan, A. L. Plant, A. M. Gotto, and L. C. Smith, “Digital imaging fluorescence microscopy: spatial heterogeneity of photobleaching rate constants in individual cells,” J. Cell Biol.100(4), 1309–1323 (1985).
[CrossRef] [PubMed]

Bullen, A.

A. Bullen and P. Saggau, “High-speed, random-access fluorescence microscopy: II. Fast quantitative measurements with voltage-sensitive dyes,” Biophys. J.76(4), 2272–2287 (1999).
[CrossRef] [PubMed]

Burstein, E. A.

E. A. Burstein and V. I. Emelyanenko, “Log-normal description of fluorescence spectra of organic fluorophores,” Photochem. Photobiol.64(2), 316–320 (1996).
[CrossRef]

Caffrey, M.

R. Koynova and M. Caffrey, “Phases and phase transitions of the phosphatidylcholines,” Biochim. Biophys. Acta1376(1), 91–145 (1998).
[CrossRef] [PubMed]

Cheng, J.-X.

L. Li, H. Wang, and J.-X. Cheng, “Quantitative coherent anti-Stokes Raman scattering imaging of lipid distribution in coexisting domains,” Biophys. J.89(5), 3480–3490 (2005).
[CrossRef] [PubMed]

Conchello, J.-A.

J. Markham and J.-A. Conchello, “Artefacts in restored images due to intensity loss in three-dimensional fluorescence microscopy,” J. Microsc.204(2), 93–98 (2001).
[CrossRef] [PubMed]

de Jong, M. R.

I. Kusters, N. Mukherjee, M. R. de Jong, S. Tans, A. Koçer, and A. J. M. Driessen, “Taming membranes: functional immobilization of biological membranes in hydrogels,” PLoS ONE6(5), e20435 (2011).
[CrossRef] [PubMed]

Demchenko, A. P.

A. P. Demchenko, Y. Mély, G. Duportail, and A. S. Klymchenko, “Monitoring biophysical properties of lipid membranes by environment-sensitive fluorescent probes,” Biophys. J.96(9), 3461–3470 (2009).
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Figures (8)

Fig. 1
Fig. 1

Schematic presentation of the effect of probe photobleaching on fluorescence emission spectrum, recorded with different sequential wavelength (λ) acquisition schemes: (a) Linear λ-sampling yields a distorted spectrum (solid circles and line in the bottom panel), compared to the expected spectrum (dotted line in the bottom panel). (b) Occasional measurements of signal at a reference λ (green points) additionally record the intensity decay rate that can be taken into account during spectral fitting. (c) Stochastic λ-sampling stores the information about bleaching dynamics together with spectral lineshape into the “saw-tooth” signal, which is due to random jumps of λ measurement points while intensity decays in time (t).

Fig. 2
Fig. 2

Schematic representation of a transition (red arrow) between two eigenstates of shifted harmonic oscillators (dashed curves represent the potentials). Fluorophore is assumed to relax from the ground vibrational state of the excited electron level (0*) into any vibrational state of the ground electron level (n). Transition probability is proportional to the overlap integral of the corresponding wave functions (ψn). Shaded wavy curves represent probability distributions (ψn2) over the reduced vibrational coordinate x. Symbols ħ and m stand for reduced Planck constant and molecular mass, respectively.

Fig. 3
Fig. 3

Comparison of the two lineshapes for spectral fitting: quantum-mechanical harmonic oscillator model (HO, black symbols) and empirical log-normal function (LN, red symbols). (a) Best fits (solid lines) to the spectrofluorimetric data of SPP268 in DPPC GUV (gray open circles) above the intensity threshold 0.2 (dotted line). (b) Goodness-of-fit (χ2) and (c) time needed for optimization (t) for spectra with different levels of added noise (SNR). To measure model robustness, average relative error (〈δp/p1〉) of the fitted parameters, compared to the values obtained for the original data set, was monitored when varying (d) intensity threshold (IT), (e) wavelength step (Δλ), and (f) SNR. Columns and error bars represent mean and standard deviation of the corresponding values, respectively, for 64 repeats of parameter optimization with noise signal generated each time anew.

Fig. 4
Fig. 4

(a) Theoretical peak position uncertainty (σλmax), calculated according to Eq. (6) for an NBD-like spectrum (w = 78 nm, a = 0.24) sampled at various SNR and wavelength steps (Δλ). One fitting parameter, Poisson noise, and λ-sampling range as in our FMS experiments were assumed. (b) Standard deviations of λMAX (σλmax), obtained from optimizations of experimental spectra across FMS images. These were acquired at various Δλ (see color legend) and exposure times to yield signals of different total SNR (SNRTOT). Light intensity through 10x objective was low enough to prevent probe photobleaching. The gray line represents the theoretically predicted λMAX precision, calculated by Eq. (8) with the same assumptions as in panel (a).

Fig. 5
Fig. 5

Comparison of wavelength sampling schemes for bleaching correction: linear without correction, linear with fitted b, linear with reference, and stochastic (see color legend in panel b). When spectral data were numerically generated (λMAX = 535 nm, w = 78 nm, a = 0.24, b = 0.02/“exposure time”; Δλ = 3 nm, Λ = 69 nm), errors of the fitted values for (a) λMAX and (b) b were monitored. For clarity of presentation, the data sets were slightly shifted along SNR-axis. Experimental FMS spectra of SPP268 solution were measured with either bleaching correction acquisition routine at various settings for exposure time and Δλ to influence SNR and SNRTOT. Photobleaching was induced by 60x magnification lens. From the results of optimization for all pixels in each λ-stack, (c) mean λMAX were compared to a reference value. (d) Standard deviations of λMAX are plotted against theoretical prediction (gray line), calculated by Eq. (8) for two-parametric fits with the same assumptions as in Fig. 4.

Fig. 6
Fig. 6

(a) Fluorescence emission spectra of SPP268 (inset) in DPPC and DOPC vesicles (blue and green line, respectively), acquired separately on a spectrofluorimeter at room temperature. (b) Representative FMS spectra, distorted due to photobleaching (circles of the appropriate colors), of the two GUV samples. Solid and dashed lines represent the model fit and bleaching-corrected spectral reconstruction (BC), respectively. Spectra were extracted from the two red points in colored images, spectrally contrasted according to (c) spectral peak position (λMAX) and (d) bleaching rate (b). Blue and green rectangles mark the areas from where the correspondingly-colored histograms of the optimized parameter values from each liposome were constructed. Similar analysis was performed also for GUV from DPPC and DPPC + chol (40 mol%) and labeled with the probe Laurdan: (e) Spectrofluorimetric data and the structure of the probe (inset). (f) Typical FMS spectra, extracted form the two red points in λMAX-contrasted images for a representative (g) DPPC and (h) DPPC + chol vesicle.

Fig. 7
Fig. 7

Spectrally contrasted images of SPP268-labeled DPPC vesicles in (a,d) gel phase, (b) liquid disordered phase, and (c) around the phase transition. Upon cooling, vesicle “1” underwent the phase transition earlier than vesicle “2”. The histograms show the distribution of λMAX for the two vesicles, marked with the accordingly-colored rectangles in the images above.

Fig. 8
Fig. 8

(a) Fluorescence intensity image of cells MCF-7 (at the bottom of the image), mixed with uni- and multilamellar vesicles DOPC (top), which were all labeled with the NBD-based probe SPP268 in the presence of cell medium. Cell cultivation and handling were the same as in our previous work [14]. (b) Spectrally contrasted image and histograms of λMAX values from the two marked areas, as obtained from stochastic λ-sampling and bleaching-corrected FMS analysis. The measured autofluorescence was low enough not to affect the analysis and results.

Equations (8)

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P 0 * n ( Δ x 2 / 2 ) n Exp ( Δ x 2 / 2 ) Γ ( n + 1 ) ,
S HO ( λ ) = P 0 * n | d n d λ | , n = E * h c / λ E 0 ,
S LN ( λ )={ exp( log2 4 a 2 [ log( 4a w ( λ λ MAX )+1 ) ] 2 ), λ> λ MAX w 4a , 0, λ λ MAX w 4a .
δp / p 1 i=1 3 | p i p i,1 | / ( 3 p i,1 ) ,
I( λ )= I 0 S LN ( λ )exp( bt ),
σ λmax = ΔλW SNR Δ F(Λ) ,
SNR TOT =SNR N =SNR Λ Δλ = SNR t EXP t TOT ,
σ λmax = ΛW SNR TOT Δ F(Λ) .

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