National Institute of Standards and Technology

3D Cell-Scaffold Contact Detection

Summary

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.

bio overview.
Biological motivation behind the cell-scaffold contact measurements.

Description

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.

A pair of cell (left) and scaffold (right) z-stacks for the microfiber scaffold type.
A pair of cell (left) and scaffold (right) z-stacks for the microfiber scaffold type.

Cell and scaffold z-stack pairs for the three types of scaffolds (spun coat, microfibers, medium microfibers).
Cell and scaffold z-stack pairs for the three types of scaffolds (spun coat, microfibers, medium microfibers).

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.

Overview
Overview of the measurement methodology for characterization of cell-scaffold contacts. The three components, modeling, validation and verification, are color-coded. The abbreviations “Stats” refers to statistical, “Geom” to geometrical, and “ROI” to region of interest.

Online visual verification of 3D segmentation and cell-scaffold contacts

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.

Major Accomplishments

Data:

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.

Publication:

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.

Contact:

Peter Bajcsy
peter.bajcsy@nist.gov
Phone: 301.975.2958

Carl Simon Jr.
carl.simon@nist.gov
Phone: 301.975.8574

Date created: October 5, 2017 | Last updated: