HE stained histological images of adipose synovium from ͑ A ͒ a knee joint of a normal rabbit, ͑ B ͒ 8 days postinoculation with S. aureus, and ͑ C ͒ 18 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͑ C ͒ demonstrates thickening of the intimal layer ͑ a ͒ , and the normal population of subintimal adipocytes has been replaced by an inflammatory cell infiltrate ͑ l ͒ consisting primarily of lymphocytes relatively evenly distributed, along with fibroblasts in a collagenous matrix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 ␮ m. 

Contexts in source publication

Context 1

... as the sources of TPF are numerous 10 biomolecules with different emission peaks and distinct TPF spectra within that bandwidth. Excitation at 730 nm elicited a stronger TPF but much weaker SHG signal than was observed with the 800-nm wavelength. The TPF signal collected from the tissue following 730-nm excitation was divided into two channels to enhance contrast using a bandpass filter ͑ 390 to 465 nm ͒ , and using the Meta detector ͑ 470 to 650 nm ͒ . These channels were pseudocolored turquoise ͑ 390 to 465 nm ͒ and orange ͑ 470 to 650 nm ͒ . The turquoise channel contains fluorescence mainly from mito- chondrial NADH, and some other intrinsic fluorophores like folic acid and retinol that have fluorescence peaks within the 390- to 465-nm bandwidth. The orange channel collected fluorescence from riboflavin and other biomolecules with fluorescence peaks beyond 500 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 to 5 mm apart. The imaging region at the periphery was chosen such that it was at least 2 mm from the cuts made during sample extraction. Images 512 ϫ 512 pixels and 1024 ϫ 1024 pixels of tissue areas from 225 ϫ 225 ␮ m to 2.25 ϫ 2.25 mm were obtained using 20 ϫ , 0.45-NA and water immersion 40 ϫ , 0.8-NA objectives. To image an area 1.125 ϫ 1.125 mm or larger, individual adjacent 225 ϫ 225- ␮ m images were acquired sequentially and combined to form the larger image. This technique, known as “tiling,” allows construction of a large, clear geo- graphical image of the synovial tissue ͑ Fig. 4 ͒ . NLOM imaging scan speeds from 6.5 to 102 ␮ sec per pixel were evalu- ated. The combination of a 40 ϫ , water immersion objective ͑ Achroplan, 0.8 NA ͒ delivering 20 mW at the tissue surface and a pixel dwell time of 6.5 ␮ sec provided images with sufficient cellular detail without thermally damaging the tissue, even during the relatively long time ͑ 7 to 15 min ͒ required to acquire a stack of up to 70 images from the same location in successive planes at 1- ␮ m intervals. The mini- mum power required for image acquisition was 18 mW de- livered to the sample with a pixel dwell time of 6.5 s. Be- low that level, the emission signals were judged to be too weak for imaging. Power levels above 34 mW used with a pixel dwell time of 12.8 ␮ s caused tissue damage. 2-D images as well as 3-D tomographic stacks of individual 2-D images obtained at 1- to 2- ␮ m intervals to a depth of up to 70 ␮ m, referred to as Z stacks, were obtained to study tissue morphology in successive image planes within a volume of tissue ͑ Fig. 2 ͒ . An HE stained histology preparation of adipose synovium from the normal knee joint of a rabbit is shown in Figs. 5 ͑ A ͒ and 6 ͑ A ͒ . 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. 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 days following inoculation with S. aureus ͓ Fig. 5 ͑ B ͔͒ 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 adipo- cytes 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 days after LPS inoculation ͓ Fig. 5 ͑ C ͔͒ 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. Figure 4 demonstrates a typical NLOM image of the synovial surface layer using 800-nm excitation and a scan rate of 12.8 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 stor- age that ranged from 3 to 25-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 to 730 nm. This is consistent with NLOM imaging 10,28 of other tissues. When using 800-nm excitation, the use of slow scan speeds corresponding to pixel dwell times of 12.8 and 25.6 ␮ sec were found to provide sufficient signal collection at each pixel to produce bright, clear images. In Figs. 6 ͑ C ͒ and 6 ͑ D ͒ , where a dwell time of 12.8 ␮ 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 be- come difficult to identify. The problem with using slow scan speeds, however, is the potential for thermal injury to the tissue. Excitation with 730 nm ͓ Fig. 7 ͑ A ͔͒ produced clear images of cells at faster scan speeds ͑ pixel dwell times of 6.4 to 12.8 ␮ sec per pixel ͒ ; however, the collagen signal was very weak. When using an 800-nm incident beam at these higher scan speeds ͓ Fig. 7 ͑ B ͔͒ , collagen still produced a clean, 28 bright SHG signal, as reported previously. A combination of the TPF channels from the 730-nm excitation image ͓ Fig. 7 ͑ A ͔͒ and the SHG channel from the 800-nm excitation image ͓ Fig. 7 ͑ B ͔͒ is shown in Fig. 7 ͑ C ͒ . Combining separate images obtained from 730- and 800-nm excitation wavelengths provided the sharpest and most detailed image of cellular and collagen composition of the tissue specimen. A number of structures of normal synovium were clearly identified by NLOM imaging. Adipocytes are seen in the NLOM image in Fig. 6 ͑ B ͒ 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 ͑ B ͔͒ are comparable in size and morphology to those demonstrated in HE preparations ͓ Fig. 6 ͑ A ͔͒ . NLOM demonstrated both venules and arterioles in synovium ͓ Figs. 6 ͑ C ͒ –6 ͑ E ͔͒ . 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 ͑ D ͔͒ . On the other hand, arteriolar walls contain a high concentration of elastin, which emits a strong TPF 10 signal. 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 ͑ E ͔͒ . Variable-length, 1 to 2 ␮ m wide, TPF-emitting filamen- 10 tous strands typical of elastin 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 ͑ F ͒ , 8 ͑ A ͒ , and 8 ͑ B ͔͒ . 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 ͒ ͑ a magnified view of the boxed region in Fig. 4 ͒ . Figure 8 ͑ A ͒ demonstrates cells of different morphologies ...

Context 2

... 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-fs pulses at a repetition rate of 80 MHz, with the center frequency tunable from 690 to 1040 nm. Excitation wavelengths of both 730 and 800 nm were used in this study, as they induce an optimal combination of imaging signals from 10,15,28 intracellular compounds and collagen, respectively. While using 800-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 nm is pseudocolored blue, while the channel containing TPF emission in the range 415 to 600 nm is pseudocolored green. The TPF signal was collected over a relatively wide bandwidth ͑ 185 nm ͒ , as the sources of TPF are numerous 10 biomolecules with different emission peaks and distinct TPF spectra within that bandwidth. Excitation at 730 nm elicited a stronger TPF but much weaker SHG signal than was observed with the 800-nm wavelength. The TPF signal collected from the tissue following 730-nm excitation was divided into two channels to enhance contrast using a bandpass filter ͑ 390 to 465 nm ͒ , and using the Meta detector ͑ 470 to 650 nm ͒ . These channels were pseudocolored turquoise ͑ 390 to 465 nm ͒ and orange ͑ 470 to 650 nm ͒ . The turquoise channel contains fluorescence mainly from mito- chondrial NADH, and some other intrinsic fluorophores like folic acid and retinol that have fluorescence peaks within the 390- to 465-nm bandwidth. The orange channel collected fluorescence from riboflavin and other biomolecules with fluorescence peaks beyond 500 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 to 5 mm apart. The imaging region at the periphery was chosen such that it was at least 2 mm from the cuts made during sample extraction. Images 512 ϫ 512 pixels and 1024 ϫ 1024 pixels of tissue areas from 225 ϫ 225 ␮ m to 2.25 ϫ 2.25 mm were obtained using 20 ϫ , 0.45-NA and water immersion 40 ϫ , 0.8-NA objectives. To image an area 1.125 ϫ 1.125 mm or larger, individual adjacent 225 ϫ 225- ␮ m images were acquired sequentially and combined to form the larger image. This technique, known as “tiling,” allows construction of a large, clear geo- graphical image of the synovial tissue ͑ Fig. 4 ͒ . NLOM imaging scan speeds from 6.5 to 102 ␮ sec per pixel were evalu- ated. The combination of a 40 ϫ , water immersion objective ͑ Achroplan, 0.8 NA ͒ delivering 20 mW at the tissue surface and a pixel dwell time of 6.5 ␮ sec provided images with sufficient cellular detail without thermally damaging the tissue, even during the relatively long time ͑ 7 to 15 min ͒ required to acquire a stack of up to 70 images from the same location in successive planes at 1- ␮ m intervals. The mini- mum power required for image acquisition was 18 mW de- livered to the sample with a pixel dwell time of 6.5 s. Be- low that level, the emission signals were judged to be too weak for imaging. Power levels above 34 mW used with a pixel dwell time of 12.8 ␮ s caused tissue damage. 2-D images as well as 3-D tomographic stacks of individual 2-D images obtained at 1- to 2- ␮ m intervals to a depth of up to 70 ␮ m, referred to as Z stacks, were obtained to study tissue morphology in successive image planes within a volume of tissue ͑ Fig. 2 ͒ . An HE stained histology preparation of adipose synovium from the normal knee joint of a rabbit is shown in Figs. 5 ͑ A ͒ and 6 ͑ A ͒ . 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. 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 days following inoculation with S. aureus ͓ Fig. 5 ͑ B ͔͒ 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 adipo- cytes 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 days after LPS inoculation ͓ Fig. 5 ͑ C ͔͒ 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. Figure 4 demonstrates a typical NLOM image of the synovial surface layer using 800-nm excitation and a scan rate of 12.8 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 stor- age that ranged from 3 to 25-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 to 730 nm. This is consistent with NLOM imaging 10,28 of other tissues. When using 800-nm excitation, the use of slow scan speeds corresponding to pixel dwell times of 12.8 and 25.6 ␮ sec were found to provide sufficient signal collection at each pixel to produce bright, clear images. In Figs. 6 ͑ C ͒ and 6 ͑ D ͒ , where a dwell time of 12.8 ␮ 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 be- come difficult to identify. The problem with using slow scan speeds, however, is the potential for thermal injury to the tissue. Excitation with 730 nm ͓ Fig. 7 ͑ A ͔͒ produced clear images of cells at faster scan speeds ͑ pixel dwell times of 6.4 to 12.8 ␮ sec per pixel ͒ ; however, the collagen signal was very weak. When using an 800-nm incident beam at these higher scan speeds ͓ Fig. 7 ͑ B ͔͒ , collagen still produced a clean, 28 bright SHG signal, as reported previously. A combination of the TPF channels from the 730-nm excitation image ͓ Fig. 7 ͑ A ͔͒ and the SHG channel from the 800-nm excitation image ͓ Fig. 7 ͑ B ͔͒ is shown in Fig. 7 ͑ C ͒ . Combining separate images obtained from 730- and 800-nm excitation wavelengths provided the sharpest and most detailed image of cellular and collagen composition of the tissue specimen. A number of structures of normal synovium were clearly identified by NLOM imaging. Adipocytes are seen in the NLOM image in Fig. 6 ͑ B ͒ 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 ͑ B ͔͒ are comparable in size and morphology to those demonstrated in HE preparations ͓ Fig. 6 ͑ A ͔͒ . NLOM demonstrated both venules and arterioles in synovium ͓ Figs. 6 ͑ C ͒ –6 ͑ E ͔͒ . 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 ͑ D ͔͒ . On the other hand, arteriolar walls contain a high concentration of elastin, which emits a strong TPF 10 signal. The overlapping TPF signals emitted both from erythrocytes and the elastin in arteriole walls made it difficult to identify individual ...