Cell-scaffold contact measurements are derived from pairs of co-registered volumetric fluorescent confocal laser scanning microscopy (CLSM) images (z-stacks) of stained cells and three types of scaffolds (i.e., spun coat, large microfiber, and medium microfiber). Our analysis of the acquired terabyte-sized collection is motivated by the need to understand the nature of the shape dimensionality (1D vs 2D vs 3D) of cell-scaffold interactions relevant to tissue engineers that grow cells on biomaterial scaffolds. This is illustrated in the figure below.
The data acquisition focuses on the measurements establishing the effect of scaffold types on cell morphology and on cell behavior. The data sets are acquired by CLSM as images (z-stacks) of cells cultured on three different scaffolds. The three scaffolds are described as follows:
| Scaffold Name and Abbreviation | Scaffold Material Description |
|---|---|
| Spun Coat (SC) | Flat films of spun-coat Poly lactic-co-glycolic acid (PLGA) |
| Large Microfibers (MF) | Electrospun PLGA microfibers (diameter equal to 2.6 µm) |
| Medium Microfibers (MMF) | Electrospun PLGA microfibers (diameter equal to 1.1 µm) |
A z-stack with two channels was collected for each of 711 cells. The two channels corresponded to cell membrane (Oregon-Green - excitation 488 nm, emission 501 nm to 570 nm) and fiber scaffold (Flammafluor648 - excitation 633 nm, emission 652 nm to 708 nm). Each z-frame in the z-stacks was exported as an 8 MB tif image with a resolution of 2048 pixels x 2048 pixels (246 µm x 246 µm) and 16 bits per pixel. Examples of z-frame tif images are shown in below.
We designed five statistical and three geometrical contact models, and then down-selected them to one from each category using a validation approach based on physically orthogonal measurements to CLSM. The two selected models were applied to 414 z-stacks with three scaffold types and all contact results were visually verified. The overview of the measurement methodology is shown in the figure below.
We built a web-based visual inspection system for inspecting the quality of 3D segmentation and cell-scaffold contact locations.
This tool is accessible online by clicking on the following link:
Online visualization verification.
The raw z-stacks and volumetric representations of segmentation results and cell-scaffold contact points are accessible from the following link:
Download web page for raw and processed z-stacks.
Publications:
Peter Bajcsy, Soweon Yoon, Stephen Florczyk, Nathan Hotaling, Mylene Simon, Piotr Szczypinski, Nicholas Schaub, Carl Simon Jr., Mary Brady, and Ram Sriram,
Modeling, Validation and Verification of Three-Dimensional Cell-Scaffold Contacts from Terabyte-sized Images,
BMC Bioinformatics, November, 2017; DOI 10.1186/s12859-017-1928-x.
Stephanie Florczyk; Nathan Hotaling, Mylene Simon, Joe Chalfoun, Allison Horenberg, Nicholas Schaub, Dongbo Wang, Piotr Szczypiński, Veronica Defelice, Peter Bajcsy, and Carl Simon,
Measuring Dimensionality of Cell-Scaffold Contacts of Primary Human Bone Marrow Stromal Cells Cultured on Electrospun Fiber Scaffolds,
Journal of Biomedical Materials Research Part A 111(1), 106-117, Oct 4th, 2022, http://doi.org/10.1002/jbm.a.37449
Peter Bajcsy
peter.bajcsy@nist.gov
Phone: 301.975.2958
Carl Simon Jr.
carl.simon@nist.gov
Phone: 301.975.8574