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Optics Express

Optics Express

  • Editor: Andrew M. Weiner
  • Vol. 21, Iss. 21 — Oct. 21, 2013
  • pp: 25291–25306
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Bleaching-corrected fluorescence microspectroscopy with nanometer peak position resolution

Iztok Urbančič, Zoran Arsov, Ajasja Ljubetič, Daniele Biglino, and Janez Štrancar  »View Author Affiliations


Optics Express, Vol. 21, Issue 21, pp. 25291-25306 (2013)
http://dx.doi.org/10.1364/OE.21.025291


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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

1. Introduction

Fluorescence microscopy has boosted advances in life sciences within the last several decades due to its high sensitivity, applicability to live-cell experiments and ability to visualize the sample [1

1. J. Pawley, Handbook of Biological Confocal Microscopy, 3rd ed. (Springer, 2006).

]. Meanwhile, a wealth of molecular information can be provided by fluorescence spectroscopic techniques through excitation/emission spectral shapes, excited state lifetimes, energy transfer efficiencies, rotational and translational correlation times, etc [2

2. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed. (Springer, 2006).

]. To localize this information within the investigated sample, many combined microspectroscopic techniques have emerged that enable molecular characterization at optical spatial resolution.

Among such hybrid methods, spectral imaging, or fluorescence microspectroscopy (FMS), has seen considerable development in recent years [3

3. 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]

,4

4. Y. Garini and E. Tauber, “Spectral imaging: methods, design, and applications,” in Biomedical Optical Imaging Technologies, R. Liang, ed. (Springer, 2013), pp. 111–161.

]. It has mostly been used for distinguishing or co-localizing fluorophores with overlapping emission spectra. To this end, several advanced techniques for spectral unmixing have been introduced [5

5. 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]

8

8. F. Fereidouni, A. N. Bader, and H. C. Gerritsen, “Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images,” Opt. Express 20(12), 12729–12741 (2012). [CrossRef] [PubMed]

]. Conversely, the applicability of these experimental systems to numerous environment-sensitive probes [9

9. 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]

] has been largely neglected. Until now only few dyes with large spectral response to local physical/chemical conditions, e.g. Laurdan [10

10. 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]

] and 3-hydroxyflavone-based probes [11

11. 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. Acta 1788(2), 495–499 (2009). [CrossRef] [PubMed]

], have been used for low spectral-resolution ratiometric imaging.

In fluorescence spectroscopy, several fluorophores with smaller spectral shifts have been extensively used. For instance, 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) enables versatile chemical modifications [12

12. S. Haldar and A. Chattopadhyay, “Application of NBD-labeled lipids in membrane and cell biology,” in Fluorescent Methods to Study Biological Membranes, Y. Mély and G. Duportail, eds., Springer Series on Fluorescence No. 13 (Springer, 2013), pp. 37–50.

,13

13. 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]

] and thus offers ample biological applications, e.g. to sense local polarity, rotational mobility, molecular packing and organization, membrane asymmetry, or temperature [12

12. S. Haldar and A. Chattopadhyay, “Application of NBD-labeled lipids in membrane and cell biology,” in Fluorescent Methods to Study Biological Membranes, Y. Mély and G. Duportail, eds., Springer Series on Fluorescence No. 13 (Springer, 2013), pp. 37–50.

]. To extend FMS to such dyes, we introduced spectral fitting [14

14. 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. Express 2(8), 2083–2095 (2011). [CrossRef] [PubMed]

,15

15. 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]

] using an empirical lineshape function (Section 3 in this paper, which follows the technical details of our work in Section 2). This approach improved spectrum peak position resolution well below filter-width and wavelength sampling step in a similar manner as lateral position determination in particle tracking [16

16. 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]

]. The analogy [17

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

] enabled us to develop a theoretical estimation for peak position resolution, which was highly consistent with our simulated and experimental data. As presented in Section 4, nanometer precision can be achieved at relatively modest signal-to-noise ratio (SNR) and wavelength sampling steps, which should be attainable by most FMS/spectral imaging systems.

To extend the applicability of FMS systems with narrow-band filters (fixed or tunable), which are more affordable and easier to integrate into existing fluorescence microscopes, we have introduced a bleaching correction routine for sequential wavelength sampling [14

14. 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. Express 2(8), 2083–2095 (2011). [CrossRef] [PubMed]

]. As shown in Fig. 1(b), it is based on measuring fluorescence intensity at a chosen reference wavelength several times during the experiment, which enables retrograde correction of the spectral lineshape by accounting for signal decay due to photobleaching. At sufficiently high SNR, the method considerably reduces bleaching-induced artifacts. At lower signal levels, however, some systematic deviations persist, which can be successfully eliminated by stochastic wavelength sampling, presented in Fig. 1(c), and improved spectral fitting [15

15. 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]

].

In Section 5, we systematically described and compared both methods for bleaching correction and spectral analysis. We showed that by careful experimental and analytical approach, peak position resolution and accuracy, described above, could be largely maintained even when using photosensitive probes. The applicability of the technique was demonstrated in Section 6 by membrane phase sensitivity of two environment- and photo-sensitive probes (an NBD-based probe and Laurdan), where spectral shifts as low as 1.5 nm were reliably detected. Additionally, the bleaching correction approach revealed a significant difference in bleaching rate between samples in different lipid phases. Hence, probe photobleaching should not be considered a disadvantage; instead, when properly recorded and analyzed, it represents additional valuable information about local molecular environment that can be exploited for bleach rate imaging [24

24. D. M. Benson, J. Bryan, A. L. Plant, A. M. Gotto Jr, 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]

26

26. 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,” Traffic 11(4), 440–454 (2010). [CrossRef] [PubMed]

].

2. Materials and methods

2.1 Materials

Phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were purchased from Avanti Polar Lipids (Alabaster, AL). Fatty acid-based NBD probe SPP268 ((2R,3S,4R,5R,6R)-2-(hydroxymethyl)-5-((7-nitrobenzo [c] [1

1. J. Pawley, Handbook of Biological Confocal Microscopy, 3rd ed. (Springer, 2006).

,2

2. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed. (Springer, 2006).

,5

5. 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]

]oxadiazol-4-yl)amino)-6-((1-tetradecyl-1H-1,2,3-triazol-4-yl)methoxy)tetrahydro-2H-pyran-3,4-diol) [13

13. 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]

] was synthesized by Dr. Stane Pajk from Faculty of Pharmacy at University of Ljubljana, while Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) was purchased from Molecular Probes (Eugene, OR). Cholesterol (chol), sucrose, 1,2-diacyl-sn-glycero-3-phospho-[1-rac-glycerol] (PG) and dimethyl sulfoxide (DMSO) were obtained from Sigma Aldrich (St. Louis, MO) while glucose was from Kemika (Zagreb, Croatia). Organic solvents chloroform and methanol were purchased from AppliChem GmbH and Merck KGaA (both from Darmstad, Germany), respectively. The organic gelator from Biomade (Groningen, The Netherlands), based on 1,3,5-cyclohexyltricarboxamide [27

27. 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 ONE 6(5), e20435 (2011). [CrossRef] [PubMed]

], was kindly provided by Dr. Ilja Küsters. All chemicals were used without further purification.

2.2 Liposome preparation

Giant unilamellar vesicles (GUV) were prepared by the gentle hydration method [28

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

] from DPPC, DOPC, and DPPC + chol (40 mol%), which at room temperature represent gel, liquid disordered, and liquid ordered phase, respectively. To each composition 15 mol% of charged PG lipids were added to induce formation of vesicles. The dry lipid film, formed on the glass tube walls by organic solvent evaporation, was prehydrated for 30 min in water vapor-saturated atmosphere at 60 °C. After the addition of preheated 0.1 M sucrose solution, the sample was left to hydrate overnight at 60 °C. Before measurements, the appropriate amount of SPP268 ethanol solution was added to the GUV suspension for final probe-to-lipid molar ratio of 1:200, while the same amount of Laurdan was added already during the preparation of the dry lipid film. The probe concentration was low enough to prevent aggregation of probe molecules or energy transfer that could affect fluorescence emission spectra.

2.3 Spectrofluorimeter measurements

Reference fluorescence emission spectra were measured at Infinite M1000 microplate reader (Tecan, Männendorf, Switzerland) at room temperature. A 96-well black plate was used in the fluorescence intensity top mode. For Laurdan and NBD samples, fluorescence was excited at 370 and 450 nm, and emission spectra recorded from 400 to 550 and from 480 to 650 nm, respectively, both excitation and emission bandwidths being 10 nm. Reference background of 0.1 M sucrose was subtracted from fluorescence emission spectra of the samples.

2.4 Quantum-mechanical spectral model – harmonic oscillator

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.
The simplest possible physical model, illustrated in Fig. 2, describes electron transition probabilities from the vibrationally-relaxed excited electron level (0*) into discrete vibrational levels of the ground electron state (n). The probability for the transition P0*→n can be determined by calculating overlap integrals between the two wave functions. Assuming identical one-dimensional harmonic oscillators, the probability reads [29

29. 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]

]
P0*n(Δx2/2)nExp(Δx2/2)Γ(n+1),
(1)
where Γ is the Gamma function [30

30. M. Abramowitz and I. A. Stegun, Handbook of Mathematical Functions: With Formulas, Graphs and Mathematical Tables (Dover, 1972).

] and Δx represents the spectral asymmetry-inducing expansion of equilibrium intramolecular distance in the excited electronic state. The spectrum (SHO), expressed by wavelength (λ), can be calculated from the following relations:
SHO(λ)=P0*n|dndλ|,n=E*hc/λE0,
(2)
where E* and E0 represent energy differences between electronic and vibrational states, respectively, h stands for the Planck constant, and c for the speed of light in vacuum. The obtained spectrum, discrete over n, can be made continuous by allowing n to take any positive real value, reasoning that various electron orbitals and vibrational modes of complex molecules contribute to the overall spectrum, which smears their discrete spectra.

2.5 Empirical spectral model – log-normal function

2.6 Comparison of the two spectral models

Both three-parametric spectral models, described above, were used to fit spectrofluorimetric data of NBD-based alkyl chain probe SPP268 in DPPC vesicles. The spectrum was normalized to its maximal signal level, and only data above the signal intensity threshold (IT) 0.2 were used. The model parameters were optimized by Mathematica’s (Wolfram Research, Champaign, IL) Nelder-Mead minimization routine. Standard χ2, reduced to the number of points and normalized to the noise level, was calculated as a measure for goodness-of-fit, while the time needed for optimization was monitored to assess computational costs.

Model robustness and consistency were tested by several trivial modifications of the fitting problem: firstly, IT was varied; secondly, wavelength step-size (Δλ) was increased by removing data points; and thirdly, random noise was added to mimic experiments with lower SNR. For a well-posed model, none of the variations should affect the optimized parameters. Therefore, average relative error (〈δp/p1〉) of parameters’ fitted values (pi), compared to those obtained for the non-modified spectrum (pi,1), was chosen to measure model robustness:
δp/p1i=13|pipi,1|/(3pi,1),
(4)
where i runs over all three parameters for either spectral model. Optimization of parameters was repeated 64 times with noise signal generated each time anew.

2.7 Bleaching spectrum model

As the log-normal model was found to be faster and more robust than the quantum-mechanical one, this lineshape function was further used for numerical simulations and to analyze FMS experiments. Photobleaching was modeled by a mono-exponential decay [34

34. T. Hirschfeld, “Quantum efficiency independence of the time integrated emission from a fluorescent molecule,” Appl. Opt. 15(12), 3135–3139 (1976). [CrossRef] [PubMed]

] of simulated intensity with bleaching rate (b), counting the time (t) from the beginning of the experiment. The bleaching spectrum signal (I) was thus described by
I(λ)=I0SLN(λ)exp(bt),
(5)
where I0 represents maximal signal level of the non-bleached spectrum. The lineshape parameters were obtained by optimized Nelder-Mead minimization [35

35. O. R., “Shaving the last 50 ms off NMinimize Web. 29 April 2012 http://mathematica.stackexchange.com/a/4877/5443,” (2012).

] of the standard reduced χ2, whereas optimal I0 was determined analytically [15

15. 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]

]. By the developed algorithm, a typical λ-stack of 512 × 512 images, averaged over 5 × 5 pixels, was analyzed in less than 5 s on a standard quad-core desktop computer.

To characterize more complex spectra due to multi-peak lineshape of the fluorophore in use, presence of multiple dyes, or significant cellular autofluorescence, the procedure allows multicomponent bleaching-corrected optimization to separate the spectral contributions [15

15. 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]

].

2.8 Numerical comparison of bleaching correction algorithms

Probe photobleaching inevitably distorts sequentially sampled spectra, as shown in Fig. 1(a). To recover the true signal, we have introduced two acquisition schemes: linear wavelength sampling with intermittent measurements at a reference wavelength [14

14. 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. Express 2(8), 2083–2095 (2011). [CrossRef] [PubMed]

], illustrated in Fig. 1(b), and stochastic wavelength sampling, depicted in Fig. 1(c). Both methods record position-dependent bleaching dynamics that can be used during spectral fitting.

Efficiencies of the two bleaching correction algorithms were first tested numerically. To mimic FMS experiments with a photosensitive NBD probe, synthetic spectra with different SNR levels were generated according to Eq. (3) (I0 = 1, λMAX = 535 nm, w = 78 nm, a = 0.24) within the range 515–585 nm. Bleaching rate and wavelength step size were varied; results for b = 0.02/“exposure time” and Δλ = 3 nm are presented. For the linear reference algorithm, six reference data points at 540 nm were also generated; to keep the total number of data the same as for the other two methods, Δλ was increased accordingly.

The generated spectra were fitted 104 times with noise signal each time generated anew and with w and a fixed to their true values. For linear acquisition where no specific information about bleaching dynamics was available, b was either set to 0 or optimized. Knowing the original parameter values (pi,0), errors of the fitted ones (pi) were calculated (δpi = |pipi,0|; pi again represents any of the optimized parameters).

2.9 FMS setup

NBD-based probe was excited by nonpolarized light from a Xe-Hg source (Sutter Lambda LS, Novato, CA) through 460/60 broad-band filter, while 377/50 was used for Laurdan (all band-pass filters and dichroics were BrightLine from Semrock, Rochester, NY). Fluorescence was detected through 550/88 and 470/100 emission filters for NBD and Laurdan, respectively. For spectral detection a narrow-band liquid-crystal tunable filter (LCTF; Varispec VIS-10-20 from CRi, Woburn, MA) was placed in front of an EMCCD camera (iXon3 897 from Andor, Belfast, UK), allowing sequential acquisition of images at different wavelengths within the transmission range of the emission filter.

For each λ-stack of images, spectra from every volume-element of the field-of-view were extracted. After the dark signal of the camera was subtracted, the spectra were corrected for transmittance of LCTF, which had been calibrated against a set of reference dyes.

2.10 FMS experiments with probe SPP268 in solution

Reference FMS experiments for fitting performance tests were conducted at room temperature with 10−4 M solution of probe SPP268 in DMSO, where NBD experienced a similar polarity as in the membrane. Objectives with 10x (air) and 60x (water immersion) magnification were used to prevent or induce significant probe photobleaching, respectively. Various settings for wavelength sampling step and exposure time were applied to vary SNR and SNRTOT, defined later by Eq. (7).

Spectra from the central region of the image (200 × 200 pix) were analyzed, as outlined above. Since the wavelength scanning range within the transmitting region of the broad-band emission filter (510–585 nm) was smaller than FWHM of NBD spectra, determination of w and especially a from FMS experiments was not efficient. They were therefore determined by fitting the spectrofluorimeter data and then kept fixed at 78 nm and 0.24, respectively, during the optimization of I0, λMAX and b. For measurements at 10x magnification, b was set to 0.

For comparison of the two wavelength acquisition schemes, mean λMAX and their standard deviations were calculated for each experiment. As the stochastic method yielded very consistent λMAX values around 543 nm, their average was taken as a reference to determine the errors at each measurement (δλMAX). SNR was determined from the brightest image in each λ-stack as standard deviation of intensity divided by mean intensity value.

2.11 FMS experiments with lipid vesicles

For easier observation, the chosen GUV suspension was 10x diluted in 0.1 M glucose solution, which caused the vesicles to settle at the bottom of the chamber due to the resulting density difference between interior and exterior of the liposomes. When mixed GUV samples were imaged, the vesicles were additionally immobilized by a transparent hydrogel, composed of an organic gelator [27

27. 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 ONE 6(5), e20435 (2011). [CrossRef] [PubMed]

], which conveniently liquefied upon shaking and solidified in a few minutes. About 60 μl of the sample was transferred into a pool, made from silicone lubrication grease (Klüber Lubrication, Munich, Germany) between a standard microscopy slide and a coverslip. Vesicles were imaged at room temperature by a 60x water immersion objective. For samples with NBD and Laurdan, stochastic wavelength scans from 510 to 582 nm and from 430 to 511 nm with 3 nm step were performed, respectively, using 100–300 ms exposure times and 200-fold electron multiplication of the signal.

For temperature-dependent experiments, the sample was sealed between two coverslips and placed on a microscopy slide with a home-made indium tin oxide (ITO) heating layer and a thermocouple for the feedback to the temperature control unit (ITC503 by Oxford Instruments, Abingdon, UK).

To minimize artifacts due to motion of vesicles during acquisition, images from each λ-stack were automatically aligned using algorithms built in Mathematica. All images were averaged over 5 × 5 pixels, or 9 × 9 for samples with Laurdan, to achieve the desired spectral resolution. For SPP268, the fitting procedure was the same as in the section 2.10, while for Laurdan w = 50 nm was used and a was allowed for optimization. To visualize the results, images were spectrally contrasted with respect to the optimized λMAX or b according to the accompanying color legends, while I0 was coded by pixel brightness [15

15. 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]

]. Parameter I0 was used to weight the contribution of each pixel when constructing histograms from subareas of the field-of-view.

3. Spectral lineshape models

To improve spectral peak position resolution below experimental λ-step and LCTF bandwidth, spectral fitting was applied in analogy with the approach used in particle tracking [16

16. 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]

]. We searched for an appropriate function that could accommodate to the main characteristics of simple fluorescence emission spectra. To adequately define peak position, width and asymmetry of a potentially noisy distribution without its extreme tails, as usually obtained by FMS, we limited our choice to three-parametric models to ensure the best fitting performance in terms of computation time, stability and parameter correlations.

In the literature, two approaches to describe fluorescence spectra have been used: deriving the spectrum from basic quantum mechanics [29

29. 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]

,36

36. F. Iachello and M. Ibrahim, “Analytic and algebraic evaluation of Franck−Condon overlap integrals,” J. Phys. Chem. A 102(47), 9427–9432 (1998). [CrossRef]

], or describing the lineshape by empirical asymmetrical functions [31

31. 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]

,32

32. 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]

]. We therefore tested the most convenient models from both classes: one based on physical description of transitions between harmonic oscillator eigenstates (HO), and the other using a log-normal lineshape (LN).

The two models were applied to fit normalized spectrofluorimeter data of SPP268 in DPPC vesicles, as presented in Fig. 3(a).
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.
Since HO model was unable to efficiently describe the spectral tails, only data above the intensity threshold IT = 0.2 were used. Above this threshold, both fitted the part of the spectrum comparably well; if some noise was added to mimic typical FMS data with SNR in the range from 10 to 100, both models achieved perfect accordance with the data with χ2-values around 1, as shown in Fig. 3(b). Though, the time needed for optimization, presented in Fig. 3(c), was significantly lower for LN model, which is of high importance as several ten thousand spectra should be fitted from pixels of each FMS λ-stack. For real-time analysis of the latter, we applied an optimized algorithm [35

35. O. R., “Shaving the last 50 ms off NMinimize Web. 29 April 2012 http://mathematica.stackexchange.com/a/4877/5443,” (2012).

] that sped up the process about 1000-fold.

To check model robustness, we monitored average relative error (〈δp/p1〉) of the three parameters, compared to the values obtained for the original data set, while varying IT, sampling step (Δλ), and SNR. The variations mimicked measurements of the same sample under different experimental conditions. As neither of the modifications changed the underlying shape of the spectrum, no variations in optimized parameters were anticipated. As seen from Figs. 3(d)–(f), the fitted parameter values of the empirical model (LN) were much less sensitive to these perturbations and scattered less than those of the physical model (HO). The instability of the latter probably originated in considerable interrelations of the parameters’ influence on the lineshape, revealed by covariance matrices. We therefore selected the better-posed LN function for our further spectral analysis.

4. Peak position resolution

According to the expression above, one can expectedly enhance the accuracy of λMAX by lowering Δλ or by increasing SNR, as shown by Fig. 4(a).
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).
A decrease in Δλ linearly increases the number of images in the λ-stack (N) and thus linearly prolongs the total experiment duration (tTOT), which does not affect SNR for filter-based FMS setups with fixed spectral bandwidth. Conversely, SNR is proportional to tEXP1/2 = (tTOT/N)1/2 – as long as camera read noise is negligible compared to Poisson shot noise. Hence, considering also the contributing powers of Δλ and SNR in Eq. (6), both ways of σλmax improvement are equally time-effective, showing that all information gathered during tTOT is equivalent.

This can be explicitly shown by introducing the total SNR (SNRTOT).:
SNRTOT=SNRN=SNRΛΔλ=SNRtEXPtTOT,
(7)
which measures the quality of information of the whole FMS experiment. Note that it can only be affected by tTOT, as SNR/tEXP1/2 depends solely on sample brightness and experimental system characteristics, i.e., illumination level and detection efficiency. Equation (7) can be used to substitute SNR and Δλ in Eq. (6) with the primary influence on λMAX resolution, SNRTOT:
σλmax=ΛWSNRTOTΔF(Λ).
(8)
Since Λ is often predestined by the experimental setup, tTOT that defines SNRTOT is the prime variable to improve λMAX resolution. Once tTOT is determined, e.g. by sample dynamics or fluorophore bleaching rate, the choice of Δλ – which in turn determines tEXP of each image in the λ-stack – can then be optimized to other experimental issues. For instance, sample movement and determination of other spectral lineshape parameters favor smaller Δλ and shorter tEXP, while larger Δλ and higher SNR are preferred for very dim samples to avoid excessive contribution of read noise. Considering σλmax, the only prerequisite is that the number of measured λ-points is larger than the number of fitted parameters [17

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

].

Within the derivation we assumed a filter-based FMS system with sequential λ-sampling and fixed spectral bandwidth. Nevertheless, Eq. (8) holds also for diffraction-based systems with variable bandwidth per spectral channel, as long as the bandwidth does not exceed approx. w/2 and significantly distorts the spectrum shape through convolution.

To verify the relation in Eq. (8), we performed FMS experiments with SPP268 in solution, using 10x lens magnification to prevent photobleaching and various wavelength sampling steps and exposure times to influence SNR and SNRTOT. Spectra from pixels across the images in each λ-stack were fitted with w and a fixed to the values obtained from spectrofluorimetric data and b = 0. As shown by Fig. 4(b), the obtained standard deviations of λMAX distributions (σλmax) confirmed that λMAX precision was mainly determined by SNRTOT, as predicted by Eq. (8), which corroborated all the approximations discussed above. For measurements with high SNRTOT where read noise was negligible, very nice accordance with the theory for one-parametric fit was obtained, even though two parameters (I0 and λMAX) were in fact extracted. This indicates that analytical determination of I0 acted as exact normalization without reducing the information available to λMAX. The explanation was further supported by results when w was also optimized; in this case σλmax were slightly higher, conforming to the theoretical curve for two parameters (data not shown).

5. Bleaching correction algorithms

If fluorescence emission intensities at various wavelengths are acquired sequentially, probe photobleaching significantly distorts the recorded spectral lineshape, as presented in Fig. 1(a) [3

3. 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]

,23

23. 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]

]. Since the acquired spectrum can be equally well described by a range of λMAX-b combinations, decoupling of the two superimposed effects is numerically ill-posed. To avoid potential misinterpretations of such data, we introduced two wavelength sampling routines that allowed us to purposely record the bleaching dynamics and take it into account during spectral fitting: linear acquisition with reference measurements [14

14. 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. Express 2(8), 2083–2095 (2011). [CrossRef] [PubMed]

], depicted in Fig. 1(b) and stochastic sampling [15

15. 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]

], illustrated in Fig. 1(c).

To evaluate both bleaching correction methods, we first applied them to numerically generated spectra, mimicking FMS experiments with an NBD probe, and compared the errors of fitted values for all optimized parameters – δλMAX and δb are presented in Figs. 5(a)and 5(b), respectively).
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.
As predicted, large errors in λMAX were observed for linear acquisition without any bleaching correction attempt. As w and a were fixed, fitting b from these data on average yielded more accurate results, but with excessively high deviations due to numerical instabilities, outlined above. Conversely, parameters scattered less for the two specialized sampling routines that both nicely replicated the true parameter set at high SNR. Figure 5(b) shows that, at lower SNR, however, the reference method often misestimated the bleaching rate from the six dedicated data points and consequently wrongly determined λMAX. In contrast, the technique with stochastic sampling, using all data points, resolved the original b value with much better precision and accuracy, leading to more reliable determination of I0 and λMAX.

Simulations at various bleaching rates and Δλ always yielded qualitatively similar results. Moreover, even if we deliberately fixed w and a to values that were slightly different than used for data generation, other parameters were still correctly resolved, further confirming the choice of a well-posed spectral model. In addition, the results substantiated our use of values obtained from fitting the spectrofluorimeter data as the best educated guess when w and a could not have been reliably determined from our FMS experiments due to narrow wavelength acquisition range, predefined by the available broad-band emission filter.

Finally, we searched for the optimal total duration of an FMS experiment with a photosensitive probe in terms of λMAX and b accuracy and precision, assuming that one can afford to arbitrarily bleach the sample within one measurement. Fast acquisition, compared to the bleaching rate of a given fluorophore, minimizes the bleaching-induced distortion of the spectrum, but also reduces SNRTOT and thus the resolution of fitted parameters. Oppositely, during a long experiment much of the signal is lost, meaning that further acquisition does not yield any useful information. Our numerical simulations, again applying various Δλ and SNR, showed that parameter reconstruction was the most effective when tTOT ≈1–1.5 b–1, i.e., when approx. 60–75% of the signal was bleached during the experiment. Similar numerical results (84%) have been obtained for optimal unmixing of spectrally-overlapping bleaching probes, assuming a diffraction-based experimental system with simultaneous λ-acquisition [38

38. 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]

].

6. Demonstration of application: lipid phase sensitivity

To demonstrate the use of stochastic bleaching correction FMS algorithm and the applicability of high spectral peak position resolution, we chose a well-known and controlled model system: spectral response to local polarity of a photosensitive NBD-based probe SPP268 was used to distinguish DPPC and DOPC GUV samples. Due to different structure of the two lipids and consequently different packing of the molecules in the bilayers, the two membranes are at room temperature found in gel and liquid disordered membrane phases, respectively [39

39. R. Koynova and M. Caffrey, “Phases and phase transitions of the phosphatidylcholines,” Biochim. Biophys. Acta 1376(1), 91–145 (1998). [CrossRef] [PubMed]

], exhibiting different polarity profiles across the bilayer [40

40. 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,” Biochemistry 33(24), 7670–7681 (1994). [CrossRef] [PubMed]

]. Since the applied fluorophore is located in the headgroup region, as depicted by the inset in Fig. 6(a), where polarity differences between phases are much smaller than in the membrane core [40

40. 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,” Biochemistry 33(24), 7670–7681 (1994). [CrossRef] [PubMed]

], only minor variations in fluorescence emission spectra were expected [12

12. S. Haldar and A. Chattopadhyay, “Application of NBD-labeled lipids in membrane and cell biology,” in Fluorescent Methods to Study Biological Membranes, Y. Mély and G. Duportail, eds., Springer Series on Fluorescence No. 13 (Springer, 2013), pp. 37–50.

]. As shown by Fig. 6(a), spectrofluorimeter measurements of bulk samples indeed showed a red shift of 3 nm for the probe in DOPC compared to DPPC sample, which was in accordance with the expected higher local polarity [41

41. 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]

] due to increased hydration of the outer membrane region [42

42. Z. Arsov and L. Quaroni, “Direct interaction between cholesterol and phosphatidylcholines in hydrated membranes revealed by ATR-FTIR spectroscopy,” Chem. Phys. Lipids 150(1), 35–48 (2007). [CrossRef] [PubMed]

].
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.

The small spectral difference was unmistakably resolved by FMS on individual vesicles. Figure 6(b) confirms that the influence of strong photobleaching was effectively fitted, resulting in clearly distinguishable spectral differences between GUV in Fig. 6(c). The observed shift around 2 nm was nicely revealed by nanometer λMAX resolution, estimated from histogram widths. Quantitative accordance with spectrofluorimetric data as well as with FMS measurements on separate GUV suspensions (data not shown) enabled us to unambiguously determine the composition of the two vesicles: DPPC for the smaller (labeled as “1”) and DOPC for the larger liposome (“2”).

Besides the spectral shift between the vesicles in different lipid phases, the method revealed a significant difference in bleaching rate, which was also in agreement with the known probe behavior. In a more polar environment, fluorescence emission spectrum of NBD shifts to higher wavelengths and lifetime decreases [41

41. 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]

]. According to the linear relationship between fluorescence lifetime and bleaching rate [34

34. T. Hirschfeld, “Quantum efficiency independence of the time integrated emission from a fluorescent molecule,” Appl. Opt. 15(12), 3135–3139 (1976). [CrossRef] [PubMed]

], the probe in DOPC should thus bleach slower. The relation was confirmed by lower b, obtained with bleaching-corrected FMS analysis for vesicle “2”, as is apparent from Fig. 6(d). In addition to spectral information, the procedure therefore provides the functionality of bleach rate imaging [24

24. D. M. Benson, J. Bryan, A. L. Plant, A. M. Gotto Jr, 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]

26

26. 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,” Traffic 11(4), 440–454 (2010). [CrossRef] [PubMed]

], representing a simple alternative to fluorescence lifetime imaging [26

26. 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,” Traffic 11(4), 440–454 (2010). [CrossRef] [PubMed]

,34

34. T. Hirschfeld, “Quantum efficiency independence of the time integrated emission from a fluorescent molecule,” Appl. Opt. 15(12), 3135–3139 (1976). [CrossRef] [PubMed]

].

To corroborate the results further, we measured FMS signal after having induced a phase transition of DPPC vesicles from gel to liquid disordered phase by heating the sample from approx. 35°C to approx. 45 °C, as presented by Figs. 7(a) and 7(b) (the transition occurs around 41 °C [39

39. R. Koynova and M. Caffrey, “Phases and phase transitions of the phosphatidylcholines,” Biochim. Biophys. Acta 1376(1), 91–145 (1998). [CrossRef] [PubMed]

]).
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.
In accordance with temperature-dependent spectrofluorimetric data (not shown), λMAX red-shifted for about 1.5 nm, as can be observed from the histograms. Figure 7(c) revealed that upon cooling, one vesicle was found to undergo the transition earlier than the other, but the change in mean λMAX was expectedly reversible, as is seen in Fig. 7(d). As a reference, we checked that no detectable effect of heating was observed for DOPC samples which do not exhibit a phase transition in this temperature region [39

39. R. Koynova and M. Caffrey, “Phases and phase transitions of the phosphatidylcholines,” Biochim. Biophys. Acta 1376(1), 91–145 (1998). [CrossRef] [PubMed]

].

To obtain even more in-depth information about particular lipid phase properties, it is also advisable to use other complementary methods, such as infrared spectroscopy [42

42. Z. Arsov and L. Quaroni, “Direct interaction between cholesterol and phosphatidylcholines in hydrated membranes revealed by ATR-FTIR spectroscopy,” Chem. Phys. Lipids 150(1), 35–48 (2007). [CrossRef] [PubMed]

,44

44. 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]

] or Raman-based imaging, e.g. coherent anti-Stokes Raman scattering imaging [45

45. 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]

] and tip-enhanced Raman scattering imaging [46

46. 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]

]. These label-free techniques are especially valuable when putative effects of probes are being evaluated and when chemical identification of membrane components is needed. Nevertheless, fluorescence methods are invaluable for many biological and biophysical applications due to their high sensitivity and applicability to live 3D samples. As such, bleaching-corrected FMS could complement the information obtained by other techniques that are required to adequately characterize complex systems.

7. Conclusion

To demonstrate the reach of the technique, we applied it to distinguish lipid vesicles in different phases with two commonly used environment-sensitive dyes, NBD and Laurdan, that shifted their spectral maxima for 1.5–3 nm. The results showed that peak position resolution, characteristic for spectrofluorimetric measurements on bulk samples, could readily be achieved at micrometer spatial scale by conventional FMS/spectral imaging systems. By this a whole new range of probes with desired biochemical characteristics, but weaker environmental response, could be exploited to characterize more complex biological systems, provided adequate reference measurements. Moreover, to distinguish various environmental effects, environment-dependent photosensitivity can be simultaneously used for bleach rate imaging, which was in our case provided automatically by bleaching-corrected FMS.

Acknowledgments

The authors thank Slovenian Research Agency (ARRS, grant nos. P1-0060, PR-03089-1, and PR-03090-1) and Centre of Excellence NAMASTE for funding, Dr. Stane Pajk for synthesizing the fluorescent probe, Alma Mehle and Maja Garvas for their help with experimental work, Prof. Igor Križaj for providing access to Tecan microplate reader, and Dr. Miha Škarabot for lending the equipment for sample temperature control.

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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]

46.

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]

OCIS Codes
(000.2170) General : Equipment and techniques
(000.4430) General : Numerical approximation and analysis
(070.4790) Fourier optics and signal processing : Spectrum analysis
(180.2520) Microscopy : Fluorescence microscopy
(300.6280) Spectroscopy : Spectroscopy, fluorescence and luminescence
(110.4234) Imaging systems : Multispectral and hyperspectral imaging

ToC Category:
Microscopy

History
Original Manuscript: August 12, 2013
Revised Manuscript: October 7, 2013
Manuscript Accepted: October 8, 2013
Published: October 16, 2013

Citation
Iztok Urbančič, Zoran Arsov, Ajasja Ljubetič, Daniele Biglino, and Janez Štrancar, "Bleaching-corrected fluorescence microspectroscopy with nanometer peak position resolution," Opt. Express 21, 25291-25306 (2013)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-21-21-25291


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