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Three dimensional multiphoton imaging of fresh and whole mount developing mouse mammary glands.

Johnson MD, Mueller SC - BMC Cancer (2013)

Bottom Line: At the margins, TEBs approach the outer collagen layer but do not penetrate it.Collagen fibril arrangement and TEB structure is well preserved during the whole mount procedure and light scattering is reduced dramatically by extracting fat resulting in improved 3D structure, particularly for SHG signals originating from collagen.Our studies demonstrated that the TEB architecture is essentially unchanged after processing.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: The applications of multiphoton microscopy for deep tissue imaging in basic and clinical research are ever increasing, supplementing confocal imaging of the surface layers of cells in tissue. However, imaging living tissue is made difficult by the light scattering properties of the tissue, and this is extraordinarily apparent in the mouse mammary gland which contains a stroma filled with fat cells surrounding the ductal epithelium. Whole mount mammary glands stained with Carmine Alum are easily archived for later reference and readily viewed using bright field microscopy to observe branching architecture of the ductal network. Here, we report on the advantages of multiphoton imaging of whole mount mammary glands. Chief among them is that optical sectioning of the terminal end bud (TEB) and ductal epithelium allows the appreciation of abnormalities in structure that are very difficult to ascertain using either bright field imaging of the stained gland or the conventional approach of hematoxylin and eosin staining of fixed and paraffin-embedded sections. A second advantage is the detail afforded by second harmonic generation (SHG) in which collagen fiber orientation and abundance can be observed.

Methods: GFP-mouse mammary glands were imaged live or after whole mount preparation using a Zeiss LSM510/META/NLO multiphoton microscope with the purpose of obtaining high resolution images with 3D content, and evaluating any structural alterations induced by whole mount preparation. We describe a simple means for using a commercial confocal/ multiphoton microscope equipped with a Ti-Sapphire laser to simultaneously image Carmine Alum fluorescence and collagen fiber networks by SHG with laser excitation set to 860 nm. Identical terminal end buds (TEBs) were compared before and after fixation, staining, and whole mount preparation and structure of collagen networks and TEB morphologies were determined. Flexibility in excitation and emission filters was explored using the META detector for spectral emission scanning. Backward scattered or reflected SHG (SHG-B) was detected using a conventional confocal detector with maximum aperture and forward scattered or transmitted SHG (SHG-F) detected using a non-descanned detector.

Results: We show here that the developing mammary gland is encased in a thin but dense layer of collagen fibers. Sparse collagen layers are also interspersed between stromal layers of fat cells surrounding TEBs. At the margins, TEBs approach the outer collagen layer but do not penetrate it. Abnormal mammary glands from an HAI-1 transgenic FVB mouse model were found to contain TEBs with abnormal pockets of cells forming extra lumens and zones of continuous lateral bud formation interspersed with sparse collagen fibers.

Conclusions: Collagen fibril arrangement and TEB structure is well preserved during the whole mount procedure and light scattering is reduced dramatically by extracting fat resulting in improved 3D structure, particularly for SHG signals originating from collagen. In addition to providing a bright signal, Carmine Alum stained whole mount slides can be imaged retrospectively such as performed for the HAI-1 mouse gland revealing new aspects of abnormal TEB morphology. These studies demonstrated the intimate contact, but relatively sparse abundance of collagen fibrils adjacent to normal and abnormal TEBS in the developing mammary gland and the ability to obtain these high resolution details subject to the discussed limitations. Our studies demonstrated that the TEB architecture is essentially unchanged after processing.

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Imaging abnormal features of developing mammary glands using optimal SHG-B/Carmine Alum/SHG-F approach. A. A three color, single slice (XY) from a Z-stack of images of 46 μm thickness is presented from a Carmine Alum whole mount (HAI-1 mouse gland). B. The three-color, 3D projection is shown (3D) as well as orthogonal views which were acquired separately for just the SHG-B and SHG-F signals using a narrower bandwidth in the ChS (META) detector for SHG-B. Arrows in the lower magnification upper panel indicate differences between the XY and 3D views of the lateral branches, which otherwise look similar indicating that they are mainly viewed in a single XY orientation relative to the stage. C. A higher magnification view of the boxed inset region in A (XY) reveals details of Carmine Alum staining and SHG-B and SHG-F. The fibers visualized using SHG are concentrated near the duct and lateral buds (asterisks in A, B and C). The dotted arrows shown in C indicate fibers (SHG-F) between and around the lateral buds. D. A higher magnification view of the boxed inset region in B reveals the 3D view of Carmine Alum staining with SHG-B and SHG-F. E. Orthogonal views including XY, XZ, and YZ reveal two lateral buds viewed as two color image of SHG-B and SHG-F (E). F. A conventional bright field image of a similar duct taken using a Nikon upright microscope equipped with a 10X/ 0.3 N. A. Nikon lens. Scale bars = 50 μm in A (top panel) and 20 μm elsewhere except F, 100 μm.
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Figure 7: Imaging abnormal features of developing mammary glands using optimal SHG-B/Carmine Alum/SHG-F approach. A. A three color, single slice (XY) from a Z-stack of images of 46 μm thickness is presented from a Carmine Alum whole mount (HAI-1 mouse gland). B. The three-color, 3D projection is shown (3D) as well as orthogonal views which were acquired separately for just the SHG-B and SHG-F signals using a narrower bandwidth in the ChS (META) detector for SHG-B. Arrows in the lower magnification upper panel indicate differences between the XY and 3D views of the lateral branches, which otherwise look similar indicating that they are mainly viewed in a single XY orientation relative to the stage. C. A higher magnification view of the boxed inset region in A (XY) reveals details of Carmine Alum staining and SHG-B and SHG-F. The fibers visualized using SHG are concentrated near the duct and lateral buds (asterisks in A, B and C). The dotted arrows shown in C indicate fibers (SHG-F) between and around the lateral buds. D. A higher magnification view of the boxed inset region in B reveals the 3D view of Carmine Alum staining with SHG-B and SHG-F. E. Orthogonal views including XY, XZ, and YZ reveal two lateral buds viewed as two color image of SHG-B and SHG-F (E). F. A conventional bright field image of a similar duct taken using a Nikon upright microscope equipped with a 10X/ 0.3 N. A. Nikon lens. Scale bars = 50 μm in A (top panel) and 20 μm elsewhere except F, 100 μm.

Mentions: Summary of imaging parameters (Zeiss LSM510/META/NLO specifications)


Three dimensional multiphoton imaging of fresh and whole mount developing mouse mammary glands.

Johnson MD, Mueller SC - BMC Cancer (2013)

Imaging abnormal features of developing mammary glands using optimal SHG-B/Carmine Alum/SHG-F approach. A. A three color, single slice (XY) from a Z-stack of images of 46 μm thickness is presented from a Carmine Alum whole mount (HAI-1 mouse gland). B. The three-color, 3D projection is shown (3D) as well as orthogonal views which were acquired separately for just the SHG-B and SHG-F signals using a narrower bandwidth in the ChS (META) detector for SHG-B. Arrows in the lower magnification upper panel indicate differences between the XY and 3D views of the lateral branches, which otherwise look similar indicating that they are mainly viewed in a single XY orientation relative to the stage. C. A higher magnification view of the boxed inset region in A (XY) reveals details of Carmine Alum staining and SHG-B and SHG-F. The fibers visualized using SHG are concentrated near the duct and lateral buds (asterisks in A, B and C). The dotted arrows shown in C indicate fibers (SHG-F) between and around the lateral buds. D. A higher magnification view of the boxed inset region in B reveals the 3D view of Carmine Alum staining with SHG-B and SHG-F. E. Orthogonal views including XY, XZ, and YZ reveal two lateral buds viewed as two color image of SHG-B and SHG-F (E). F. A conventional bright field image of a similar duct taken using a Nikon upright microscope equipped with a 10X/ 0.3 N. A. Nikon lens. Scale bars = 50 μm in A (top panel) and 20 μm elsewhere except F, 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750743&req=5

Figure 7: Imaging abnormal features of developing mammary glands using optimal SHG-B/Carmine Alum/SHG-F approach. A. A three color, single slice (XY) from a Z-stack of images of 46 μm thickness is presented from a Carmine Alum whole mount (HAI-1 mouse gland). B. The three-color, 3D projection is shown (3D) as well as orthogonal views which were acquired separately for just the SHG-B and SHG-F signals using a narrower bandwidth in the ChS (META) detector for SHG-B. Arrows in the lower magnification upper panel indicate differences between the XY and 3D views of the lateral branches, which otherwise look similar indicating that they are mainly viewed in a single XY orientation relative to the stage. C. A higher magnification view of the boxed inset region in A (XY) reveals details of Carmine Alum staining and SHG-B and SHG-F. The fibers visualized using SHG are concentrated near the duct and lateral buds (asterisks in A, B and C). The dotted arrows shown in C indicate fibers (SHG-F) between and around the lateral buds. D. A higher magnification view of the boxed inset region in B reveals the 3D view of Carmine Alum staining with SHG-B and SHG-F. E. Orthogonal views including XY, XZ, and YZ reveal two lateral buds viewed as two color image of SHG-B and SHG-F (E). F. A conventional bright field image of a similar duct taken using a Nikon upright microscope equipped with a 10X/ 0.3 N. A. Nikon lens. Scale bars = 50 μm in A (top panel) and 20 μm elsewhere except F, 100 μm.
Mentions: Summary of imaging parameters (Zeiss LSM510/META/NLO specifications)

Bottom Line: At the margins, TEBs approach the outer collagen layer but do not penetrate it.Collagen fibril arrangement and TEB structure is well preserved during the whole mount procedure and light scattering is reduced dramatically by extracting fat resulting in improved 3D structure, particularly for SHG signals originating from collagen.Our studies demonstrated that the TEB architecture is essentially unchanged after processing.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: The applications of multiphoton microscopy for deep tissue imaging in basic and clinical research are ever increasing, supplementing confocal imaging of the surface layers of cells in tissue. However, imaging living tissue is made difficult by the light scattering properties of the tissue, and this is extraordinarily apparent in the mouse mammary gland which contains a stroma filled with fat cells surrounding the ductal epithelium. Whole mount mammary glands stained with Carmine Alum are easily archived for later reference and readily viewed using bright field microscopy to observe branching architecture of the ductal network. Here, we report on the advantages of multiphoton imaging of whole mount mammary glands. Chief among them is that optical sectioning of the terminal end bud (TEB) and ductal epithelium allows the appreciation of abnormalities in structure that are very difficult to ascertain using either bright field imaging of the stained gland or the conventional approach of hematoxylin and eosin staining of fixed and paraffin-embedded sections. A second advantage is the detail afforded by second harmonic generation (SHG) in which collagen fiber orientation and abundance can be observed.

Methods: GFP-mouse mammary glands were imaged live or after whole mount preparation using a Zeiss LSM510/META/NLO multiphoton microscope with the purpose of obtaining high resolution images with 3D content, and evaluating any structural alterations induced by whole mount preparation. We describe a simple means for using a commercial confocal/ multiphoton microscope equipped with a Ti-Sapphire laser to simultaneously image Carmine Alum fluorescence and collagen fiber networks by SHG with laser excitation set to 860 nm. Identical terminal end buds (TEBs) were compared before and after fixation, staining, and whole mount preparation and structure of collagen networks and TEB morphologies were determined. Flexibility in excitation and emission filters was explored using the META detector for spectral emission scanning. Backward scattered or reflected SHG (SHG-B) was detected using a conventional confocal detector with maximum aperture and forward scattered or transmitted SHG (SHG-F) detected using a non-descanned detector.

Results: We show here that the developing mammary gland is encased in a thin but dense layer of collagen fibers. Sparse collagen layers are also interspersed between stromal layers of fat cells surrounding TEBs. At the margins, TEBs approach the outer collagen layer but do not penetrate it. Abnormal mammary glands from an HAI-1 transgenic FVB mouse model were found to contain TEBs with abnormal pockets of cells forming extra lumens and zones of continuous lateral bud formation interspersed with sparse collagen fibers.

Conclusions: Collagen fibril arrangement and TEB structure is well preserved during the whole mount procedure and light scattering is reduced dramatically by extracting fat resulting in improved 3D structure, particularly for SHG signals originating from collagen. In addition to providing a bright signal, Carmine Alum stained whole mount slides can be imaged retrospectively such as performed for the HAI-1 mouse gland revealing new aspects of abnormal TEB morphology. These studies demonstrated the intimate contact, but relatively sparse abundance of collagen fibrils adjacent to normal and abnormal TEBS in the developing mammary gland and the ability to obtain these high resolution details subject to the discussed limitations. Our studies demonstrated that the TEB architecture is essentially unchanged after processing.

Show MeSH
Related in: MedlinePlus