Limits...
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.

Show MeSH

Related in: MedlinePlus

Live GFP-mouse mammary gland compared with the identical whole mounted gland. GFP-mouse mammary gland from a 4.5 week old mouse (gland number 3) was prepared as described in Methods and imaged using a Zeiss LD PAPO 25x/0.8 lens (Table 1). A. SHG-B/SHG-F were imaged in A and SHG-B/ BP 500-550/SHG-F were imaged in B. Orthogonal views are compared. In the live tissue XZ view (on top left), blue represents the unfiltered ChD transmitted signal which includes SHG-F (white arrow) and the GFP fluorescence (blue of epithelial cells, dotted arrow). In whole mount tissue (at right), the SHG-F (blue, white arrow) is more intense. The signal from unfiltered ChD also includes the Carmine Alum fluorescence (remaining blue associated with epithelial cells). The XY view illustrates SHG-B (red) of the surface fibrillar layer (Z = 1 and Z = 6, respectively, for live and whole mount). B. Orthogonal views made at different Z-depths illustrate increased information provided by combination of SHG signals (SHG-B, red and ChD unfiltered, including both SHG-F plus GFP in the live and SHG plus Carmine Alum fluorescence in the whole mount). Arrows indicate points of comparison between live and whole mount tissue. Asterisks indicate a fiber-associated vessel. C. The TEB shown in A-B is included in this lower magnification image taken using a Zeiss Neofluar 10x/0.30 lens (asterisk, green GFP, red, SHG-B). D. A kymograph was generated from a line with average 100 pixel width. The line was similarly placed on the image of the live and whole mount TEB and the image merged. SHG-B from the whole mount appears in red, and the SHG-B from the live appears in green. A-B, Scale bar = 50 μm, C, Scale bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750743&req=5

Figure 4: Live GFP-mouse mammary gland compared with the identical whole mounted gland. GFP-mouse mammary gland from a 4.5 week old mouse (gland number 3) was prepared as described in Methods and imaged using a Zeiss LD PAPO 25x/0.8 lens (Table 1). A. SHG-B/SHG-F were imaged in A and SHG-B/ BP 500-550/SHG-F were imaged in B. Orthogonal views are compared. In the live tissue XZ view (on top left), blue represents the unfiltered ChD transmitted signal which includes SHG-F (white arrow) and the GFP fluorescence (blue of epithelial cells, dotted arrow). In whole mount tissue (at right), the SHG-F (blue, white arrow) is more intense. The signal from unfiltered ChD also includes the Carmine Alum fluorescence (remaining blue associated with epithelial cells). The XY view illustrates SHG-B (red) of the surface fibrillar layer (Z = 1 and Z = 6, respectively, for live and whole mount). B. Orthogonal views made at different Z-depths illustrate increased information provided by combination of SHG signals (SHG-B, red and ChD unfiltered, including both SHG-F plus GFP in the live and SHG plus Carmine Alum fluorescence in the whole mount). Arrows indicate points of comparison between live and whole mount tissue. Asterisks indicate a fiber-associated vessel. C. The TEB shown in A-B is included in this lower magnification image taken using a Zeiss Neofluar 10x/0.30 lens (asterisk, green GFP, red, SHG-B). D. A kymograph was generated from a line with average 100 pixel width. The line was similarly placed on the image of the live and whole mount TEB and the image merged. SHG-B from the whole mount appears in red, and the SHG-B from the live appears in green. A-B, Scale bar = 50 μm, C, Scale bar = 100 μm.

Mentions: Stereofluorescence microscopy of live mouse mammary gland, ex vivo. GFP-mice were sacrificed and the 3rd inguinal gland was excised. A a-c. Carmine Alum-stained glands used for multiphoton imaging of normal TEBs in this study (see Figures 4, 8, and 11) were imaged using bright field optics, first with a Nikon SMZ1500 stereofluorescence (A a) and then using a Nikon E600 upright microscope (A b, Nikon 20X/ 0.5 N.A., and c, Nikon 40 X /0.95 N.A.). Scale bars = 50 μm. B a-c. Another mammary gland from a GFP-mouse was imaged using the stereofluorescence microscope. Fat cells, just visible in B a-b at lower magnifications, are viewed surrounding the ductal epithelium obscuring cellular details of the terminal end bud (TEB) in B c.


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

Johnson MD, Mueller SC - BMC Cancer (2013)

Live GFP-mouse mammary gland compared with the identical whole mounted gland. GFP-mouse mammary gland from a 4.5 week old mouse (gland number 3) was prepared as described in Methods and imaged using a Zeiss LD PAPO 25x/0.8 lens (Table 1). A. SHG-B/SHG-F were imaged in A and SHG-B/ BP 500-550/SHG-F were imaged in B. Orthogonal views are compared. In the live tissue XZ view (on top left), blue represents the unfiltered ChD transmitted signal which includes SHG-F (white arrow) and the GFP fluorescence (blue of epithelial cells, dotted arrow). In whole mount tissue (at right), the SHG-F (blue, white arrow) is more intense. The signal from unfiltered ChD also includes the Carmine Alum fluorescence (remaining blue associated with epithelial cells). The XY view illustrates SHG-B (red) of the surface fibrillar layer (Z = 1 and Z = 6, respectively, for live and whole mount). B. Orthogonal views made at different Z-depths illustrate increased information provided by combination of SHG signals (SHG-B, red and ChD unfiltered, including both SHG-F plus GFP in the live and SHG plus Carmine Alum fluorescence in the whole mount). Arrows indicate points of comparison between live and whole mount tissue. Asterisks indicate a fiber-associated vessel. C. The TEB shown in A-B is included in this lower magnification image taken using a Zeiss Neofluar 10x/0.30 lens (asterisk, green GFP, red, SHG-B). D. A kymograph was generated from a line with average 100 pixel width. The line was similarly placed on the image of the live and whole mount TEB and the image merged. SHG-B from the whole mount appears in red, and the SHG-B from the live appears in green. A-B, Scale bar = 50 μm, C, Scale bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Live GFP-mouse mammary gland compared with the identical whole mounted gland. GFP-mouse mammary gland from a 4.5 week old mouse (gland number 3) was prepared as described in Methods and imaged using a Zeiss LD PAPO 25x/0.8 lens (Table 1). A. SHG-B/SHG-F were imaged in A and SHG-B/ BP 500-550/SHG-F were imaged in B. Orthogonal views are compared. In the live tissue XZ view (on top left), blue represents the unfiltered ChD transmitted signal which includes SHG-F (white arrow) and the GFP fluorescence (blue of epithelial cells, dotted arrow). In whole mount tissue (at right), the SHG-F (blue, white arrow) is more intense. The signal from unfiltered ChD also includes the Carmine Alum fluorescence (remaining blue associated with epithelial cells). The XY view illustrates SHG-B (red) of the surface fibrillar layer (Z = 1 and Z = 6, respectively, for live and whole mount). B. Orthogonal views made at different Z-depths illustrate increased information provided by combination of SHG signals (SHG-B, red and ChD unfiltered, including both SHG-F plus GFP in the live and SHG plus Carmine Alum fluorescence in the whole mount). Arrows indicate points of comparison between live and whole mount tissue. Asterisks indicate a fiber-associated vessel. C. The TEB shown in A-B is included in this lower magnification image taken using a Zeiss Neofluar 10x/0.30 lens (asterisk, green GFP, red, SHG-B). D. A kymograph was generated from a line with average 100 pixel width. The line was similarly placed on the image of the live and whole mount TEB and the image merged. SHG-B from the whole mount appears in red, and the SHG-B from the live appears in green. A-B, Scale bar = 50 μm, C, Scale bar = 100 μm.
Mentions: Stereofluorescence microscopy of live mouse mammary gland, ex vivo. GFP-mice were sacrificed and the 3rd inguinal gland was excised. A a-c. Carmine Alum-stained glands used for multiphoton imaging of normal TEBs in this study (see Figures 4, 8, and 11) were imaged using bright field optics, first with a Nikon SMZ1500 stereofluorescence (A a) and then using a Nikon E600 upright microscope (A b, Nikon 20X/ 0.5 N.A., and c, Nikon 40 X /0.95 N.A.). Scale bars = 50 μm. B a-c. Another mammary gland from a GFP-mouse was imaged using the stereofluorescence microscope. Fat cells, just visible in B a-b at lower magnifications, are viewed surrounding the ductal epithelium obscuring cellular details of the terminal end bud (TEB) in B c.

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