A confocal microscope (Zeiss LSM 710, Oberkochen, Germany) was used to image the cervices. A femtosecond laser (MaiTai DeepSee, eHP, Spectraphysics, Newport Corporation, CA, USA) producing 70-fs centered at 780 nm was used to illuminate the sample, and a 20x 0.8 NA objective lens and motorized stage were used to image the entire cross-section of the tissue. Images were then automatically stitched in Zen software (Zeiss, Oberkochen, Germany). Imaging the entire cross-section allowed navigation of the anatomical regions for 3D imaging. A 40x 1.2 NA water-immersion objective acquired three volumetric images (212x212x50 μm) from each of the outer zone, inner zone, and septum (n=9 images from each cervix), scanning selected regions with a 330-500 nm step size (50 μm total depth). Each volumetric image was divided into 4 equal sub-images (50x50x50 μm) to better capture the heterogeneity of the samples. Together, 36 data points were collected from each cervix, resulting in 324 total measurements from cervical regions at different stages of pregnancy. Samples were placed on a No. 1.5 cover glass-bottom dish (MatTek, Ashland, MA) with PBS buffer solution to keep samples hydrated during imaging.
No 1.5 cover glass bottom dish
Manufactured by MatTek
The No. 1.5 cover glass-bottom dish is a laboratory equipment item designed for cell culture and microscopy applications. It features a glass bottom that provides a clear surface for observation and imaging.
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Lab products found in correlation
2 protocols using no 1.5 cover glass bottom dish
1
Cervical Tissue Imaging by Confocal Microscopy
2
Cervix Tissue Imaging with SHG Microscopy
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A custom SHG microscope imaged the cervix samples. A Ti:Sapphire laser illuminated the samples producing 100-fs centered at 780 nm. A 10× 0.25 NA objective lens on an inverted microscope (Olympus, Tokyo, Japan) collected the SHG signal for the 2D images. To construct an image of the whole cross-section of the cervix, a motorized stage scanned the field of view (535 μm × 535 μm) with a step size of 500 μm and the acquired images were stitched together. In addition to the SHG images, a set of brightfield images of the whole cross-section were acquired for co-registration. 3D image stacks were obtained in three locations in the ring, three locations in the near-septum, and one location in the septum. We used a 60× 1.0 NA water immersion objective lens to collect the 3D-SHG images and the z step size was 350 nm. The samples were placed on a No. 1.5 cover glass-bottom dish (MatTek, Ashland, MA) with buffer solution to remain hydrated during imaging.
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