2.1.

Microscope and Imaging

The SHG imaging system consists of a laser scanning head (Olympus Fluoview 300) mounted on an upright microscope (Olympus BX61), coupled to a mode-locked Titanium Sapphire laser. All measurements were performed with a laser fundamental wavelength of $900\mathrm{nm}$ with average power of 5 to $50\mathrm{mW}$ at the specimen. The system is designed for simultaneous SHG detection of the forward and backward components. In the former, a long working distance $40\times 0.8$ N.A. water-immersion objective and a 0.9 N.A. condenser provide excitation and signal collection, respectively. The backward component is collected through the objective in a non-descanned configuration. In both geometries, the SHG signal is isolated with a longwave pass dichroic mirror and $10\text{-}\mathrm{nm}$ bandpass filters $\left(450\mathrm{nm}\right)$ . The signals are detected by a pair of identical photon-counting photomultiplier modules (Hamamatsu). The SHG wavelength $\left(450\mathrm{nm}\right)$ was confirmed with a fiber optic spectrometer (Ocean Optics). There is no detectable autofluorescence for collagen at this excitation wavelength.

Three-dimensional (3-D) SHG image stacks were quantitatively analyzed with ImageJ software (http://rsb.info.nih.gov/ij/). A coumarin dye slide emitting two-photon excited fluorescence at the SHG wavelength was used to calibrate both signal collection channels to account for uneven losses in optical paths and relative collection efficiency of the two detectors. Since the forward-to-backward fluorescence ratio from a dye slide is assumed to be one, it becomes the normalization factor for the two channels.

The input polarization is controlled by a set of half- and quarter-wave plates. We determined de facto the polarization of excitation light at the focal plane by matching SHG maxima and minima to those previously measured for linear (myofibrils) and spherical (dye-labeled spherical cells) specimens. For measurements not involving polarization analysis, circular polarization of the laser fundamental was used for the excitation. We adapted both acquisition channels for analysis of the polarization of the emitted SHG signals by addition of collimation lenses and Glan laser polarizers. Images were taken with the analyzing polarizers in the parallel and orthogonal positions to the excitation light to calculate the resulting anisotropy.

2.2.

Tissue Preparation

Mice carrying the oim mutation are maintained in the B6C3Fe-a/a(C57BL/6JLe X C3HeB/FeJLe) hybrid background.¹⁰ These mice were additionally crossbred with a pOBCol3.6GFP transgenic aCD-1 outbred strain in our facility. The oim/oim mice can be distinguished from the WT by their body phenotype (smaller size and weight and presence of limb deformities due to fractures). The genotype was confirmed by a previously described PCR analysis.¹¹ Imaging measurements were made within $3\mathrm{h}$ of sample excision. The overall thicknesses of the biopsies were $\sim 100\mathrm{\mu }\mathrm{m}$ and contained the epidermis, dermis, and adipose layers.

Femurs from 3-month-old oim/oim and WT mice were fixed in 4% paraformaldehyde for 3 days followed by decalcification in 15% EDTA for 3 to 4 days. The bones were then soaked in 30% sucrose in PBS for 1 day. All the processes of fixation, decalcification, and cryoprotection were done at $4°\mathrm{C}$ under constant agitation. Samples were embedded in frozen embedding medium (Thermo Shandon) at $-70°\mathrm{C}$ , and $5\text{-}\mathrm{\mu }\mathrm{m}$ -thick full-length sections were made with the assistance of the CryoJane tape system (Instrumedics, Inc.).

The oim cell line was obtained from an immortalized primary culture of calvarial cells from an oim mouse.¹⁰ The corresponding control cell line was the normal pre-osteoblast MC3T3 line (ATCC). Cells were grown to confluence for 3 weeks in a 6-well plate, fed with alpha minimal essential medium, β-glycerol phosphate, and ascorbic acid $(25\mathrm{\mu }\mathrm{g}∕\mathrm{ml})$ to induce collagen secretion.

3.1.

Collagen Secreted in Tissue Culture

As an initial test of our hypothesis that SHG will be able to differentiate between normal and OI tissues, we began by comparing our results with that of a previously established model. Kobayashi used electron microscopy to visualize secreted collagen by human fibroblasts in tissue culture and observed pronounced differences in normal and OI assembly.¹² Here we compare the morphology of collagen secreted from immortalized MC3T3 pre-osteoblasts (control) and oim calvarial cells in tissue culture. Cells were grown for 21 days and imaged in a fully hydrated environment. Representative SHG images collected in the forward direction of the secreted collagen matrix from normal and oim cells are shown in Fig. 1 . The collagen fibrils secreted from MC3T3 cells in Fig. 1a forms a tightly woven mesh, where the empty spaces are the location of the cells, which are transparent in this modality. In strong contrast, the oim collagen network in Fig. 1b, is characterized by longer, less densely packed fibrils. These results are in qualitative agreement with Kobayashi ¹² Based on this similarity, we suggest that SHG may be used as a diagnostic tool to detect abnormal collagen assembly following a skin biopsy. Additionally, SHG may provide insight into the collagen secretion process, as these experiments can be performed for the same cells over a course of days to weeks.

Fig. 1

Comparison of the collagen fibril morphology secreted from (a) MC3T3 pre-osteoblasts and (b) an oim calvarial derived line in tissue culture. The collagen fibrils are more well-packed for the wild type control. Scale bar $=50\mathrm{\mu }\mathrm{m}$ .

3.2.

Bone Morphology

We examined whether the fibril morphology and organization of the collagen in WT and oim bone could be differentiated by SHG imaging. Figures 2a and 2b show representative images from cryosections of WT and oim, respectively, collected from the same region of the femur. While the collagen fibrils in bone lack the spatial regularity of tendon (shown later in Fig. 4), these images suggest that a more regular fibrillar structure is present in the WT. An initial assessment of the organization can be obtained by quantitatively comparing the integrated SHG intensities of the WT and oim. Integration of 10 image sets shows that the WT is characterized by $\sim$ two-fold larger SHG intensity than the diseased state. As the SHG intensity is sensitive to both the local collagen concentration and the assembly, it is instructive to determine the cause of the intensity difference. While we cannot nondestructively measure the collagen concentration in these cryosections, later we show that this is similar for oim and WT skin. Thus, if we assume that the relative concentration of type I collagen is similar across tissue types, these results suggest that the decreased intensity from diseased bone arises from reduced organization. We note that these measurements were necessarily performed on fixed bone sections, as fresh bone undergoes a rapid autolysis, resulting in distortion of the tissue structure and consequently the optical properties. As shown in previous studies, such fixation does not significantly alter the fibrillar structure,¹³ and this method is thus appropriate for comparison of tissues.

Fig. 2

Representative SHG images from cryosections of demineralized femur. Panels (a) and (b) are the wild type (WT) and oim, respectively. The WT has more regular fibrillar structure and stronger SHG intensity. Scale bar $=50\mathrm{\mu }\mathrm{m}$ .

Fig. 4

Comparison of the crimping in (a) WT and (b) oim tendon SHG imaging. The oim has approximately a twofold increase in periodicity, suggesting that it has a decreased elastic modulus. Scale $\mathrm{bar}=50\mathrm{\mu }\mathrm{m}$ .

We can also make use of the SHG signal polarization anisotropy to further characterize the collagen assembly. As we have previously described, this measurement provides a determination of the order of the fibrils in each focal volume.^{1, 14} Here the laser polarization is fixed at the optimal angle $\left(45\mathrm{deg}\right)$ relative to the long axis of the bone,⁵ and two sequential SHG images are acquired with a polarizer oriented at 0 and $90\mathrm{deg}$ with respect to the input polarization. The results are shown in Fig. 3 , where the top and bottom panels are the WT and oim, respectively. The WT images are dominated by parallel fibrils, where the parallel and perpendicular images are similar in morphology, suggesting that the signal comes primarily from highly aligned molecules in the assembly. In contrast, the organization is much more randomized for the oim bone, with no predominant fibril direction. This is further exemplified by the dissimilarity of the morphology in the parallel and perpendicular images, where many orthogonally oriented features appear for these two polarization states (see fibrils denoted by arrow). This suggests an underlying disorder in the collagen molecules that assemble into the fibrils. We have previously quantified such organization by calculating the resulting SHG anisotropy, $\beta$ , by:

Fig. 3

SHG polarization anisotropy of WT (top row) and oim (bottom row) femur. The laser polarization was $45\mathrm{deg}$ with respect to the axis of the bone, and sequential images were obtained with a Glan polarizer parallel and perpendicular to the excitation. The oim has fibrillar components in both polarization states, indicating that the matrix is less organized than the WT. Scale $\mathrm{bar}=50\mathrm{\mu }\mathrm{m}$ .

3.3.

Tendon

Previous scanning electron microscope (SEM) studies by McBride showed that the fascicle cross sections of oim tendons were significantly smaller than those of the WT.¹⁵ We further examine this issue to determine whether SHG can differentiate morphological and structural aspects at the smaller scale of assembly of fibrils and fibers in tendon. Forward-collected SHG images for the WT and oim tendon are shown in Figs. 4a and 4b , respectively. We first measure the crimping, or the “waviness,” in the tendons where the periodicity of the crimp is related to the material elastic modulus. The SHG images show that the characteristic frequency is much higher in the oim tissue. Average periodicities were $114\pm 15\mathrm{\mu }\mathrm{m}$ ( $n=33$ fibrils) for the WT and $49\pm 6\mathrm{\mu }\mathrm{m}$ ( $n=56$ fibrils) for the oim. We attribute the increased crimping in the oim to a decreased elastic modulus versus the WT. This result is consistent with decreased stiffness in oim bone and skin reported by other workers.^{16, 17}

Next we investigate whether the oim tendons are also comprised of smaller or less-packed fibrils. To this end, we exploit the directionality of the SHG emission. Backward-detected SHG can arise from either direct backward coherent emission or from multiple scattering of initially forward-directed photons. The first scenario is highly dependent upon the fibril diameters and packing. Fibrils in tissues much smaller than the SHG wavelength produce a symmetrical forward and backward emission distribution.^{18, 19} When the dipoles of adjacent molecules form aligned structures comparable to or larger than $\lambda$ (in the axial dimension), the emission becomes highly forward directed. Due to the coherent nature of the SHG process, different fibril morphology can be observed in the forward and backward channels. Specifically, smaller features can be better visualized in the backward channel. This effect has been observed in collagen of tendon and cornea by Williams ³ and Han, ²⁰ respectively, and more recently for fibrillar cellulose by Nadiarnykh ¹⁴

We compared the forward and backward signals with concurrent polarization anisotropy analysis to obtain the highest level of sensitivity to the supramolecular collagen structure. This approach yielded few discernible differences in the forward-collected geometry (not shown). In fact, calculation of the anisotropy parameter, $\beta$ , results in a range of $\sim 0.3$ to 0.75 for both normal and diseased tissues. However, marked morphological differences are observed in the backward-collected geometry. Figures 5a and 5b show the parallel and perpendicular components of the backward-propagating SHG for the WT and oim, respectively. These images for the WT are highly similar. In contrast, the oim parallel channel displays smaller, segmented fibrils as well as longer, continuous fibrils. While differing in morphology, calculation of the anisotropy also results in a similar range of values to that in the forward channel.

Fig. 5

Backward SHG polarization anisotropy of (a) WT and (b) oim tendon. The laser polarization was $45\mathrm{deg}$ with respect to the axis of the bone, and sequential images were obtained with a Glan polarizer parallel and perpendicular to the excitation. Smaller features are seen in the backward component of the oim tendon, resulting from a shorter coherence length and subsequent destructive interference of this coherent SHG component. These smaller fibrils are not observed in the forward channel (not shown). Scale $\mathrm{bar}=20\mathrm{\mu }\mathrm{m}$ .

To explain this appearance, we need to consider the fibril sizes. Our SEM analysis of the distribution of fibril sizes is shown in the histogram in Fig. 6 , where the most probable values for the oim (b) and WT (a) are $\sim 70$ and $160\mathrm{nm}$ , respectively. Elsewhere, we have provided a general treatment of the morphology observed in SHG images based on fibril size and packing, which predicts that the forward and backward channels will have different coherence lengths, where the visualization of segmented fibrils can arise from destructive interference in the latter.¹⁹ Based on this work, we suggest that the contrast from these discontinuous-looking segmented fibrils is likely coherent (i.e., direct backward emission) arising from the smaller oim fibrils. We can then account for this segmented morphology primarily appearing in the parallel channel and the absence of these features in the perpendicular direction. We showed earlier that the forward SHG signal in these tendons is primarily polarized parallel to the laser electric field, and also that the forward and backward anisotropy in a fibrillar specimen were similar.¹⁴ Thus, it is expected that the initial SHG backward emission here will have similar anisotropy. The backward-collected image is comprised of both coherent and incoherent multiple scattered components, and we suggest that the perpendicular image arises largely from multiple scattering of the forward signal, which does not display the segmented contrast. Upon scattering, the polarization of the signal will become randomized, and the signal will be captured in both backward polarization channels. We suggest that segmented appearing fibrils are not observed in the WT SHG images because of the larger diameter and concurrent longer coherence length. While both tissues are characterized by a similar range of anisotropies, the use of polarization analysis is essential to reveal the backward-coherent polarized emission on top of the depolarized multiple scattered component. Specifically, this uniquely shows the segmented appearance of the smaller fibrils in the oim tendon.

Fig. 6

Histogram of the fibril sizes as determined by SEM analysis of (a) WT and (b) oim tendon.

Unlike in bone, we cannot make a quantitative assessment of the relative SHG intensities in these samples because these fascicles are sufficiently thick ( $\sim 50$ to $100\mathrm{\mu }\mathrm{m}$ ) to support multiple-scattering events (the mean free path in such tissues is $\sim 10$ to $20\mathrm{\mu }\mathrm{m}$ ). As a result, the absolute intensity in each case will depend on the scattering coefficients at both the fundamental and SHG wavelengths, and due to differences in assembly, these parameters will be different in each tissue. However, we can exploit the directionality of the SHG signal propagation as a means to probe the density of the fibrillar packing. The forward-to-backward ratio (F/B) that arises from multiple scattering of the forward signal will depend on the magnitude¹⁴ of the reduced scattering coefficient ${\mu }_s^{\prime }$ . We have not yet been able to measure these coefficients in mouse tendon but have measured the F/B intensities in WT and oim tendon. A histogram of the results obtained by integrated $z$ projection of the SHG intensity is shown in Fig. 7 , where the mean F/B values for the normal and diseased mice are $2.1\pm 0.6$ and $3.5\pm 0.6$ , respectively. The increased scattering in the WT (i.e., lower F/B) is also consistent with the higher degree of similarity in the polarized SHG images in Fig. 5a, where both channels contained a significant multiple-scattered component. Conversely, the more forward directed signal for the oim is consistent with a less dense matrix, as the mean free path will be longer and there is lower probability for multiple collisions. We therefore suggest that this finding is consistent with sparser packing of fibrils in the matrix and the mechanical properties in vivo, and that this approach of measuring propagation could be used as a diagnostic tool.

Fig. 7

Histogram of the measured F/B ratio of oim and WT tendon. The oim is more forward directed, indicative of a lower scattering coefficient and less dense matrix.

3.4.

Skin

Our long-term goal is to develop SHG imaging as a clinical diagnostic tool. For both ex vivo and in vivo imaging, skin is the most accessible tissue, and to this end, we examine whether SHG can quantitatively differentiate WT and oim skin. Representative images are shown in Figs. 8a and 8b , respectively, where these were taken at depths into the dermis of $\sim 20$ microns (plus $\sim 10\mathrm{\mu }\mathrm{m}$ of the epidermis). Based on our SEM analysis, the average fibril sizes in the oim and WT skin are $\sim 70$ and $100\mathrm{nm}$ , respectively, and cannot be resolved in the SHG microscope. However, on inspection, these images suggest that the fibrils are less ordered for the diseased skin. As the SHG contrast depends on the square of the concentration of the sources as well as their molecular organization, we need to determine the contributions of these effects. To this end, the tissues were stained with Sirius Red and Fast Green FCF to assess the collagen and total protein content, respectively. These histological sections are shown in Figs. 8c and 8d, respectively. We first note that the Sirius Red staining appears more highly packed in the dermal assembly for the WT and is consistent with the tighter packing seen in the SHG image. Quantitative measurements of the extracted Sirius Red showed that the collagen content in the oim was 85% that of the WT. Given this similarity, we ascribe the higher degree of SHG contrast difference between WT and oim skin to reduced collagen organization in the diseased tissue.

Fig. 8

Morphology of WT and oim skin determined by (a) SHG and (b) histological staining with Sirius Red and Fast Green FCF. Both contrasts show denser packing of the collagen fibrils for the WT relative to the oim. Scale $\mathrm{bar}=50\mathrm{\mu }\mathrm{m}$ . (Color online only.)

We must be able to extract quantitative differences to apply the method as a disease diagnostic. The complex morphology of skin does not lend itself to polarization anisotropy. Indeed, attempts to determine $\beta$ led to a random distribution, even when performed over several ranges of size scales in the images. However, we can readily perform relative assessments of the organization and packing by measurement of the F/B as described earlier for tendon as a means of comparing the scattering properties. These measurements obtained by integrated average $z$ projections of 3-D image stacks show F/B values of $2.6\pm 0.3$ and $1.6\pm 0.2$ for the oim and WT, respectively. These values follow the same trends observed in tendon, being indicative of a smaller reduced scattering coefficient, and similarly indicating that the oim tissue is less organized than the WT. This result suggests that SHG imaging of skin can be used as a quantitative assessment of this disorder.