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Optics Express

Optics Express

  • Editor: Michael Duncan
  • Vol. 14, Iss. 17 — Aug. 21, 2006
  • pp: 7810–7820
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Multimode nonlinear optical imaging of the dermis in ex vivo human skin based on the combination of multichannel mode and Lambda mode

Shuangmu Zhuo, Jianxin Chen, Tianshu Luo, Dingsong Zou, and Jingjun Zhao  »View Author Affiliations


Optics Express, Vol. 14, Issue 17, pp. 7810-7820 (2006)
http://dx.doi.org/10.1364/OE.14.007810


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Abstract

A Multimode nonlinear optical imaging technique based on the combination of multichannel mode and Lambda mode is developed to investigate human dermis. Our findings show that this technique not only improves the image contrast of the structural proteins of extracellular matrix (ECM) but also provides an image-guided spectral analysis method to identify both cellular and ECM intrinsic components including collagen, elastin, NAD(P)H and flavin. By the combined use of multichannel mode and Lambda mode in tandem, the obtained in-depth two photon-excited fluorescence (TPEF) and second-harmonic generation (SHG) imaging and TPEF/SHG signals depth-dependence decay can offer a sensitive tool for obtaining quantitative tissue structural and biochemical information. These results suggest that the technique has the potential to provide more accurate information for determining tissue physiological and pathological states.

© 2006 Optical Society of America

1. Introduction

An emerging application of multiphoton microscopy (MPM) is the observation of unstained samples based on intrinsic sources of nonlinear signals [1

1. W.R. Zipfel, R.M. Williams, and W.W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21, 1369–1377 (2003). [CrossRef] [PubMed]

, 2

2. W.R. Zipfel, R.M. Williams, R. Christie, A.Y. Nikitin, B.T. Hyman, and W.W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. USA. 100, 7075–7080 (2003). [CrossRef] [PubMed]

] such as two photon-excited fluorescence (TPEF) and second-harmonic generation (SHG). It can provide high contrast and optical sectioning capabilities for involving nonlinear light-matter interactions. The ability of MPM to produce images deep in optically thick preparations is crucial for intravital tissue microscopy. Moreover, SHG enables direct imaging of anisotropic biological structures, such as collagen [3–5

3. P.J Campagnola and L.M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol. 21, 1356–1360 (2003). [CrossRef] [PubMed]

]. SHG can produce high-resolution, high contrast images of tissue morphology [2

2. W.R. Zipfel, R.M. Williams, R. Christie, A.Y. Nikitin, B.T. Hyman, and W.W. Webb, “Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation,” Proc. Natl. Acad. Sci. USA. 100, 7075–7080 (2003). [CrossRef] [PubMed]

, 6

6. A. Zoumi, A. Yeh, and B.J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. USA. 99, 11014–11019 (2002). [CrossRef] [PubMed]

] and has also be applied to study dynamics in tissue physiology, such as monitoring collagen modification in tumor growth [7

7. E. Brown, T McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R.K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9, 796–800 (2003). [CrossRef] [PubMed]

], and optically recording action potential change in neuron cells [8

8. D.A. Dombeck, M. Blanchard-Desce, and W.W. Webb, “Optical recording of action potentials with second-harmonic generation microscopy,” J. Neurosci. 24, 999–1003 (2004). [CrossRef] [PubMed]

]. Recently, because these imaging (TPEF and SHG) modalities involve different contrast mechanisms and can provide in tandem complementary information about tissue structure and function, the combination of TPEF and SHG has attracted more attention. These imaging modalities are easy to implement simultaneously and differ only in optical filter selection and detector placement. The combination of TPEF and SHG has been implemented for the study of cells [9

9. P.J. Campagnola, M.D Wei, A. Lewis, and L.M. Loew, “High-resolution nonlinear optical imaging of live cells by second harmonic generation,” Biophys. J. 77, 3341–3349 (1999). [CrossRef] [PubMed]

, 10

10. P.J. Campagnola, H.A. Clark, W.A. Mohler, A. Lewis, and L.M. Loew, “Second-harmonic imaging microscopy of living cells,” J. Biomed. Opt. 6, 227–286 (2001). [CrossRef]

], thin tissue sections [11

11. P.J. Campagnola, A.C. Millard, M. Terasaki, P.E. Hoppe, C.J. Malone, and W.A. Mohle, “Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 81, 493–508 (2002). [CrossRef]

], and for the more practical case of thick, unstained living specimens [6

6. A. Zoumi, A. Yeh, and B.J. Tromberg, “Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence,” Proc. Natl. Acad. Sci. USA. 99, 11014–11019 (2002). [CrossRef] [PubMed]

, 12

12. Y.C. Guo, H.E. Savage, F. Liu, S.P. Schantz, P.P. Ho, and R.R. Alfano, “Subsurface tumor progression investigated by noninvasive optical second harmonic tomography,” Proc. Natl. Acad. Sci. USA. 96, 10854–10856 (1999). [CrossRef] [PubMed]

].

In this study, we demonstrate for the first time the multimode nonlinear imaging technique to carry out the selective visualization of ECM and the identification of dermis intrinsic components by combining multichannel mode with Lambda mode. The technique is illustrated by imaging human dermis using a mode-locked femtosecond Ti: sapphire laser coupled to a Zeiss LSM 510 META laser scanning microscopy. We also show that the technique can provide some quantitative tissue structural and biochemical information. Furthermore, we qualitatively interpret the origin of the backward SHG signals from tissue.

2. Materials and methods

All the experiments described in this paper were based on ex-vivo human skin. Human skin was obtained from Affiliated Xiehe Hospital of Fujian Medical University. The specimens were stored at a bottle of liquid nitrogen (-196°C) before they were used. The skin samples were excided perpendicular to the epidermal layer so that each section comprised a complete transverse cross-section of the epidermal and dermal layers and sandwiched between the microscope slide and a piece of the cover glass. To avoid dehydration or shrinkage during the whole imaging process, the specimen was sprinkled with PBS solution (PH 7.4). The dermal part of ex-vivo human skin is our region of interest.

In this study, the multimode nonlinear optical imaging based on the combination of multichannel mode and Lambda mode was performed on a Zeiss LSM 510 META laser scanning microscopy equipped with a mode-locked femtosecond Ti: sapphire laser (110fs, 76MHz), tunable from 700nm to 980nm (Coherent Mira 900-F), as shown in Fig. 1.

Fig. 1. Schematic of multimode nonlinear optical imaging experimental setup.

The polarization direction of the laser light is the horizontal polarization. An Acousto-Optic Modulator (AOM) is used to control the laser intensity attenuation. A Plan-Apochromat 63× (N.A.=1.4) oil immersion objective (Zeiss) was employed for focusing the excitation beam and for collecting of the backward signals. We detected all signals in the backward direction. The signals were directed by a main dichroic beam splitter (MDBS) to the META detector. The META detector with 32-gated photon counting module is used to collect x-y images at a series of emission wavelengths, covering from 377nm to 716nm. Each photon counting module can cover a spectral range of 10.7nm. An IR beam block filters ( Zeiss KP650), which is in front of META detector, was be used to ensure that excitation light was filtered out and only emission signals were recorded. The LSM 510 META laser scanning microscopy is controlled via a standard high-end Pentium PC and linked to the electronic control system via an ultrafast SCSi interface. This system has two especial imaging modes: multichannel mode and Lambda mode. These two modes use the same detector that is META detector to acquire the imaging. The multichannel mode has eight independent-channels and each channel can selectively be set to detect emission signals within the random range from 377 to 716nm to achieve imaging. The Lambda mode permits to simultaneously record the spectral resolved image and the corresponding spectra by creating emission lambda stacks. In this study, we have combined multichannel mode with Lambda mode to carry out multimode nonlinear imaging. In multichannel mode, we selected two independent-channels from eight channels to achieve SHG (387–409nm) and TPEF (447–628nm) imaging of human dermis, respectively. The SHG/TPEF images were subsequently recorded and generally attained within a few seconds. In our experiments, if the two channels (SHG and TPEF) were recorded simultaneously, which they bear the same system parameters, we found that the integrated total intensity of SHG is about 1/5 of that of TPEF (as indicated in the Fig. 5 b). Thus, when SHG imaging is on optimal, it is more likely that the much higher relative TPEF intensity obscure the SHG signal; when TPEF imaging is on optimal, the SHG signal will become much weaker. The acquired SHG/TPEF image bears low contrast and signal-to-background. So, in this study we used two independent channels and they can acquire images with the optimal system parameters, including the laser intensity, the detection gain, amplifier offset, and amplifier gain, which can enhance the image contrast. This is an advantage of the multichannel mode. At the same time, in Lambda mode we acquire the spectral resolved TPEF/SHG image and the corresponding spectra in the same region as using the multichannel mode. Using our setting, about 1s of exposure were necessary for the alternation of two modes. The acquisition of a single 512×512 pixels image was generally achieved within a few seconds. But, note that all the images presented in this article are not 512×512 pixels in size because of taking into account the size of images and discussed contents. The average laser power at the specimen was maintained at <6mW and no photobleaching was observed at this low power level.

3. Results and discussion

3.1. Multimode nonlinear optical imaging of human skin

Fig. 2. TPEF/SHG nonlinear imaging of human dermis using the multichannel mode. The images were acquired by using two independent-channels: (a) Typical SHG image (green color-coded, 387 – 409nm) for excitation power PSHG = 6.0mW. The signal mainly arises from collagen and (b) TPEF image (red color-coded, 447 – 628nm) for PTPEF = 2.6mW. Elastin, NAD(P)H and flavin are responsible for the signals. (c) TPEF/SHG image was obtained by overlaying two channels, in which the contrast has been enhanced to outline the various structures seen in the image. The excitation wavelength λex was 800nm. The images are 135×315 pixels in size 12-bit pixel depth.

Table 1. System parameters of two independent-channels in the multihannel mode

table-icon
View This Table

Multichannel mode has also capability to generate high-contrast structural images deep inside highly scattering tissues. It was found that detailed, high-contrast features could be resolved in TPEF/SHG optical sections throughout the full ~ 60μm thickness of a freshly dissected, unfixed sample of human dermis (Fig. 3 a). Figures 3 b–h shows TPEF/SHG images from human dermis at depths of 0, 10, 20, 30, 40, 50, 60 μm, for λex =800nm. As represented in Fig. 3 b–h, the distribution and content of ECM components are visible. Optical sections at various depths can better reveal fine details of the internal tissue structure. The signal progressively degrades with increasing depth, but the TPEF/SHG images still bear high-contrast. However, the maximum imaging depth reached here is only limited by the working distance of microscopy objective and the low value of excitation power used (PSHG = 6.0mw, PTPEF = 2.6mw). Recently, some groups have reported the maximum depth of two-photon imaging can reach up to one millimeter [29

29. P. Theer, M.T. Hasan, and W. Denk, “Two-photon imaging to a depth of 1000 microns in living brains by use of a Ti:Al203 regenerative amplifier,” Opt. Lett. 28, 1022–1024 (2003). [CrossRef] [PubMed]

], while leaving the tissue intact. Further improving our system may permit deeper imaging.

Fig. 3. TPEF/SHG images obtained from human dermis at various depths using the multichannel mode. A dissected sample of human dermis was optically sectioned through its full thickness of ~60μm. (a) Graphic representation of the image volume of tissue. xy (top), yz (left), and xz (right) sections from the boundaries of the volume are shown. Note that dimensions are indicated, and are not portrayed proportionally. (b–h) TPEF/SHG images of human dermis at depths of 0, 10, 20, 30, 40, 50 and 60 μm. The images are 265×320 pixels in size 12-bit pixel depth. Scale bar = 20μm. (λex = 800nm, PSHG = 6.0mW, PTPEF = 2.6mW).

Fig. 4. The spectral resolved TPEF/SHG image of human dermis from the same region in Fig. 2 and the corresponding spectra from regions of interest (ROI). They simultaneously were obtained by using the Lambda mode. The image is 135×315 pixels in size 12-bit pixel depth. (λex = 800nm, P = 5mW)

In the Lambda-mode, we could also investigate the dependence of the SHG and TPEF signals intensity on imaging depth in turbid tissues. The spectral resolved TPEF/SHG images from human dermis at various depths were acquired. The emission spectra were obtained by taking the image of the whole optical section plane and averaging the intensity from each pixel within the image. To suppress the 470 nm autofluorescence peak of NAD(P)H, 830 nm excitation wavelength was used instead of 800 nm. Figure 5 (a) shows the emission spectra from human dermis at depths 0, 20, 40, 60 μm, while Fig. 5 (b) gives integrated total intensity of SHG and TPEF spectra versus penetration depth, z, for λex = 830nm. To ensure the reproducibility of the results, the experiment was repeated three times. The SHG and TPEF signals were observed to decay exponentially with depth. Multiple light scattering decreases the number of excitation photons reaching the focus plane. Multiple light scattering also decreases the number of SHG and TPEF photons collected by the objective lens. Thus, the SHG and TPEF signals decay with depth. The detected signal intensity, I, decays as a function of depth, z, according to:

(I)z=(I)0exp(Az)

where A is an attenuation coefficient that is a function of the sample absorption and scattering properties at the both the excitation and emission wavelengths. The inverse of A yields an attenuation length, latt . Figure 5 (c) and (d) show the plot of the natural logarithm of SHG and TPEF integrated total intensity versus depth, z, with latt(SHG) and latt(TPEF) ≈ 25.6μm. The SHG (purple light) wavelength is shorter than that of TPEF (blue light), the sample absorption and scattering properties of SHG is bigger [30

30. H.J. van Staveren, C.J.M. Moes, J. van Marie, S.A. Prahl, and M.J.C. van Gemert, “Light scattering in intralipid-10% in the wavelength range of 400–1100 nm,” Appl .Opt. 30, 4057–4514 (1991).

, 31

31. A.K. Dunn, V.P. Wallace, M. Coleno, M.W. Berns, and B.J. Tromberg, “Influence of optical properties on two-photon fluorescence imaging in turbid samples,” Appl. Opt. 39, 1194–1201 (2000). [CrossRef]

]. So, the attenuation of SHG is more significant than TPEF, which is consistent with the above results. In addition, it is well known that refractive index mismatch induced spherical aberration is a ubiquitous problem for in-depth optical biological imaging. However, recently Dong’s group has used it to determine the refractive indices of uniformly luminescent specimens by measuring the signals decay profile as a function of imaging depth [32

32. P.T. Fwua, W. Loa, and C.Y. Dong, “Refractive index determination from multiphoton and confocal spherical aberration microscopy,” in Multiphoton Microscopy in the Biomedical Sciences V, A. Periasamy and P.T.C. So, eds., Proc. SPIE. 5700, 70–77 (2005). [CrossRef]

]. In other words, the signals decay profile as a function of imaging depth can reflect changes of the refractive indices of specimens. Here, the SHG and TPEF signals decay profile as a function of imaging depth also can use to monitor changes of the refractive indices of biological tissue. These finding suggest that the SHG and TPEF depth-dependent decay can provide a general estimate of optical properties, which are critical parameter for deep-tissue imaging and reflect various physiologic processes [33

33. T. Collier, D. Arifler, A. Malpica, M. Follen, and R. Richards-Kortum, “Determination of epithelial tissue scattering coefficient using confocal microscopy,” IEEE J.Sele.Top. Qua. Elec. 9, 307–313 (2003). [CrossRef]

]. The SHG and TPEF depth-dependent decay may have the potential to be as sensitive tools for obtaining quantitative tissue information.

Fig. 5. Emission spectra obtained from human dermis at various depths using the Lambda mode. Each spectrum is the average of measurement acquired at the whole optical section plane. (a)SHG and TPEF spectra obtained at depths of 0, 20, 40, 60μm. (b) Integrated total intensity of SHG and TPEF spectra versus penetration depth. (c, d)Plot of the natural logarithm of SHG and TPEF integrated total intensity versus penetration depth, Z, respectively. Error bars represent calculated standard deviations. (λex=830nm, P = 5mW).

To sum up, the multimode nonlinear optical imaging technique can allow direct visualization of ECM components and the identification of both cellular and ECM intrinsic components. The combined use of multichannel mode and Lambda mode in tandem can obtain quantitative information regarding the biomorphology and biochemistry of tissue.

3.2. Origin of the backward SHG signal

4. Conclusion

We have developed a multimode nonlinear optical imaging technique based on the combination of multichannel mode and Lambda mode and applied it to image human skin. The experimental results demonstrates that the technique can permit high-contrast visualization of ECM intrinsic components, and identify both cellular and ECM intrinsic components including collagen, elastin, NAD(P)H and flavin. Our findings indicate that in-depth TPEF/SHG imaging and TPEF/SHG signals depth-dependence decay provide a means for extracting quantitative structural and biochemical information from tissue by the combined use of multichannel mode and Lambda mode in tandem.

Until now, few other techniques can provide a direct visualization of ECM intrinsic components, and identify both cellular and ECM intrinsic components. The technique reported here opens many possibilities of studying physiological activity of intact tissues. The technique provides a new tool to map the distribution of ECM components and monitor cellular redox state in intact tissue, providing quantitative information regarding biomorphology and biochemistry of tissue, and may prove useful to clinical diagnostics as well as to basic biological research.

Acknowledgments

The project was supported by the National Natural Science Foundation of China (No. 60508017), Program for New Century Excellent Talents in Fujian Province University, the Natural Science Foundation of Fujian Province of China (No. A0510015), the Innovation Foundation for Young Technological Talents of Fujian Province (NO.2003J027), and the Scientific Program of the Educational Hall of Fujian Province (NO.JA05215).

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30.

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A.K. Dunn, V.P. Wallace, M. Coleno, M.W. Berns, and B.J. Tromberg, “Influence of optical properties on two-photon fluorescence imaging in turbid samples,” Appl. Opt. 39, 1194–1201 (2000). [CrossRef]

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OCIS Codes
(170.6510) Medical optics and biotechnology : Spectroscopy, tissue diagnostics
(170.6900) Medical optics and biotechnology : Three-dimensional microscopy
(180.5810) Microscopy : Scanning microscopy
(190.4160) Nonlinear optics : Multiharmonic generation
(190.4180) Nonlinear optics : Multiphoton processes

ToC Category:
Medical Optics and Biotechnology

History
Original Manuscript: May 26, 2006
Revised Manuscript: July 19, 2006
Manuscript Accepted: July 27, 2006
Published: August 21, 2006

Virtual Issues
Vol. 1, Iss. 9 Virtual Journal for Biomedical Optics

Citation
Shuangmu Zhuo, Jianxin Chen, Tianshu Luo, Dingsong Zou, and Jingjun Zhao, "Multimode nonlinear optical imaging of the dermis in ex vivo human skin based on the combination of multichannel mode and Lambda mode," Opt. Express 14, 7810-7820 (2006)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-14-17-7810


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References

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