In vivo and ex vivo imaging of intra-tissue elastic fibers using third-harmonic-generation microscopy

Elastin is an essential and widespread structural protein in charge of the integrity on tissues and organs. In this study, we demonstrate that elastin is a major origin of the third-harmonic-generation (THG) contrast under Cr:forsterite laser excitation operating at 1230nm, with selective visualization inside many tissues such as lung tissues and arteries. In vivo imaging of the nude mouse elastic cartilage beneath the hypodermis by epiTHG microscopy keeps the high resolution and contrast in all three dimensions. Combined with second-harmonic-generation microscopy, THG microscopy exhibits the ability to show the extraordinary proliferation of elastic fibers for the ophthalmic disease of pterygium and the capability of distinguishable visualization from collagen. ©2007 Optical Society of America OCIS codes: (180.6900) Three-dimensional microscopy; (190.4160) Multiharmonic generation; (190.4180) Multiphoton processes; (170.3880) Medical and biological imaging References and links 1. S. 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Introduction
Elastin is an important structural protein which is arranged as fibers in the extracellular matrix (ECM) with the nature of elasticity and resilience to ensure the structural integrity on tissues and organs.Elastic fiber is widespread inside the human body including large arteries, lung, elastic cartilage, ligament, tendon, and skin where it confers the properties of stretching and elastic recoil.With aging, injury or the effect of the acquired diseases, the abnormality of elastic fibers becomes apparent morphologically or quantitatively such as degradation, excessiveness, and aberrance resulting in many diseases [1,2].Recently, third-harmonicgeneration (THG) microscopy has been emerged as an important imaging modality in biological researches with the advantages including intrinsic optical sectioning capability due to the nature of third-order nonlinearity and no energy release due to the characteristic of virtual-state-transition.In our previous in vivo studies of vertebrate embryos under Cr:forsterite laser excitation operating at 1230nm [3], complex developmental processes throughout the 1.5-mm-thick zebrafish embryo could all be continuously visualized in vivo, and no optical damage was revealed after a long-term continuous observation with 100-mW average incident power onto one embryo.In another study of in vivo hamster oral cavity with epi-THG microscopy under the same laser source [4], THG can provide high spatial resolution images of oral mucosa and sub-mucosa in all three dimensions as well as blood flow in the capillary without complex physical biopsy procedures.After 3-hours continuous observation under the epi-THG microscope in the same area, no evidence of coagulation necrosis in buccal squamous epithelium and sub-epithelial stroma appeared under the examination of conventional microscopy.Both results indicate the least-invasive property of the higher harmonic generation processes under 1230nm the Cr:forsterite laser excitation.Much ink has been spent on the application of THG microscopy for the observations of various structures in bio-tissues [5][6][7][8][9][10], embryos [3], hamster oral cavity in vivo [4] as well as the visualization of intracellular Ca 2+ dynamics [11] and the distribution of micrometer-sized lipid bodies [12].These studies imply that to explore and ensure the origins of the THG contrasts inside the bio-tissues is highly desirable.In this paper, we report that elastic fiber is one of the dominating origins of the THG contrast under the Cr:forsterite laser excitation operating at 1230nm, and this enhanced THG emission from elastic fibers can provide selective visualization inside many tissues.Our study was first explored by simultaneous imaging of the THG microscopy with the two-photon microscopy of the endogenous fluorescence from the elastic molecules in unstained human lung tissues and rat aortas.Our investigation was further conformed by simultaneous three-photon fluorescence microscopy using exogenous staining with the eosin dye, which could selectively discriminate the elastic fibers [13][14][15].Then, we demonstrate the in vivo imaging of the elastin distribution in the unstained elastic cartilage in a live nude mouse by using epi-THG microscopy combining with the endogenous 2PF microscopy simultaneously.Finally, we show that this THG-based technique can certainly reveal the unusual proliferation of elastic fibers for the ophthalmic disease of pterygium.

Nonlinear scanning microscopy
Figure 1(a) shows the setup of our nonlinear laser scanning microscope.For the purpose to assist future clinical examinations, we only collect the backward propagating signals in this study.The setup here is mainly modified from our previous system for in vivo optical biopsy of hamster oral cavity [4].The THG microscopy performed for this study is based on a homebuilt femtosecond Cr:forsterite laser centered at 1230 nm with a 110 MHz repetition rate and a 100-fs pulsewidth [16].The laser output was initially shaped and collimated by a telescope and then coupled into a modified beam scanning system (Olympus Fluoview300) connecting to an upright microscope (Olympus BX-51).An IR water-immersion objective (Olympus LUMplanFL/IR 60X/NA 0.9/Working distance 2mm) was used to focus the laser beam into the test tissue or the live nude mouse ear.For a 512×512 resolution, the maximum scanning rate of Fluoview300 is 1000 lines per second corresponding to about two frames per second.The employed objective acts as the collection lens to collect the backward propagating THG, second-harmonic-generation (SHG), and multi-photon excited fluorescence including endogenous or exogenous signals, which are separated from the laser beam with a dichroic mirror (865dcxru, Chroma technology).A color filter in the system further ensures filtering out the fundamental laser wavelength.Different fluorescence wavelengths and optical harmonics are further separated with another dichroic mirror (490DRXR, Chroma technology) and directed into two different PMTs with appropriate interference filters.The interference filters used for THG, SHG and endogenous fluorescence from the elastin are D410/30X, Z615/10X and HQ650/45X (Chroma technology), and the filter used for exogenous fluorescence from eosin is HQ575/50X (Chroma technology).The average laser power was 150 mW corresponding to 1.3nJ pulse energy illuminated on the test tissue or animal.In theory, the lateral resolution of THG microscopy is 410nm [9,17].

Tissue preparation and animals
The human lung tissues were stored in liquid nitrogen and obtained from the tissue bank of National Taiwan University Hospital.The rat aortas were excised and also frozen by liquid nitrogen.They both were cut into 120um thin sections for intrinsic imaging and followed by eosin staining for extrinsic imaging.In the in vivo study, nude mouse is used as our animal model.The experimental protocols were approved by the National Taiwan University Institutional Animal Care and Use Committee (NTU-IACUC).After anaesthetization, the nude mouse ear is fixed as shown in Fig. 1(b).Fig. 1(c) is an example to rest the nude mouse in our microscope system with a thermal blanket maintaining the body temperature of the test animal.In the experiment of pterygium, surgical specimens of pterygium and normal conjunctiva specimens from patients were obtained from the Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan.The specimens were fixed in 3.7% formaldehyde in 0.1 mol/L of phosphate-buffered-saline (PH 7.4) at 4°C for storage before imaging.Elastin is considered as one of the endogenous fluorescent sources for the two-photon fluorescence (2PF) microscopy under Ti:sapphire laser excitation operating at about 800nm.In the last few years, several articles have been devoted to study the structural and functional properties of elastic fibers in the vascular wall by using the endogenous 2PF microscopy [18][19][20].In a similar way, to make sure whether elastin still fluoresces under 1230nm excitation, we measured the nonlinear emission spectra of the purified elastin powders extracted from the human lung and aorta, either in the solid form or being dissolved in liquid.Under 1230 nm excitation, as shown in Fig. 2, all the nonlinear emission peaks of the elastin powder spectra are with the same central wavelength around 655nm.Besides, the measured fluorescence power was found to be in quadratic dependence on the excitation power, confirming its twophoton nature.In addition, the two-photon excitation action cross-section (σ TPE , defined as the product of the two-photon absorption cross-section and the fluorescence quantum yield) [21] of purified elastin in the solution was measured.The measurement of the σ TPE followed the method performed by Blab et al. previously [21].We used the reported value of σ TPE of HcRed excited with 1230nm [22] as a reference and considered the difference of transmission of optics in different wavelengths to calibrate the value of σ TPE of elastin based on the measured power of two-photon fluorescence of elastin excited with 1230nm laser beam.The value of the σ TPE is 0.125 ± 0.0044GM (1GM=10 -50 cm 4 s/photon) with an excitation wavelength of 1230nm, indicating that elastin is a low efficiency two-photon fluorescence protein.

Ex vivo THG and endogenous fluorescence imaging of elastic fibers
Figure 3 is the intra-tissue emission spectrum measured from the area of the elastic fiber in a rat aorta.From Fig. 3, the THG signal emitted from the elastic fiber is about two orders of magnitude larger than the 2PF signal with the central wavelength around 655nm, which is the same as the spectral measurement of elastin powders.This result implies that the strong THG emissions from elastin could be a promising contrast to provide selective imaging.Then, we recorded the endogenous 2PF and THG images simultaneously in the intact human lung tissues and the rat aorta samples.In the image of human lung tissue [indicated by yellow arrows in Fig. 4(a)], some THG signals (blue) excellently correlating with endogenous 2PF (magenta) are the sites of elastic fibers, revealing the alveolar structure.In the image of rat aorta, what has to be noticed is that the parallel THG signals is filled with the endogenous 2PF signals from elastic fibers [indicated by yellow arrows in Fig. 4(b)]; in other words, it should be the exact location of elastic fibers between the parallel THG signals, characterizing the lamellar structure in the artery media.These experiments highly suggest that it is the elastic fibers responsible for some of the dominating THG signals.

Ex vivo THG and exogenous fluorescence imaging of elastic fibers
To further prove the origin of THG signals from the elastin fibers, we stained the human lung tissue and the rat aorta with added probe, eosin, to exactly label the elastin.Eosin, a specific and strongly fluorescent elastin marker, has the ability to selectively visualize elastin fibers in different tissues for the fluorescence microscope, and the elastin endogenous fluorescence contributes no or only little comparatively [14].For the reason given above, we also measured the nonlinear fluorescence spectrum of eosin under 1230 nm excitation and found the emission peak value is 555nm (as shown in Fig. 5).According to the measured eosin spectrum, we recorded exogenous three-photon fluorescence and THG images [Fig.4(c) and 4(d)] at the same time within tissues.The stained images (orange) are perfectly correlated with the endogenous fluorescence images and have the identical relation to the THG images (The overlapping area shows white color).This result further verifies that one significant contrast origin of THG in the biological tissues comes from elastin.

In vivo THG and endogenous fluorescence imaging of elastic cartilage
One principal advantage of THG microscopy is its minimal invasiveness for the study of live samples with a sub-micron resolution [3,4].In our ex vivo experiments we demonstrate that one significant contrast origin of THG in the biological tissues comes from elastin.It is pertinent to consider whether this technique could be feasible for imaging the elastin in vivo.Therefore, we recorded the in vivo images in the unstained elastic cartilage of the live nude mouse ear combining THG microscopy and endogenous 2PF microscopy simultaneously.As shown in Fig. 6, deep into the skin the interconnecting sheets of elastin material can be identified by means of the overlap of the weak 2PF and the strong epi-THG images.Besides, it must be noted that the cell membrane contributes the distinct rounded images probably on account of the great refractive index difference between the chondrocyte and the lacuna, which is the space the chondrocyte occupies.Figure 7 shows an example movie of a sequential set of horizontally sectioned 2PF and epi-THG images taken from the elastic cartilage of the nude mouse ear beneath the hypodermis.Similar to the endogenous multiphoton fluorescence microscopy and SHG microscopy, THG microscopy is with contrasts contributing from several major origins and is thus with a possibility of false positive examination of elastin.As shown in Fig. 8, there are some THG signals from chondrocytes instead of elastic fibers.We attempted to solve this problem by examining the polarization of the generated THG radiation from elastic fibers under linearly polarized incident radiation excitation.With a fixed polarized incident light, we examined the polarization of the THG signals with a polarizer in front of the detection.Figure 9 shows the measured THG intensity by rotating the angle θ of the THG polarizer to that of the excitation polarization for different elastin fiber orientations.Our polarization study indicated that the generated THG polarization is in parallel to the polarization of the excitation light, independent of the fiber orientations, similar to the case of an isotropic medium.It is thus unable to differentiate the elastic fiber from other isotropic media by means of polarization management.Our study also suggests that the THG enhancement in elastin fibers could be due to the two-photon resonance [23] of the elastin molecules rather than the fibril structures [24].

Imaging elastosis of the conjunctiva by using epi-THG microscopy
With a strong THG contrast from elastin fibers, one should be able to see the variation of the THG signal intensity from elastosis.Elastosis is the abnormal proliferation of elastic fibers within tissues [25].Pterygium, a common disease associated with elastosis in ophthalmology, is characterized by the degeneration of collagens which are replaced by thickened tortuous elastotic fibers, mainly owing to the excessive sun exposure [26].The lesion growing from conjunctiva extending to the corneal surface will distort the corneal structure and even obscure the optical center of the cornea.SHG has been utilized for biological imaging application [27][28][29][30], possessing the same advantages of no energy deposition and intrinsic sectioning capability as THG for imaging within tissues.It has been established that collagen is one of the major contrast origins of SHG [30][31][32].Collagen and elastin are two major components of the ECM.The system setup of THG microscopy can be easily integrated with SHG microscopy based on the same laser system [22] to provide a possible way for the visualization of the elastin and collagen changes inside the diseased conjunctiva.To verify this possibility, we acquired the multimodal images of the normal conjunctiva and pterygium excised from patients, respectively.From previous studies [33], SHG signal mainly maps the distribution of collagen fibers within the normal conjunctiva without the existence of elastic fiber, as shown in Fig. 10(a).In contrast to the SHG signals revealing the collagen distribution, THG signals predominantly reflecting the distribution of elastin fibers shows the evidence of elastosis [as shown in Fig. 10(b)].Our study provides the clear evidence that high density elastic fibers could be imaged by THG microscopy just as expected in a known diseased conjunctiva with hyperplastic elatic fibers, and THG microscopy offers a new way to distinguish elastin and collagen fibers without staining, in combination of the SHG microscopy.This combined SHG and THG microscopy could be ideal as least invasive tool for the study of elastosis [34][35][36][37][38].

Conclusion
Our study concludes that elastin is a major origin of THG contrast in varieties of tissues such as arteries and lung tissues, under Cr:forsterite laser excitation operating at 1230nm.Met the noninvasive requirement for in vivo studies, THG microscopy is a 3-D morphological imaging tool not only providing the submicron resolution for detailed histological information due to its interface-sensitive nature but also leaving no energy deposition to examined tissues due to the virtual-state-transition characteristic.Easily integrated with other nonlinear optical microscopy such as SHG and 2PF signals with the same laser system, THG microscopy could provide a new way to distinguish elastin and collagen fibers without staining.

Fig. 1 .
Fig. 1.System setup.(a) Experimental diagram of THG and multiphoton microscopes.(b) Fixation of the nude mouse ear.(c) Acquire images of the test animal under the microscope with a thermal blanket.

Fig. 2 .
Fig. 2. Normalized emission spectra of elastin powder under 1230 nm nonlinear excitation.All emission spectra are with a central wavelength around 655nm.The emission spectra from elastin powder of aorta and lung are denoted in black and red colors respectively; while the emission spectra from elastin solution of aorta and lung are denoted in green and blue colors respectively.

Fig. 3 .
Fig.3.Intra-tissue emission spectrum measured from the area of the elastic fiber in a rat aorta.SHG signal comes from the collagen closely adjacent to the elastic fiber in the artery media.

Fig. 4 .
Fig. 4. THG and multi-photon fluorescence imaging of human lung tissue and rat aorta.(a) and (b) are simultaneous THG (blue) and endogenous 2PF (magenta) images of a human lung tissue and a rat aorta, respectively.(c) and (d) are simultaneous THG (blue) and eosin-stained exogenous fluorescence (orange) images of a human lung tissue and a rat aorta, respectively.The co-localization of blue and orange colors shows white color.All yellow arrows within four images indicate the location of elastic fibers.Scale bar: 20µm.

Fig. 6 .
Fig. 6.In vivo horizontal sections of nude mouse elastic cartilage using THG (blue) and endogenous 2PF (magenta) microscopes.(a) Endogenous 2PF image of elastic cartilage adjusted at the same contrast level with Fig. 6(c).PMT acquisition voltage: 3000V.(b)Endogenous 2PF image adjusted at a very low contrast level.PMT acquisition voltage: 3000V.(c) THG image.PMT acquisition voltage: 1200V.(d) Simultaneous THG and endogenous 2PF image.Yellow arrows indicate one of the locations of the elastic fibers.Elastic fibers in the elastic cartilage shows high contrast, high spatial resolution, and distinguishable intensity using in vivo THG microscopy.Scale bar: 20µm.

Fig. 7 .
Fig. 7. (690 KB) A movie of in vivo depth-resolved horizontal sections in elastic cartilage of the nude mouse ear.This movie is composed of 15 horizontal images.The optical depth difference between adjacent images is 2.1 μm.Image size: 80μm×80μm.

Figure 8
Figure 8 is a movie of 3-D reconstruction of a sequential set of horizontally sectioned images provided by the in vivo epi-THG microscopy.The 3-D reconstruction comprises 25 horizontal sections and has 2.1-μm interval between two adjacent images.The network of elastic fibers can be found to be arranged in a 3-D honeycomb configuration.It could be concluded that epi-THG microscopy is feasible to offer high resolution 3-D intra-tissue histological information of the unstained elastic cartilage in vivo with least invasiveness to provide an ideal new platform for studies of supporting tissues in the future.

Fig. 8 .
Fig. 8. (2.19 MB) A movie of 3-D reconstruction of a sequential set of horizontally sectioned in vivo images from the elastic cartilage of the nude mouse ear.Green arrows indicate one of the locations of the elastic fibers.This movie is composed of 25 horizontal images.The optical depth difference between adjacent images is 2.1 μm.Image size: 120μm×120μm.

Fig. 9 .
Fig. 9. Normalized THG intensity after a linear polarization as a function of the polarizer angle θ relative to the linear polarization angle of the incident excitation.The square data points represent the case that the excitation polarization is parallel (0°) to the elastic fiber.The triangular data points represent the case that the excitation polarization is 45° to the elastic fiber.