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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

  • Editor: Gregory W. Faris
  • Vol. 4, Iss. 12 — Nov. 10, 2009
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Three distinct sarcomeric patterns of skeletal muscle revealed by SHG and TPEF Microscopy

Gaëlle Recher, Denis Rouède, Patrick Richard, Antoine Simon, Jean-Jaques Bellanger, and François Tiaho  »View Author Affiliations


Optics Express, Vol. 17, Issue 22, pp. 19763-19777 (2009)
http://dx.doi.org/10.1364/OE.17.019763


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Abstract

We have extensively characterized the sarcomeric SHG signal as a function of animal species (rat versus xenopus), age (adult versus larval) and tissue preparation (fixed or fresh) and we found that the main feature of this signal is a single peak per mature sarcomere (about 85% of all sarcomeres). The remaining (15%) was found to be either double peak per mature sarcomere or mini sarcomeres (half of a sarcomere) using α-actinin immuno detection of the Z-band. The mini sarcomeres are often found in region of pitchfork-like SHG pattern. We suggest that double peak SHG pattern could indicate regions of sarcomeric proteolysis whereas pitchfork-like SHG pattern could reveal sarcomeric assembly.

© 2009 Optical Society of America

1. Introduction

Second harmonic generation (SHG) imaging microscopy has recently emerged as a powerful and unique non-destructive imaging tool for high-resolution, high-contrast, three-dimensional studies of endogenous proteins such as myosin, collagen, tubulin (microtubule), glial fibrillary acidic protein, starch, cellulose and holds promise for both basic research and clinical pathology [1

1. P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J 82, 493–508 (2002). [CrossRef]

8

8. S. Psilodimitrakopoulos, S. Santos, I. Amat-Roldan, A. K. N. Thayil, D. Artigas, and P. Loza-Alvarez, “In vivo, pixel-resolution mapping of thick filaments’ orientation in nonfibrilar muscle using polarization-sensitive second harmonic generation microscopy,” J. Biomed. Opt. 14, 014001 (2009). [CrossRef] [PubMed]

]. SHG imaging microscopy relies on a nonlinear interaction of tightly focused ultrashort laser pulses with noncentrosymmetric quasi crystalline arrangement of optically hyperpolarizable molecules (harmonophores) causing scattered coherent radiation at twice the fundamental frequency. SHG is essentially an instantaneous phemomenon compared to fluorescence and all the incoming energy is converted into radiated photons. No nonradiative processes are involved in SHG and in consequence live cells do not suffer from phototoxic effects or photobleaching and therefore viability is extended. Moreover SHG imaging microscopy offers intrinsic optical studies of 3D endogeneous proteins which often required exogenous immunofluorescence labeling with poor optical contrast and invasiveness [9

9. D. E. Rudy, T. A. Yatskievych, P. B. Antin, and C. C. Gregorio, “Assembly of thick, thin, and titin filaments in chick precardiac explants,” Dev. Dyn. 221, 61–71 (2001). [CrossRef] [PubMed]

]. Finally polarization dependence of the SHG signal can be exploited as a new source of contrast to gain information from orientation and submicrometric organization of endogenous proteins [4

4. F. Tiaho, G. Recher, and D. Rouède, “Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy,” Opt. Express 15, 12286–12295 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-19-12286. [CrossRef] [PubMed]

, 10

10. C. Odin, T. Guilbert, A. Alkilani, O. P. Boryskina, V. Fleury, and Y. Le Grand, “Collagen and myosin characterization by orientation field second harmonic microscopy,” Opt. Express 16, 16151–16165 (2008), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-16-20-16151. [CrossRef] [PubMed]

].

Fig. 1 Schematic diagram illustrating the major sarcomeric components of striated muscles. (a) Global view of two myofibrils (thickness ≈ 1 μm) formed by contractile sarcomeres (width ≈ 2.2 μm) which assemble laterally and display a pattern of alternating light (I) and dark (A) bands. (b) View of the sarcomere showing overlapping arrays of bipolar myosin thick filaments and actin thin filaments. Thick and thin filaments are transversally interconnected at the M- and Z- band respectively. Myomesin or M-protein (M-band) involved in these connections are not represented. Thick filaments are longitudinally connected at the Z- and M- band by titin and for simplicity only the connection at the Z-band is represented. (c) The myosin molecule is an hexamere of two heavy chains and two pairs of light chains (not presented) and has a length of about 160 nm. The heavy chain α-helix tails of each myosin molecule form a coiled-coil super helix dimer. (d) Schematic representation of the antiparallel assembly of myosin molecules at the M-band free of heads (width ≈ 120–200 nm). Myosin heads point away from the filament center and myosin tails have antiparallel overlapping (width 85 –130 nm).

Coherent emission from myosin molecules of thick filaments is responsible of the characteristic periodical sarcomeric SHG signal observed in striated muscles. Surprisingly the SHG pattern is not always identical and is either one-band or two-band centered at the midlle of the sarcomere. Single-band sarcomeric SHG pattern has been observed in gastrocnemius of adult xenopus [4

4. F. Tiaho, G. Recher, and D. Rouède, “Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy,” Opt. Express 15, 12286–12295 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-19-12286. [CrossRef] [PubMed]

], frog tibialis anterior muscle [15

15. F. Vanzi, M. Capitanio, L. Sacconi, C. Stringari, R. Cicchi, M. Canepari, M. Maffei, N. Piroddi, C. Poggesi, V. Nucciotti, M. Linari, G. Piazzesi, C. Tesi, R. Antolini, V. Lombardi, R. Bottinelli, and F. S. Pavone, “New techniques in linear and non-linear laser optics in muscle research,” J. Muscle Res. Cell Motil. 27, 469–479 (2006). [CrossRef] [PubMed]

], mouse quadriceps or gastrocnemius muscles [16

16. S. V. Plotnikov, A. M. Kenny, S. J. Walsh, B. Zubrowski, C. Joseph, V. L. Scranton, G. A. Kuchel, D. Dauser, M. Xu, C. C. Pilbeam, D. J. Adams, R. P. Dougherty, P. J. Campagnola, and W. A. Mohler, “Measurement of muscle disease by quantitative second-harmonic generation imaging,” J Biomed. Opt. 13, 044018 (2008). [CrossRef] [PubMed]

, 17

17. F. Légaré, C. Pfeffer, and B. R. Olsen, “The role of backscattering in SHG tissue imaging,” Biophys. J. 93, 1312–1320 (2007). [CrossRef] [PubMed]

], mouse and human hind lamb muscles [18

18. E. Ralston, B. Swaim, M. Czapiga, W. L. Hwu, Y. H. Chien, M. G. Pittis, B. Bembi, O. Schwartz, P. Plotz, and N. Raben, “Detection and imaging of non-contractile inclusions and sarcomeric anomalies in skeletal muscle by second harmonic generation combined with two-photon excited fluorescence,” J. Struct. Biol. 162, 500–508 (2008). [CrossRef] [PubMed]

], veal cutlet muscles [10

10. C. Odin, T. Guilbert, A. Alkilani, O. P. Boryskina, V. Fleury, and Y. Le Grand, “Collagen and myosin characterization by orientation field second harmonic microscopy,” Opt. Express 16, 16151–16165 (2008), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-16-20-16151. [CrossRef] [PubMed]

], nematode body-wall muscles [8

8. S. Psilodimitrakopoulos, S. Santos, I. Amat-Roldan, A. K. N. Thayil, D. Artigas, and P. Loza-Alvarez, “In vivo, pixel-resolution mapping of thick filaments’ orientation in nonfibrilar muscle using polarization-sensitive second harmonic generation microscopy,” J. Biomed. Opt. 14, 014001 (2009). [CrossRef] [PubMed]

]. Double-band sarcomeric SHG pattern has been observed in C-elegans body-wall muscles [1

1. P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J 82, 493–508 (2002). [CrossRef]

, 3

3. W. Mohler, A. C. Millard, and P. J. Campagnola, “Second harmonic generation imaging of endogenous structural proteins,” Methods 29, 97–109 (2003). [CrossRef] [PubMed]

], frog heart muscles [19

19. T. Boulesteix, E. Beaurepaire, M. P. Sauviat, and M. C. Schanne-Klein, “Second-harmonic microscopy of unstained living cardiac myocytes: measurements of sarcomere length with 20-nm accuracy,” Opt. Lett. 29, 2031–2033 (2004). [CrossRef] [PubMed]

], mouse tibialis anterior muscle [20

20. M. Both, M. Vogel, O. Friedrich, F. von Wegner, T. Kunsting, R. H. Fink, and D. Uttenweiler, “Second harmonic imaging of intrinsic signals in muscle fibers in situ,” J. Biomed. Opt. 9, 882–892 (2004). [CrossRef] [PubMed]

], mouse leg and chicken heart [21

21. S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys. J. 90, 693–703 (2006). [CrossRef]

], rabbit psoas muscles [15

15. F. Vanzi, M. Capitanio, L. Sacconi, C. Stringari, R. Cicchi, M. Canepari, M. Maffei, N. Piroddi, C. Poggesi, V. Nucciotti, M. Linari, G. Piazzesi, C. Tesi, R. Antolini, V. Lombardi, R. Bottinelli, and F. S. Pavone, “New techniques in linear and non-linear laser optics in muscle research,” J. Muscle Res. Cell Motil. 27, 469–479 (2006). [CrossRef] [PubMed]

], drosophilla flight muscles [22

22. C. Greenhalgh, N. Prent, C. Green, R. Cisek, A. Major, B. Stewart, and V. Barzda, “Influence of semicrystalline order on the second-harmonic generation efficiency in the anisotropic bands of myocytes,” Appl. Opt. 46, 1852–1859 (2007). [CrossRef] [PubMed]

, 23

23. N. Prent, C. Green, C. Greenhalgh, R. Cisek, A. Major, B. Stewart, and V. Barzda, “Intermyofilament dynamics of myocytes revealed by second harmonic generation microscopy,” J. Biomed. Opt. 13, 041318 (2008). [CrossRef] [PubMed]

]. Such change of the sarcomeric SHG pattern is puzzling since it is well known from electron microscopy (EM) studies that the size of thick filaments is constant and that of the M-band is between (120–200 nm) in vertebrates [12

12. R. Craig and J. L. Woodhead, “Structure and function of myosin filaments,” Curr. Opin. Struct. Biol. 16, 204–212 (2006). [CrossRef] [PubMed]

14

14. S. G. Page and H. E. Huxley, “Filament Lengths in Striated Muscle,” J. Cell Biol. 19, 369–390 (1963). [CrossRef] [PubMed]

, 24

24. T. Shimizu, J. E. Dennis, T. Masaki, and D. A. Fischman, “Axial Arrangement of the Myosin Rod in Vertebrate Thick Filaments: Immunoelectron Microscopy with a Monoclonal Antibody to Light Meromyosin,” J. Cell Biol. 101, 1115–1123 (1985). [CrossRef] [PubMed]

]. In order to understand the sarcomeric SHG pattern and the conditions of its change, we undertook a quantitative study of the sarcomeric SHG signal taking into account the influence of animal species (rat versus xenopus), age (adult versus larval) and tissue preparation (fixed or fresh) using high optical resolution objectives. We found that the sarcomeric SHG signal could be either one peak or two peaks independently of the experimental conditions. Proportion of sarcomeres with one peak is the most frequent result and has been measured to be about 85%. Using α-actinin immuno detection of the Z-band, we observed that SHG intensity profiles with two peaks per sarcomere could originate from either mini sarcomeres (half of a sarcomere) or mature sarcomeres. We show that double peak SHG pattern is dramatically increase by muscle proteolysis. We discuss the biological meaning of the double peak SHG patterns.

2. Experimental methods

2.1. Tissue preparation

Muscle tissues were obtained from gastrocnemius of adult Xenopus laevis (national breeding facility of xenopus animals in Rennes, France) and wistar rats (Janvier, Le Genest-St-Isle-France). At least five adult xenopus and rats were used. The tissues were fixed with paraformaldehyd (PFA) 4% (by incubation of the tissue block or by perfusion of the fixative with heparin in the whole animal prior to dissection of the muscle) or either fresh. For fixed tissues, muscles were dissected, fixed over night at 4°C then rinsed at least three times with the appropriate saline buffer (e. g. Phosphate Buffer Saline (PBS) for rat tissues and Mark’s Modified Ringer (MMR) for xenopus tissues). For proteolysis analysis of muscle tissues, stage 50 tadpoles were euthanized in MS222 (0.5 mg/mL). They were either immediately fixed (J0) in PFA (4 % at 4°C) or led in breeding water at room temperature (18–22°C) for 24 H (J1) or 48 H (J2) before being fixed according to the same procedure. They were rinsed after one night of fixation in MMR saline buffer before slicing. 100 or 200 μm thick tissues were obtained by slicing muscles directly glued on the stage of a vibroslicer (LEICA, VT 1200S). Particular care has been taken to cut the slices along the main axis of the myofibrils. Slices were mounted between two coverslips in mounting medium (Vectashild, Vector Burlingame, CA) before SHG imaging. For α-actinin immunostaining, 10–15 μm cryostat sections, mounted on gelatin coated coverslips, were permeabilized with buffer containing 0.5% triton and 5% fetal calf serum for 10 minutes and incubated at 4°C over night in MMR containing 0.1% triton, 5% fetal calf serum and α-actinin antibody (1:100, mouse monoclonal IgM, ab9465, Abcam, Cambridge, MA, USA). Slices were rinsed three times in MMR and incubated at 4°C over night in Ringer containing 0.1% triton, 5% fetal calf serum and secondary antibody (1:100, Alexa Fluor 488 goat anti-mouse IgG, A11029, Molecular Probes, Eugene, OR, USA). After being rinsed three times, the tissue was covered with a drop of mounting medium (Vectashield, Vector Burlingame, CA) and a second coverslip was laid on. After being sealed, preparations were imaged.

2.2. Imaging system

Images were acquired on PIXEL (http://pixel.univ-rennes1.fr/) facility of GIS EUROPIA, University of Rennes1, France) based on either a Leica TCS SP2 confocal scanning head coupled to a DMIRB inverted microscope or an Olympus FV1000 confocal scanning head coupled to a BX61WI upright microscope. Each setup is equipped with a MAITAI Spectra Physics femtosecond laser. High numerical aperture objective either Leica 63X, oil immersion, NA 1.4, HCX PLAPO or Olympus 60X, water immersion, NA1.2, UplanApo/IR or Olympus NA1.1, LUMFl were used for applying 10–20 mW of 940 nm excitation at the sample. The theoretical optical resolution was about 400 nm, 480 nm and 520 nm for the three objectives respectively [25

25. Max Born and Emil Wolf, Principles of Optics: Electromagnetic Theory of Propagation, Interference and Diffraction of Light (Pergamon Press, 6th Edition, Oxford, 1980). [PubMed]

]. The SHG signal was collected in a forward direction using either a multi-immersion S1 (NA = 0.90–1.4) Leica condenser or an Olympus WI-UCD universal condenser (NA = 0.80). A BG39 bandpass filter and a 470 nm IR filter (10 nm FWHM) were placed before the PMT. The dichroic filter wheel of the microscope was removed and replaced by a computer control PR50CC Newport rotation stage (precision 0.1°) equiped with an achromatic zero-order Quartz-MgF2 half-wave plate in order to adjust the polarization angle of the incident IR electric field without movement of the biological specimen. The images were quantitatively analyzed with open source ImageJ software (http://rsb.info.nih.gov/ij/). All the specimens were positionned on the fixed X,Y stage of the microscope with light propagating in the Z direction. The Olympus setup was used only for the experiments concerning the effect of proteolysis on SHG signal.

2.3. Quantification of single-band sarcomeres

To quantify the percentage of sarcomeres presenting SHG signal with one peak or two peaks per sarcomere, we have used two methods described in Appendix based respectively on a FFT analysis followed by a two-band pass Gabor filtering (Appendix 1) and an intensity profile analysis (Appendix 2). The values of Table 1 were obtained from 5 to 24 thick slices randomly chosen from at least five animals for each experimental condition with randomized acquisition fields.

Table 1. Quantification of sarcomeric SHG patterns in xenopus and rat muscles. The first two lines represent the mean percentage and standard deviation (± SD) of single-band (SB) sarcomeres from xenopus gastrocnemius (X Gastroc), xenopus tail (X tail) and rat gastrocnemius (R Gastroc) muscles. The tissues were fixed with PFA 4% (incubated or perfused) or either fresh. Note that the mean percentage has been determined using the Gabor filtering method (Gabor) or the intensity profile analysis (IPA) presented in Appendix 1 and Appendix 2 respectively. The values were obtained from 5 to 24 thick slices (40×40×10 μm3) from at least five animals for each experimental condition. The mean sarcomere size (± SD) and the average (± SD) full width at half maximum (FWHM) of the sarcomeric SHG signal were determined for both single-band (SB) and double-band (DB) sarcomeres for each experimental condition.

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3. Experimental results

It has already been shown that the sarcomeric SHG pattern can present either one band [4

4. F. Tiaho, G. Recher, and D. Rouède, “Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy,” Opt. Express 15, 12286–12295 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-19-12286. [CrossRef] [PubMed]

, 8

8. S. Psilodimitrakopoulos, S. Santos, I. Amat-Roldan, A. K. N. Thayil, D. Artigas, and P. Loza-Alvarez, “In vivo, pixel-resolution mapping of thick filaments’ orientation in nonfibrilar muscle using polarization-sensitive second harmonic generation microscopy,” J. Biomed. Opt. 14, 014001 (2009). [CrossRef] [PubMed]

, 10

10. C. Odin, T. Guilbert, A. Alkilani, O. P. Boryskina, V. Fleury, and Y. Le Grand, “Collagen and myosin characterization by orientation field second harmonic microscopy,” Opt. Express 16, 16151–16165 (2008), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-16-20-16151. [CrossRef] [PubMed]

, 16

16. S. V. Plotnikov, A. M. Kenny, S. J. Walsh, B. Zubrowski, C. Joseph, V. L. Scranton, G. A. Kuchel, D. Dauser, M. Xu, C. C. Pilbeam, D. J. Adams, R. P. Dougherty, P. J. Campagnola, and W. A. Mohler, “Measurement of muscle disease by quantitative second-harmonic generation imaging,” J Biomed. Opt. 13, 044018 (2008). [CrossRef] [PubMed]

18

18. E. Ralston, B. Swaim, M. Czapiga, W. L. Hwu, Y. H. Chien, M. G. Pittis, B. Bembi, O. Schwartz, P. Plotz, and N. Raben, “Detection and imaging of non-contractile inclusions and sarcomeric anomalies in skeletal muscle by second harmonic generation combined with two-photon excited fluorescence,” J. Struct. Biol. 162, 500–508 (2008). [CrossRef] [PubMed]

] or two bands [1

1. P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J 82, 493–508 (2002). [CrossRef]

, 3

3. W. Mohler, A. C. Millard, and P. J. Campagnola, “Second harmonic generation imaging of endogenous structural proteins,” Methods 29, 97–109 (2003). [CrossRef] [PubMed]

, 15

15. F. Vanzi, M. Capitanio, L. Sacconi, C. Stringari, R. Cicchi, M. Canepari, M. Maffei, N. Piroddi, C. Poggesi, V. Nucciotti, M. Linari, G. Piazzesi, C. Tesi, R. Antolini, V. Lombardi, R. Bottinelli, and F. S. Pavone, “New techniques in linear and non-linear laser optics in muscle research,” J. Muscle Res. Cell Motil. 27, 469–479 (2006). [CrossRef] [PubMed]

, 19

19. T. Boulesteix, E. Beaurepaire, M. P. Sauviat, and M. C. Schanne-Klein, “Second-harmonic microscopy of unstained living cardiac myocytes: measurements of sarcomere length with 20-nm accuracy,” Opt. Lett. 29, 2031–2033 (2004). [CrossRef] [PubMed]

23

23. N. Prent, C. Green, C. Greenhalgh, R. Cisek, A. Major, B. Stewart, and V. Barzda, “Intermyofilament dynamics of myocytes revealed by second harmonic generation microscopy,” J. Biomed. Opt. 13, 041318 (2008). [CrossRef] [PubMed]

]. In order to elucidate the mechanism underlying this variation, we have extensively characterized the SHG signal from 100 μm thick slices of xenopus and rat muscles. Typical SHG images from both tissues are shown in Fig. 2(a,b). In most regions, sarcomeric SHG signals are bright straight bands regularly spaced orthogonally to the long axis of each myofibril oriented along the horizontal X axis of the microscope stage. One can also noticed in some regions the presence of a regular pattern with the same spacing but presenting two bands (see arrowheads). Corresponding SHG intensity profiles presented in Fig. 2(c,d) along indicated segments of Fig. 2(a,b) clearly show that within the same spacing (≈ 2 μm) the SHG pattern present either one peak or two peaks. We undertook immuno staining of α-actinin which is considered as a specific molecular marker of Z-band [21

21. S. V. Plotnikov, A. C. Millard, P. J. Campagnola, and W. A. Mohler, “Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres,” Biophys. J. 90, 693–703 (2006). [CrossRef]

, 26

26. J. W. Sanger, S. M. Kang, C. C. Siebrands, N. Freeman, A. P. Du, J. S. Wang, A. L. Stout, and J. M. Sanger, “How to build a myofibril,” J. Muscle Res. Cell Motil. 26, 343–354 (2005). [CrossRef]

]. TPEF signal was found to periodically alternate with sarcomeric SHG signal as illustrated in Fig. 3 demonstrating that SHG signal can effectively be either one band or two bands per sarcomere. Two methods were used to quantify the proportion of each pattern. The first one (see Appendix 1) is based on a FFT analysis followed by a local two band-pass Gabor filtering [27

27. S. G. Mallat, A wavelet tour of signal processing (Academic Press, 2nd Edition, London, 1999).

] which enhances the spectral components associated to sarcomeres with one band or two bands. A post-filtering decision stage enables to automatically classify each pixel in one of three classes: background, single-band sarcomere and double-band sarcomere. The second one (see Appendix 2) is based on the detection of the maximum of the SHG intensity profile corresponding to a transition between a positive and a negative first discrete derivative. This later method classify SHG pattern in two categories: pattern with one band per sarcomere or pattern with two bands per sarcomere. The results, given in Table 1 for the analyzed samples, show that single-band sarcomeric SHG pattern is the predominant feature (85%) of most sarcomeres for adult rat and xenopus. For xenopus, this result is independent of the method of tissue preparation and is identical for two weeks old tadpole fresh tail muscles. Note that the two independently designed methods yield the same result demonstrating their efficiency.

Fig. 2 Typical SHG images and intensity profiles from adult gastrocnemius muscles. (a) Optical sections illustrating SHG images from xenopus (a) and rat (b) muscles. The scale bar is 2 μm and the full image is 40×40 μm. A Gaussian Blur filter (radius=2) was applied for each image. Arrowheads and arrows indicate respectively bright double-band sarcomeric SHG signal and bright pitchfork SHG pattern. (c, d) SHG intensity profiles along the indicated segments in respectively (a) and (b). The left and right profiles of images (c) and (d) correspond respectively to the horizontal black and white lines drawn in (a) and (b). Note that the optical resolution was not sufficient to discriminate indiviual myofibrils that are horizontally aligned.
Fig. 3 Typical sarcomeric TPEF and SHG signal of gastrocnemius muscles. Single-band (a,b) and double-band (c,d) sarcomeres from xenopus (a,c) and rat (b,d). The upper and lower panels of (a–d) correspond respectively to α-actinin TPEF and myosin SHG signals. Scale bar (2 μm).

Fig. 4 SHG images illustrating the effect of 1 day proteolysis on sarcomeric SHG patterns of xenopus tail muscles. (a) Control without proteolysis (J0). (b) After 24H proteolysis (J1). Each image is 13×13 μm.

Table 2. Quantification of the effect of proteolysis on sarcomeric SHG patterns in xenopus tail muscles. The values represent the mean percentage and standard deviation (± SD) of single-band (SB) sarcomeres from xenopus tail (X tail) muscles. J0 is the control without proteolysis, J1 and J2 correspond respectively to 24 H and 48H proteolysis. The values were obtained from 5 to 12 thick slices from two or three animals for each experimental condition.

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A close look of Fig. 2(a–b) indicates that two distinct double-band SHG patterns can be observed. The first one is a genuine double-band sarcomeric SHG pattern (see arrowheads in Fig. 2(a,b)) as illustrated in Fig. 3(a–d) and the second one is a pitchfork-like pattern (see arrows in Fig. 2(a,b)). The quantification method did not discriminate between these two patterns because of their same spatial frequency and it should be noted that these patterns represent the 15% value of double-band sarcomeres given in Table 1. We have discussed above the possible mechanisms leading to a double-band sarcomeric SHG pattern and one can wonder if the pitchfork-like pattern could be also generated by the same mechanisms? In order to characterize this pattern, we undertook immuno staining of α-actinin (marker of the Z-band). The corresponding TPEF signal was found to periodically alternate with SHG signal as illustrated in Fig. 5 with 50% reduction of both the distance between two adjacent Z-bands (see arrows) and of the width of the SHG signal of the corresponding A-bands (see arrowheads) revealing the presence of mini sarcomeres. We were unable with α-actinin immuno staining to mark all the Z-bands of the thick tisues due to antigene accessibility and photo-bleaching. Nethertheless each time TPEF immuno staining revealed mini sarcomeres they were found in regions of pitchfork-like SHG pattern. It is therefore reasonnable to associate pitchfork-like SHG pattern with mini sarcomeres. On can also notice that in regions of pitchfork-like pattern, we observed misalignment and disconnection of α-actinin fluorescence staining and continuity of the myosin SHG signal between adjacent myofibrils with a visible angle (≈ 30°) (see arrowheads in Fig. 5). This angle suggests that the orientation of thick filaments between mini and mature sarcomeres could be different. To probe this orientation, we undertook a polarization dependence study of the SHG signal in a region containing pitchfork-like structures (see Fig. 6(a)). Indeed polarization dependence of the SHG signal is a very sensitive probe of the orientation of harmonophores [4

4. F. Tiaho, G. Recher, and D. Rouède, “Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy,” Opt. Express 15, 12286–12295 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-19-12286. [CrossRef] [PubMed]

, 6

6. S. W. Chu, S. Y. Chen, G. W. Chern, T. H. Tsai, Y. C. Chen, B. L. Lin, and C. K. Sun, “Studies of χ(2)/χ(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy,” Biophys. J. 86, 3914–3922 (2004). [CrossRef] [PubMed]

, 34

34. I. Freund, M. Deutsch, and A. Sprecher, “Connective tissue polarity. Optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon,” Biophys. J. 50, 693–712 (1986). [CrossRef] [PubMed]

] and would detect any change in the orientation of myosin molecules. As expected, the SHG intensity varies (see Fig. 6(b)) with the orientation of the incident IR electric field identically inside and outside the pitchfork-like structure suggesting identical orientation of A-band thick filaments between adjacent myofibrils. The SHG intensity in three selected ROIs ▴, • and ▪ localized respectively in the lower, upper and median arms of the pitchfork-like structure of Fig. 6(a) was plotted in Fig. 6(c) as a function of the polarization angle of the incident IR electric field. The result shows that the variation of the SHG intensity is identical for all ROI’s. We have adjusted the experimental data with the following theoretical model [4

4. F. Tiaho, G. Recher, and D. Rouède, “Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy,” Opt. Express 15, 12286–12295 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-19-12286. [CrossRef] [PubMed]

] describing the evolution of the SHG intensity I2ω for polar filaments
I2ω[sin22α+(sin2α+χ33χ15cos2α)2].
(1)
In this equation, α is the angle between the incident IR electric field and the main axis of the myofibrils which corresponds to the X axis of the laboratory coordinates (X, Y, Z) (see the inset of Fig. 6(c)). This alignement was performed outside the regions of pitchfork-like pattern. In the above equation χ33, χ31=χ15 are the two independent macroscopic nonlinear susceptibility coefficients assuming a dominant axial hyperpolarizability coefficient and a cylindrical symmetry of myosin molecules along the main axis X of the myofibrils. We have used a nonlinear least-squares fit with the Levenberg-Marquardt method [35

35. W. H. Press, B. P. Flannery, S. A. Teukolsky, and W. T. Veterlin, Numerical Recipe (Cambridge University Press, Cambridge, 1986).

] in order to adjust the fitting parameter χ33/χ15 with the experimental data of Fig. 6(c). We found that the fitting parameter χ33/χ15=0.56 was identical for all ROIs and in good agreement from the values found for myosin [4

4. F. Tiaho, G. Recher, and D. Rouède, “Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy,” Opt. Express 15, 12286–12295 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-19-12286. [CrossRef] [PubMed]

]. From this result, we conclude that the orientation of the A-band thick filaments is identical in all branches of the pitchfork which is in good agrement with the presence of mini sarcomeres revealed by immuno staining.

Fig. 5 TPEF α-actinin analysis of regions with pitchfork-like SHG pattern of gastrocnemius muscles and diagram of myofibrils with mature and mini sarcomeres. Upper (TPEF) and lower (SHG) panels are respectively from xenopus (a) and rat (b). Note the progressive alignment of the Z-band from left to right in (a) suggesting maturation of the myofibril. Scale bar (2μm).
Fig. 6 Polarization dependence study of the SHG signal in regions with pitchfork-like structures. (a) Typical SHG pitchfork-like pattern. (b) SHG optical sections illustrating the effect of five different incident polarization angles α (0°, 50°, 90°, 130°, 180°) on the emitted signal from the same field of view. The arrows indicate the polarization of the incident electric field for each panel (the upperward vertical arrow represents 0°). (c) Normalized SHG signal as a function of the incident polarization angle α for ROIs ▪, ▴ and • indicated in (a). Note the correspondance between the ROIs in (a) and legend symbols in (c). The long axis of myosin filaments was oriented along the X axis of the laboratory coordinates (X, Y, Z) as shown in the inset.

What could be the physiological revelance of these mini sarcomeres? The mechanism of sarcomeric proteins assembly into myofibrils is currently a matter of debate but one of the three proposed models developed by Sanger et al. [26

26. J. W. Sanger, S. M. Kang, C. C. Siebrands, N. Freeman, A. P. Du, J. S. Wang, A. L. Stout, and J. M. Sanger, “How to build a myofibril,” J. Muscle Res. Cell Motil. 26, 343–354 (2005). [CrossRef]

] suggests that the size of sarcomeres changes during myofibrillogenesis and could proceed as a transition through three categories of fibrils: premyofibrils, nascent myofibrils and mature myofibrils [36

36. D. Rhee, J. M. Sanger, and J. W. Sanger, “The premyofibril: evidence for its role in myofibrillogenesis,” Cell Motil. Cytoskeleton 28, 1–24 (1994). [CrossRef] [PubMed]

, 37

37. J. W. Sanger, J. Wang, B. Holloway, A. Du, and J. M. Sanger, “Myofibrillogenesis in skeletal muscle cells in zebrafish,” Cell Motil Cytoskeleton , Epub ahead of print (2009). [CrossRef] [PubMed]

]. These authors suggest that premyofibrils and nascent myofibrils are mini sarcomeres. Based on the preferential localization of mini-sarcomeres in regions of pitchfork-like SHG pattern, we propose that these latter could correspond to regions of myofibrillogenesis.

4. Conclusion

In this report we have shown that the main feature of the sarcomeric SHG signal is a single peak per sarcomere (about 85% of all sarcomeres). α-actinin immuno detection of the Z-band has revealed that the remaining (15%) was either double peak per mature sarcomere or mini sarcomeres (half of a sarcomere). Mini sarcomeres are found in region of pitchfork-like SHG pattern suggesting that this pattern could be a signature of myofibrillogenesis. We show that double peak sarcomeric SHG pattern found in normal physiological conditions is enhanced during proteolysis suggesting that this pattern could be a signature of muscle degradation.

5. Appendix 1

Gabor filtering method

In order to detect single- and double- band sarcomeres, a local spatial frequency analysis is used and manually applied to a stack. It is based on two band-pass Gabor filtering [27

27. S. G. Mallat, A wavelet tour of signal processing (Academic Press, 2nd Edition, London, 1999).

] which enhance the spectral components associated to single-band (frequency f1) and double-band sarcomeres (frequency f2). A post-filtering decision stage enables to automatically classify each pixel in one of three classes: background, single-band sarcomere and double-band sarcomere. The overall process is detailed in the following considering as an example the SHG image of Fig. 7(a).

Fig. 7 Single-band and double-band frequency estimation by FFT analysis. (a) Before processing FFT’s the SHG image (512×512), corresponding to that of Fig. 2(a) after a Gaussian Blur filter (radius=1), is manually rotated in order to orientate horizontally the main direction of myofibrils. The full image is 40×40 μm. (b) SHG intensity profile Ii(x) for lines i=208 and i=248. (c) The modulus of the FFT, |Ii(x)^| is given for lines i=208 and i=248. Line i = 208 contains single- and double- band sarcomeres while line i = 448 contains only single-band sarcomeres. (d) In order to highlight main frequencies components, |Ii(x)^| are then averaged on all lines i providing |I(x)^|¯. Estimation of the single band frequency f1 is done by the selection on |I(x)^|¯ of the peak corresponding to the fundamental frequency (bold circle on (d)).

Single- and double- band frequencies estimation

The intensity profile Ii(x) is performed for each line i and the result is presented in Fig. 7(b) for lines i=208 and i=448. The single-band frequency f1 is then estimated based on the Fast Fourier Transform (FFT) [38

38. A. Oppenheim and R.W. Schafer, Digital Signal Processing (Prentice-Hall, Englewood Cliffs, 1975).

] of each SHG intensity profile (See Fig. 7(c)) and is deduced from the average of all FFT’s of all lines (See Fig. 7(d)). The double band frequency f2 is defined as f2 = 2 f1. For example, for the SHG image of Fig. 7(a), we found f1 = 0.55 μm−1. Note that axis x is defined as the direction of the main filament axis.

Gabor filters description

Two band-pass Gabor filters gf1 (x) and gf2 (x) [27

27. S. G. Mallat, A wavelet tour of signal processing (Academic Press, 2nd Edition, London, 1999).

], centered on frequencies f1 and f2 are used (Fig. 8(a,b)) to detect significant energies around each frequency at a current position on line i of the SHG image. The filter is defined by a complex impulse response function gf (x) = gr(x, f) + igi(x, f) = W(x)(cos(2πfx) + isin(2πfx)) equal to a complex harmonic signal at spatial frequency f (f = f1 or f = f2) multiplied by a Gaussian-shaped envelope W(x)=1σ2πexp(x2σ2). The dispersion parameter σ determines the Gabor filter spatial resolution around the origin and its inverse σ−1 determines frequency dispersion around central frequency f. The SHG image is filtered line by line by gf1 (x) and gf2 (x). The responses of these two filters to the intensity profile Ii(x) of each line i provide local information on spectral components energies around f1 and f2 and hence on the possible presence of single- or double- band sarcomeres (See Fig. 8(c,d)). Therefore, filtering each line of the SHG image by the Gabor filters result in two filtered images (not presented) If1 and If2 highlighting single- and double- band location in this image. The gray value level of each pixel (i, j) of these filtered images are defined by If (i, j) = |Ii * gf (j)| for f = f1 and f = f2. In order to enhance signal to noise ratio If1 and If2 are smoothed with a 2D spatial rectangular mask 24×7 pixels leading to two smoothed images ISf1 and ISf2 presented in Fig. 8(e,f).

Fig. 8 Gabor filter analysis. Impulse responses of Gabor filters associated to (a) single band frequency gf1 (x) = gr(x, f1) + igi(x, f1) and (b) double band frequency gf2 (x) = gr(x, f2) + igi(x, f2). A high value of σ provides a precise spectral analysis but a poor spatial resolution. A good compromise is a value of σ of 1f124pixels. Response of the two Gabor filters gf1 (x) and gf2 (x) for intensity profiles Ii(x) of lines i=208 (c) and i=448 (d) of Fig. 7(b). Operator * represents the convolution of the intensity profile with the impulse response of the Gabor filter. Single- and double- band frequency filtering f1 and f2 are respectively in full and dash line. Line i=208 contains single- and double- band sarcomeres while line i = 448 contains only single band sarcomeres. (e) and (f) represent the result of the smoothing after Gabor filtering at frequencies f1 and f2 applied to the SHG image of Fig. 7(a). (g) and (h) represent the result of the decision step for respectively single-band and double-band sarcomeres. Lines i=208 and i=448 considered in (c,d) are drawn in white in (g,h). In order to limit side effects caused by Gabor filtering, two vertical bands of width 24 pixels ( 1f124pixels) are not considered for decision in (g) and (h).

Decision

The goal of the decision stage is to decide for each pixel (i, j) (depending on the two values ISf1(i,j) and ISf2(i,j)) if it belongs to a single-band sarcomere, a double-band sarcomere or to background. The following decision rule is used:
  • if ISf1(i,j)+ISf2(i,j)<T1: no sarcomere (the responses to the two Gabor filters are low);
  • if ISf1(i,j)+ISf2(i,j)T1 and ISf1(i,j)/ISf2(i,j)T2: single-band sarcomere;
  • otherwise: double-band sarcomere.
Thresholds T1 and T2 are typically 10 and 2. The value of T1 is determined empirically based on the rejection of the background. T2 > 1 enables to consider the fact that profiles corresponding to double-band sarcomeres (frequency f2) contain also patterns with single-band sarcomere (frequency f1). The percentage of single-band sarcomeres of Table 1 is given by the ratio of the number of pixels belonging to single-band sarcomeres over the number of pixels belonging to single- or double- band sarcomeres. From the decision step two masks are made in order to separate pattern with single- or double- band sarcomeres in the SHG image. The result of the two masks on Fig. 7(a) is presented in Fig. 8(g,h)

6. Appendix 2

Intensity profile analysis

An algorithm was built, and a corresponding ImageJ macro was designed, to automatically compute in a stack the percentage of myofibrils with one peak per sarcomere. Care was taken to orientate horizontally the main direction of the myofibrils parallel to the X axis (See Fig. 9). Firstly a gaussian filter (radius=4) was applied to each slice k of the stack. The number of peaks Ni,k of the pixel intensity profile is computed for line i of slice k. Adjacent lines i were separated by a distance of 1 to 2 μm in order to analyze only different myofibrils (the typical myofibril diameter is about 1 μm). Identification of a peak is based on the detection of points corresponding to a transition between a positive and a negative first discrete derivative. The minimum value mink Ni,k is determined for line i from all slices k of the entire stack. The average minkNi,k¯ is computed from all values of mink Ni,k of all lines i and considered as the reference number for myofibrils with one peak per sarcomere. A decision threshold (t) is taken in order to classify each myofibril as myofibril with one peak or two peaks per sarcomere using the following rule: myofibril of line i of slice k corresponds to myofibril with one peak per sarcomere if Ni,k(1+t)×minkNi,k¯ (typically t = 0.2). The percentage of myofibrils with one peak per sarcomere which is reported in Table 1 is estimated for the whole stack as follow
card({(i,k):Ni,k(1,t)×minkNi,k¯})card({(i,k)})×100,
where card(E) denotes number of elements in E.

Fig. 9 Description of the method of intensity profile analysis. 3D schematic view of an entire stack where the first image of the stack corresponds to that of Fig. 2(a). Each image is oriented wih the myofibril main axis oriented along the X axis of the laboratory. The number of peaks Ni,k of the pixel intensity profile is determined for line i of slice k. The minimum value mink Ni,k is determined for line i from all slices k and the average minkNi,k¯ is deduced from all lines i.

Acknowledgments

We thanks Région Bretagne, Rennes Métropole, Conseil Général d′Ille-et-Vilaine Ministére de l’enseignement supérieur et de la recherche for their financial support, Fabrice Senger, Hélène Mereau for their technical help and Emmanuel Schaub for helpful discussions.

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OCIS Codes
(170.0170) Medical optics and biotechnology : Medical optics and biotechnology
(180.0180) Microscopy : Microscopy
(190.4160) Nonlinear optics : Multiharmonic generation

ToC Category:
Medical Optics and Biotechnology

History
Original Manuscript: July 23, 2009
Revised Manuscript: September 25, 2009
Manuscript Accepted: October 7, 2009
Published: October 16, 2009

Virtual Issues
Vol. 4, Iss. 12 Virtual Journal for Biomedical Optics

Citation
Gaëlle Recher, Denis Rouède, Patrick Richard, Antoine Simon, Jean-Jacques Bellanger, and François Tiaho, "Three distinct sarcomeric patterns of skeletal muscle revealed by SHG and TPEF Microscopy," Opt. Express 17, 19763-19777 (2009)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-17-22-19763


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References

  1. P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, "Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues," Biophys. J 82, 493-508 (2002). [CrossRef]
  2. P. J. Campagnola and L. M. Loew, "Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms," Nature Biotechnology 21, 1356-1360 (2003). [CrossRef] [PubMed]
  3. W. Mohler, A. C. Millard, and P. J. Campagnola, "Second harmonic generation imaging of endogenous structural proteins," Methods 29, 97-109 (2003). [CrossRef] [PubMed]
  4. F. Tiaho, G. Recher, and D. Rouède, "Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy," Opt. Express 15, 12286-12295 (2007), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-15-19-12286. [CrossRef] [PubMed]
  5. G. Cox, N. Moreno, J. Feijó, "Second-harmonic imaging of plant polysaccharides," J. Biomed. Opt. 10, 024013 (2005). [CrossRef] [PubMed]
  6. S. W. Chu, S. Y. Chen, G. W. Chern, T. H. Tsai, Y. C. Chen, B. L. Lin and C. K. Sun, "Studies of x(2)/x(3) tensors in submicron-scaled bio-tissues by polarization harmonics optical microscopy," Biophys. J. 86, 3914- 3922 (2004). [CrossRef] [PubMed]
  7. Y. Fu, H. Wang, R. Shi, and J. X. Cheng, "Second harmonic and sum frequency generation imaging of fibrous astroglial filaments in ex vivo spinal tissues," Biophys J. 92, 3251-3259 (2007). [CrossRef] [PubMed]
  8. S. Psilodimitrakopoulos, S. Santos, I. Amat-Roldan, A. K. N. Thayil, D. Artigas, and P. Loza-Alvarez, "In vivo, pixel-resolution mapping of thick filaments’ orientation in nonfibrilar muscle using polarization-sensitive second harmonic generation microscopy," J. Biomed. Opt. 14, 014001 (2009). [CrossRef] [PubMed]
  9. D. E. Rudy, T. A. Yatskievych, P. B. Antin and C. C. Gregorio, "Assembly of thick, thin, and titin filaments in chick precardiac explants," Dev. Dyn. 221, 61-71 (2001). [CrossRef] [PubMed]
  10. C. Odin, T. Guilbert, A. Alkilani, O. P. Boryskina, V. Fleury, and Y. Le Grand, "Collagen and myosin characterization by orientation field second harmonic microscopy," Opt. Express 16, 16151-16165 (2008), http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-16-20-16151. [CrossRef] [PubMed]
  11. J. C. Sparrow, and F. Schock, "The initial steps of myofibril assembly: integrins pave the way," Nat. Rev. Mol. Cell Biol. 10, 293-298 (2009). [CrossRef] [PubMed]
  12. R. Craig and J. L. Woodhead, "Structure and function of myosin filaments," Curr. Opin. Struct. Biol. 16, 204-212 (2006). [CrossRef] [PubMed]
  13. R. Craig, and R. Padron, Molecular structure of the sarcomere (McGraw-Hill, New York, 2004).
  14. S. G. Page, and H. E. Huxley, "Filament Lengths in Striated Muscle," J. Cell Biol. 19, 369-390 (1963). [CrossRef] [PubMed]
  15. F. Vanzi, M. Capitanio, L. Sacconi, C. Stringari, R. Cicchi, M. Canepari, M. Maffei, N. Piroddi, C. Poggesi, V. Nucciotti, M. Linari, G. Piazzesi, C. Tesi, R. Antolini, V. Lombardi, R. Bottinelli, and F. S. Pavone, "New techniques in linear and non-linear laser optics in muscle research," J. Muscle Res. Cell Motil. 27, 469-479 (2006). [CrossRef] [PubMed]
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