2.1.

Normal Synovium

Specimens of normal synovial tissue for histology and NLOM imaging were obtained from New Zealand white rabbits weighing $4\text{to}5\mathrm{Kg}$ that were terminated under Institutional Animal Care and Use Committee (IACUC)-approved projects performed in other laboratories where the knee joints were not affected.

2.2.

Animal Injection Preparation

New Zealand white rabbits (Western Oregon Rabbit Company, Philomath, Oregon) weighing $\sim 4\mathrm{Kg}$ were used in the research study, which was approved by the IACUC of the University of California, Irvine. Prior to intra-articular injection of an experimental agent, rabbits were immobilized with an intramuscular injection of $25\text{-}\mathrm{mg}∕\mathrm{Kg}$ ketamine HCl. Following immobilization, 2 to 5% isoflurane was administered via face mask throughout the preparation of the inoculation site, the placement of the fentanyl patch, and during intra-articular injection. The injection site was shaved and sterilized using a chlorhexidine acetate scrub, cleaned with alcohol, and sprayed with 2% chlorhexidine acetate solution. An intra-articular injection of the experimental agent was made into the knee joint by inserting a $25\text{-gauge}$ needle into the joint space from the lateral aspect through a window identified by palpation of the lateral margin of the patella, the proximal lateral margin of the tibia, and the cranio-distal margin of the lateral femoral condyle.

For relief of anticipated knee pain a transdermal fentanyl patch $(25\mu \mathrm{g}∕\mathrm{h})$ (Mylan Pharmaceuticals Incorporated, Morgantown, West Virginia) was applied to freshly shaved skin on the dorsum in the intrascapular region.²⁴ Fentanyl patches were removed and replaced every $4\text{days}$ . Each new patch was applied to an adjacent freshly shaved area of the dorsal intrascapular region.

2.3.

Lipopolysaccharide-Induced Arthritis Model

Arthritis of an immune-mediated etiology was induced in a standard rabbit model using two intra-articular injections of $50\text{-}\mu \mathrm{g}$ lipopolysaccharide from E. coli 0111, B4 (L5293, Sigma Chemical Company, Saint Louis, Missouri) suspended in $0.1\mathrm{ml}$ of sterile saline.²⁵ Three animals received an injection of LPS in the right knee on day 0 and again on day 4, and then one animal each was euthanized and tissue harvested on days 4, 11, and 18.

2.4.

Septic Arthritis Model

Septic arthritis was induced in a standard rabbit model^{26, 27} using one intra-articular injection of penicillin-sensitive Staphylococcus aureus (ATCC 25923) (American Type Culture Collection, Rockville, Maryland) maintained and prepared by the Department of Pathology, College of Health Sciences at the University of California, Irvine. A single injection of a bacterial inoculum titrated to ${10}^4$ colony forming units/ml sterile saline and $0.1\mathrm{ml}$ ( ${10}^3$ CFU) was made into the right knee of each of the three rabbits in the same manner as the injection procedure described for the LPS model. The day of injection was designated day 0, and one animal each was euthanized and tissue harvested on days 2, 4, and 8 postinjection.

2.5.

Specimen Collection and Preparation

Rabbit models have been used extensively in human joint disease research because of the similarities that exist in pathology and pathogenesis, and the ease with which the relatively large rabbit knee joint can be studied in contrast to the mouse joint. In this study, within three hours following euthanasia, synovium was obtained from the knee joints of 12 normal adult male New Zealand white rabbits and three treated rabbits in each of two induced arthritis groups.

Harvested rabbit knee joints were dissected on the medial and lateral aspects to expose each meniscus, and a tissue specimen was obtained from each side of the joint, providing a total of two tissue specimens from each joint. The meniscus was lifted from the tibial surface and both the meniscus and the attached joint capsule were removed from each side of the joint without changing the shape or size of the synovial membrane. Each meniscus remained attached to the joint capsule adjacent to it so that the tissue could be manipulated without disrupting the synovium (Fig. 3 ), and so that the inner surface of the joint capsule where the synovium is located could always be identified. The synovial lining obtained from the site of attachment to the meniscus is adipose synovium. Each specimen was approximately $15\times 15\mathrm{mm}$ .

Fig. 3

(a) Meniscus with (b) synovium covered joint capsule attached to its lateral margin, as dissected from a rabbit knee joint. Retention of the meniscus allowed orientation and easy manipulation of the joint capsule tissue without disrupting the synovium.

Each sample of adipose synovium was placed in a fluorescence microscopy dish (Fluorodish, World Precision Instruments, Incorporated, Sarasota, Florida) containing a drop of 0.9% saline solution to maintain tissue hydration. As the NLOM imaging system uses an inverted microscope, the synovium, which is on the inside surface of the joint capsule, was carefully placed face-down flat, without any gross folds or under any tension, on the bottom of the microscopy dish, and imaging signals were collected in the backward direction. The specimen was covered with a piece of saline-soaked gauze to further prevent dehydration during imaging. Each microscopy dish with a synovium sample was stored in a sealed bag at $4°\mathrm{C}$ until imaged. All samples were imaged within $3\mathrm{h}$ of collection except for one normal specimen that was imaged $25\mathrm{h}$ after collection.

2.6.

Histology

Following NLOM imaging, each specimen was fixed in 10% buffered formalin at room temperature for $12\mathrm{h}$ , and then rinsed and stored in phosphate-buffered saline solution. Each specimen was subsequently dehydrated, cleared, imbedded in paraffin, cut into $6\text{-}\mu \mathrm{m}$ -thick sections, and placed onto slides. The mounted slide sections were deparaffinized and the slide preparations stained with H&E (Sigma-Aldrich, Saint Louis, Missouri).

The stained slide sections were imaged using light microscopy (Olympus BH-2, Olympus America, Incorporated, Melville, New York) and a MicroFire digital imaging system (Olympus America, Incorporated) coupled to a computer and recorded using image capture software (PictureFrame, Optronics, Goleta, California). Synovial images from light microscopy were then compared to NLOM images to define the structures and cells imaged within each specimen.

2.7.

Nonlinear Optical Microscopy

NLOM was performed using a Zeiss LSM 510 NLO Meta system (Carl Zeiss MicroImaging, Incorporated, Thornwood, New York) with a Coherent Chameleon Ti:sapphire femtosecond laser (Coherent, Incorporated, Santa Clara, California) as the excitation source. The Chameleon laser source provided $140\text{-}\mathrm{fs}$ pulses at a repetition rate of $80\mathrm{MHz}$ , with the center frequency tunable from $690\text{to}1040\mathrm{nm}$ . Excitation wavelengths of both 730 and $800\mathrm{nm}$ were used in this study, as they induce an optimal combination of imaging signals from intracellular compounds and collagen, respectively.^{10, 15, 28}

While using $800\text{-}\mathrm{nm}$ excitation, light emission from the tissue was separated into two channels using the grating-based Meta detector available in the Zeiss LSM 510 NLO system. The channel containing SHG emission between 395 and $405\mathrm{nm}$ is pseudocolored blue, while the channel containing TPF emission in the range $415\text{to}600\mathrm{nm}$ is pseudocolored green. The TPF signal was collected over a relatively wide bandwidth $\left(185\mathrm{nm}\right)$ , as the sources of TPF are numerous biomolecules with different emission peaks¹⁰ and distinct TPF spectra within that bandwidth. Excitation at $730\mathrm{nm}$ elicited a stronger TPF but much weaker SHG signal than was observed with the $800\text{-}\mathrm{nm}$ wavelength. The TPF signal collected from the tissue following $730\text{-}\mathrm{nm}$ excitation was divided into two channels to enhance contrast using a bandpass filter $\left(390\text{to}465\mathrm{nm}\right)$ , and using the Meta detector $\left(470\text{to}650\mathrm{nm}\right)$ . These channels were pseudocolored turquoise $\left(390\text{to}465\mathrm{nm}\right)$ and orange $\left(470\text{to}650\mathrm{nm}\right)$ . The turquoise channel contains fluorescence mainly from mitochondrial NADH, and some other intrinsic fluorophores like folic acid and retinol that have fluorescence peaks within the $390\text{-}\text{to}465\text{-}\mathrm{nm}$ bandwidth. The orange channel collected fluorescence from riboflavin and other biomolecules with fluorescence peaks beyond $500\mathrm{nm}$ .¹⁰

As our purpose was to image synovium in its native state, no fluorescent stains were used on the tissue. To thoroughly survey each tissue specimen, images were obtained from: 1. the meniscus-synovium interface, 2. the central region of the synovial specimen, and 3. the region along the periphery where the adipose synovium attaches to articular cartilage. These imaging regions were $3\text{to}5\mathrm{mm}$ apart. The imaging region at the periphery was chosen such that it was at least $2\mathrm{mm}$ from the cuts made during sample extraction.

Images $512\times 512\text{pixels}$ and $1024\times 1024\text{pixels}$ of tissue areas from $225\times 225\mu \mathrm{m}\text{to}2.25\times 2.25\mathrm{mm}$ were obtained using $20\times$ , $0.45\text{-}\mathrm{NA}$ and water immersion $40\times$ , $0.8\text{-}\mathrm{NA}$ objectives. To image an area $1.125\times 1.125\mathrm{mm}$ or larger, individual adjacent $225\times 225\text{-}\mu \mathrm{m}$ images were acquired sequentially and combined to form the larger image. This technique, known as “tiling,” allows construction of a large, clear geographical image of the synovial tissue (Fig. 4 ). NLOM imaging scan speeds from $6.5\text{to}102\mu \sec$ per pixel were evaluated. The combination of a $40\times$ , water immersion objective (Achroplan, $0.8\mathrm{NA}$ ) delivering $20\mathrm{mW}$ at the tissue surface and a pixel dwell time of $6.5\mu \sec$ provided images with sufficient cellular detail without thermally damaging the tissue, even during the relatively long time $\left(7\text{to}15\min \right)$ required to acquire a stack of up to 70 images from the same location in successive planes at $1\text{-}\mu \mathrm{m}$ intervals. The minimum power required for image acquisition was $18\mathrm{mW}$ delivered to the sample with a pixel dwell time of $6.5\mu \mathrm{s}$ . Below that level, the emission signals were judged to be too weak for imaging. Power levels above $34\mathrm{mW}$ used with a pixel dwell time of $12.8\mu \mathrm{s}$ caused tissue damage.

Fig. 4

A NLOM tiled image of the synovial surface layer constructed from sequentially acquired $225\times 225\text{-}\mu \mathrm{m}$ images using $800\text{-}\mathrm{nm}$ excitation. (a) The SHG emission from collagen is in blue, (b) TPF, in green, is mostly from intracellular sources, and (c) spaces between synovial folds lack any fluorescence signal. Scale bar is $100\mu \mathrm{m}$ . The box-enclosed area is further magnified in Fig. 8 to show cellular details. (Color online only.)

Fig. 8

Magnified NLOM images showing synovial cells and elastin fibers from (A) the box-enclosed area of Fig. 4, and (B) from Fig. 6. Rounded synoviocytes, $10\text{to}20\mu \mathrm{m}$ in diameter (a), and elongated synoviocytes, $40\text{to}50\mu \mathrm{m}$ long (b), can be distinguished in (A). Long, thin elastin-like fibers (c) in (A) and (B) are observed to be morphologically distinct from the elongated synoviocytes in (A). Scale bars are $50\mu \mathrm{m}$ .

2-D images as well as 3-D tomographic stacks of individual 2-D images obtained at $1\text{-}\text{to}2\text{-}\mu \mathrm{m}$ intervals to a depth of up to $70\mu \mathrm{m}$ , referred to as Z stacks, were obtained to study tissue morphology in successive image planes within a volume of tissue (Fig. 2).

3.1.

Histology

An HE stained histology preparation of adipose synovium from the normal knee joint of a rabbit is shown in Figs. 5, 6 . The cross section of synovium demonstrates both intima and subintima. The sharp transition between the intima and subintima and the characteristic morphological details of each of these layers of adipose-type synovium are easily observed. The intima is seen as a densely populated cellular region that is one to three cells thick, while the subintima is composed primarily of adipocytes and small numbers of capillaries, small venules, and arterioles set in a randomly distributed matrix of loosely packed thin collagen fibrils.

Fig. 5

HE stained histological images of adipose synovium from (A) a knee joint of a normal rabbit, (B) $8\text{days}$ postinoculation with S. aureus, and (C) $18\text{days}$ postinoculation with LPS from E. coli. (A) In the cross section of normal adipose synovium, the intima (a) is seen as a densely populated cellular region that is one to three cells thick, while the subintima (b) is composed primarily of adipocytes (c) and small numbers of capillaries (d), small venules (e), and arterioles set in a randomly distributed matrix of loosely packed thin collagen fibrils (f). The sharp transition between the intima and subintima and the characteristic morphological details of each layer are easily observed. The cross section of adipose synovium from the S. aureus infected knee (B) demonstrates that a denuded intimal layer (g) and the adipocytes (c) of the thickened and edematous subintima have been largely replaced by an inflammatory cell infiltrate (h) consisting primarily of heterophils. Capillaries (i) are congested and their walls are thickened. Fibroblasts (j) are beginning to produce a delicate fibrillar matrix, while the meshwork of pre-existing collagen fibers is randomly displaced. The image of the inflammatory reaction that is present in the adipose synovium of the rabbit knee joint following LPS inoculation (C) demonstrates thickening of the intimal layer (a), and the normal population of subintimal adipocytes has been replaced by an inflammatory cell infiltrate (l) consisting primarily of lymphocytes relatively evenly distributed, along with fibroblasts in a collagenous matrix containing congested capillaries (i), arterioles (k), and venules (e). In some areas, edema (m) separates the matrix of collagen fibrils. Scale bars are $50\mu \mathrm{m}$ .

Fig. 6

Important synovial structures as demonstrated by (A) HE sectioning and (B) through (F) NLOM imaging using $800\text{-}\mathrm{nm}$ excitation. All images except for (D) are presented in the same scale for ease of comparison. The HE section shows the thin intimal layer (a) and the much thicker subintima (b). Collagen (c) appears as a widespread wavy pattern in (A) and is identified particularly well by its strong blue SHG signature in (B) through (F). Adipocytes (d) appear as empty spaces in the extracellular collagen matrix in the HE stained section (A) and as dark oval structures enclosed by the blue SHG signal of the collagen fibers in the NLOM image (B). Rouleaux formation of erythrocytes in undulating venuoles (e) set in a background meshwork of blue collagen fibrils of the subintima are seen in (C) and (D). (D) Image is a magnification of the area inside the box in (C), and demonstrates the visualization of individual erythrocytes. An arteriole (f) is seen in (A) and in (E), where the green colored TPF signal of elastin fibers outline the wall of a linearly oriented arteriole. Arteriole walls have elastin that provides a strong TPF signal, while the primarily collagen-containing venule walls produce only a SHG signal that highlights erythrocytes within the vascular lumen (e). Linearly directed elastin fibers appearing as green filaments (g) set in a meshwork of less-well-oriented blue collagen fibers are observed in synovial tissue along the insertion line of the adipose synovium with the meniscus (F). Scale bars are $50\mu \mathrm{m}$ for all images. (Color online only.)

The three animal subjects in the septic arthritis group demonstrated a polymorponuclear inflammatory response with loss of normal intimal and subintimal morphology as anticipated, which was progressive with increasing time from joint inoculation, and could be characterized in both NLOM and HE stained histology sections. The image of the HE stained histologic section of the adipose synovium of the rabbit knee at $8\text{days}$ following inoculation with S. aureus [Fig. 5] represents the most advanced septic pathology of the study, and reveals an inflammatory reaction with a denuded intimal surface and a thickened and edematous subintima. The adipocytes found in normal adipose synovium have been replaced by an inflammatory cell infiltrate consisting primarily of heterophils. Capillaries are congested and their walls are thickened. The meshwork of pre-existing collagen fibers is randomly displaced and fibroblasts produce a delicate fibrillar matrix.

The three animal subjects in the LPS arthritis group demonstrated intimal thickening with a mononuclear inflammatory response that was progressive with increasing time from joint inoculation, the characteristics of which could be distinguished in both NLOM images and HE stained histology sections. A photomicrograph of an HE stained histology section of the inflammatory reaction that is present in adipose synovium of the rabbit knee $18\text{days}$ after LPS inoculation [Fig. 5] represents the most advanced immune-mediated pathology of the study, and demonstrates a thickened intima. The normal population of adipocytes has been replaced by an inflammatory cellular infiltrate consisting primarily of lymphocytes evenly distributed along with fibroblasts in a collagenous matrix. The matrix also contains congested capillaries, arterioles, and venules that in some areas are separated by edema.

3.2.

Nonlinear Optical Microscopy Imaging of Synovium

Figure 4 demonstrates a typical NLOM image of the synovial surface layer using $800\text{-}\mathrm{nm}$ excitation and a scan rate of $12.8\mu \sec$ per pixel. The SHG (blue) signal reveals the dense matrix of collagen and the TPF (green) signal from intracellular compounds highlights individual cells. NLOM imaging of cells and tissues was not affected by periods of tissue storage that ranged from $3\text{to}25\text{-}\mathrm{h}$ postdissection.

The imaging signals from the tissue showed quadratic dependence on excitation intensity as expected for TPF and SHG sources. The tissue emission spectra measured in the spectral imaging mode of the Zeiss LSM 510 NLO Meta system helped further characterize the nature of the SHG signal. For different wavelengths of excitation, the sharp SHG peak always occurred at exactly half the incident wavelength, as anticipated. With no correction for the spectral dependence of optical component performance, the SHG signal from extracellular collagen weakened and the TPF from tissue constituents brightened as the excitation wavelength was decreased from $800\text{to}730\mathrm{nm}$ . This is consistent with NLOM imaging of other tissues.^{10, 28}

When using $800\text{-}\mathrm{nm}$ excitation, the use of slow scan speeds corresponding to pixel dwell times of 12.8 and $25.6\mu \sec$ were found to provide sufficient signal collection at each pixel to produce bright, clear images. In Figs. 6, 6, where a dwell time of $12.8\mu \sec$ per pixel was used, individual erythrocytes within blood vessels [Fig. 6, part e] are clearly visible. When the scan speed is increased on the same tissue sample, erythrocytes lose clear definition and become difficult to identify. The problem with using slow scan speeds, however, is the potential for thermal injury to the tissue.

Excitation with $730\mathrm{nm}$ [Fig. 7 ] produced clear images of cells at faster scan speeds (pixel dwell times of $6.4\text{to}12.8\mu \sec$ per pixel); however, the collagen signal was very weak. When using an $800\text{-}\mathrm{nm}$ incident beam at these higher scan speeds [Fig. 7], collagen still produced a clean, bright SHG signal, as reported previously.²⁸ A combination of the TPF channels from the $730\text{-}\mathrm{nm}$ excitation image [Fig. 7] and the SHG channel from the $800\text{-}\mathrm{nm}$ excitation image [Fig. 7] is shown in Fig. 7. Combining separate images obtained from 730- and $800\text{-}\mathrm{nm}$ excitation wavelengths provided the sharpest and most detailed image of cellular and collagen composition of the tissue specimen.

Fig. 7

Comparison of synovial images of the same location acquired using (A) $730\text{-}\mathrm{nm}$ excitation and (B) $800\text{-}\mathrm{nm}$ excitation at a pixel dwell time of $12.8\mu \sec$ . At this scan speed, cells (a) appear clearer with $730\text{-}\mathrm{nm}$ excitation, while collagen (b) is most clearly distinguished when using $800\text{-}\mathrm{nm}$ excitation. (C) An improved image was created by combining the TPF channels from (A) and SHG channel from (B). Scale bars are $20\mu \mathrm{m}$ .

3.3.

Identification of Morphologic Characteristics of Normal Synovial Tissue

A number of structures of normal synovium were clearly identified by NLOM imaging. Adipocytes are seen in the NLOM image in Fig. 6 as large ovoid black spaces with their cell margins marked by a TPF signal, surrounded by collagen fibers identified by their characteristic SHG signal. Adipocytes appear dark in NLOM due to the large volume of lipid content, which does not fluoresce. The adipocytes [Fig. 6, part d] of the NLOM image [Fig. 6] are comparable in size and morphology to those demonstrated in HE preparations [Fig. 6].

NLOM demonstrated both venules and arterioles in synovium [Figs. 6, 6, 6]. Venule walls, which contain collagen, emitted an SHG signal, while the erythrocytes within the lumen emitted a TPF signal. The two signals produced sufficient contrast to clearly identify erythrocytes inside the venules [Fig. 6]. On the other hand, arteriolar walls contain a high concentration of elastin, which emits a strong TPF signal.¹⁰ The overlapping TPF signals emitted both from erythrocytes and the elastin in arteriole walls made it difficult to identify individual erythrocytes within arterioles [Fig. 6].

Variable-length, $1\text{to}2\mu \mathrm{m}$ wide, TPF-emitting filamentous strands typical of elastin¹⁰ were observed in the subintima of the synovium, generally in association with the dense collagen matrix, in regions adjacent to the attachment of the joint capsule to bone [Figs. 6, 8, 8 ]. The elastin fibers were not highly organized but do appear to have some common directionality in the regions where they are present. While the elastin fibers were clearly observed where they lie in a longitudinal orientation to the imaging plane, because of their narrow diameter, it is possible that their presence was not recognized where they lie perpendicular to the imaging plane.

Cells were identified by the green fluorescence from their cytoplasm silhouetting dark nuclei, as seen in Fig. 8 (a magnified view of the boxed region in Fig. 4). Figure 8 demonstrates cells of different morphologies imaged in synovial tissue by NLOM. Synoviocytes in HE sections only demonstrated an oval morphology, while NLOM imaging [Fig. 8] allowed identification of both round [Fig. 8, part a] and elongated [Fig. 8, part b] synoviocytes as well as the collagen matrix (blue).

Images with structural detail of tissue could be obtained to a depth of $70\mu \mathrm{m}$ from the synovial surface. Image stacks of normal adipose-type synovium acquired using $730\text{-}\mathrm{nm}$ excitation clearly demonstrate the structural distinction between the intima and subintima. The dense layer of cells within the intima produces a strong TPF signal, while the loosely collagenous matrix of the subintima that has poor SHG initiation at $730\mathrm{nm}$ remains relatively dark. The depth at which there is a transition from intima to subintima could be determined by viewing successive image planes from a Z stack for an abrupt change in cellular and matrix composition.

3.4.

Identification of Morphologic Characteristics of Pathologic Synovial Tissue

All NLOM images of septic inflammatory and LPS-induced synovitis tissue demonstrated increased cellular content when compared to normal tissue at equivalent locations and depths (Figs. 9 and 10 ). This was particularly evident in areas below the intimal layer, where normal subintimal tissue has substantially fewer cells than the intima. Histologic sections of the pathology specimens demonstrated a marked inflammatory response characterized by mixed inflammatory cells, macrophages and fibroblasts in the septic specimens [Fig. 9], and a generalized and predominantly lymphocytic infiltration with pronounced intimal thickening observed in the synovium of the LPS-treated joints [Fig. 9]. NLOM images of LPS and septic specimens revealed increased intracellular fluorescence consistent with the elevated level of metabolic activity of cells actively engaged in the inflammatory response [Figs. 9, 9, 9, 9]. There was a conspicuous absence of collagen SHG observed in the septic tissue [Figs. 9, 10], while collagen SHG, although reduced, continued to be observed in synovial tissue of the LPS-treated joints [Figs. 9, 10]. The larger size of the inflammatory cell types (heterophils and macrophages) with lobulated nuclei, and the elongated morphology of the fibroblasts was clearly evident in the NLOM images of the septic tissue [Figs. 9, 9, 10], while lymphocytes that are morphologically smaller in size with an oval nucleus, with minimal to no evidence of fibroblast activity, and the continued observation of some collagen SHG characterized the pathology of the tissue from LPS-treated joints [Figs. 9, 9, 10].

Fig. 9

Images of rabbit synovial tissue, (A), (B), and (C) normal, (D), (E), and (F) $8\text{days}$ postbacterial inoculation, and (G), (H), and (I) $18\text{days}$ post-LPS intra-articular injection, demonstrating HE stained histology sections in (A), (D), and (G), NLOM using $730\text{-}\mathrm{nm}$ excitation in (B), (E), and (H), and NLOM using $800\text{-}\mathrm{nm}$ excitation in (C), (F), and (I). All NLOM images were acquired in a plane parallel to and at a depth of $15\mu \mathrm{m}$ from the specimen surface [dotted line, in (A)] and thus primarily image the inflammatory response within the intima. Histologic sections of the pathology specimens demonstrate a marked inflammatory response characterized by mixed inflammatory cells, macrophages, and fibroblasts in the septic specimens in (D) and a generalized and predominantly lymphocytic infiltration with pronounced intimal thickening observed in the synovium of the LPS-treated joints in (G). NLOM images of both pathology specimens in (E), (F), (H), and (I) reveal increased intracellular fluorescence consistent with the elevated level of metabolic activity of cells actively engaged in the inflammatory response. There is a conspicuous absence of collagen SHG observed in the septic tissue in (F), while collagen SHG (blue) continues to be observed in synovial tissue of the LPS-treated joints in (I). The larger size of the inflammatory cell types (heterophils and macrophages) with multilobulated nuclei and the elongated morphology of the fibroblasts is clearly evident in the NLOM images of the septic tissue in (E) and (F), while in lymphocytes that are morphologically smaller in size with an oval nucleus, minimal to no evidence of fibroblast activity and the continued observation of some collagen SHG characterize the pathology of the tissue from LPS-treated joints in (H) and (I). Scale bars are $50\mu \mathrm{m}$ . (Color online only.)

Fig. 10

NLOM images of rabbit synovial tissue demonstrating the enhanced image quality achieved by combining images acquired by sequential $730\text{-}\mathrm{nm}$ and then $800\text{-}\mathrm{nm}$ excitation. The images illustrated were obtained from the subintima in a plane parallel to and at a depth of $45\mu \mathrm{m}$ from the surface of (A) and (B) normal, (C) $8\text{days}$ postbacterial inoculation, and (D) $18\text{days}$ post-LPS intra-articular injection specimens. At a depth of $45\mu \mathrm{m}$ , the imaging plane is below the intimal layer of the normal synovium and demonstrates subintimal tissue predominately consisting of (A) adipocytes or (B) collagen matrix with little other cellular content, while both the (C) septic and (D) LPS specimens demonstrate a substantial increase in cellular content consistent with the inflammatory processes. The septic tissue demonstrates large cells with multilobular nuclei characteristic of rabbit heterophils and macrophages, along with elongated cells characteristic of fibroblasts, while the cellular infiltrate observed in the LPS specimens are smaller cells with oval nuclei characteristic of an inflammatory response predominated by lymphocytes. SHG emission of collagen, viewed as blue fibrils in the NLOM images, is not observed in the subintimal tissue of the septic specimens in (C), while it continues to be observed, although in reduced amounts, in LPS specimens in (D). Scale bars are $50\mu \mathrm{m}$ . (Color online only.)