Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.
Part 1: Experimental Procedure
Video data contain an abundance of information. However, this information is often difficult to extract and when done manually by humans and may require many hours of personnel time to complete. Manual analysis by humans is also subject to variation in interpretation and error. Video bioinformatics involves the use of computer software to mine specific data from video images. This method of analysis is rapid and can eliminate error that occurs when analysis is done manually by humans. The purpose of this article is to demonstrate a method for quantifying human embryonic stem cell colony growth using a video bioinformatics method.
In this paper, time lapse video images were collected using a Nikon BioStation CT incubation unit that allows multiple fields of cells to be imaged over time (Fig. 1). The methods described in this report are applicable to video data collected by any video microscopic set up.
In our experimental design, we plated H9 hESC in 12-well tissue culture plates for 48 hours. During this interval, colonies were allowed to fully attach and spread on Matrigel. Then, plates with attached hESC colonies were transferred to the BioStation CT and incubated for an additional 48 hours. While in the BioStation, images of the colonies were collected at 7 minute intervals and were later used to create time-lapse video sequences. Videos for each colony were then analyzed to quantify colony growth using video bioinformatics recipes developed with the CL-Quant software. The analyses done using recipes created with the CL-Quant software were checked for truthfulness manually using Adobe Photoshop.
Part 2: Preparation of attached hESC colonies
Part 3: Video Bioinformatics
CL-Quant software was used to create three “recipes” that when run in sequence will determine the pixel number or area in microns for each colony over time. The three recipes are built in sequence and for this application include segmentation, enhancement, and measurement.
Analyzing hESC colony growth using the CL-Quant software/recipe development:
Part 4: Checking recipe accuracy using the Photoshop software
In order to validate the recipe created with (CL-Quant software), the same data can be analyzed manually with Adobe Photoshop. For this analysis, every 10th frame (every 70 min time point) was analyzed to measure colony size over 48 hours.
Representative Results
Figure 1: Film strip of a hESC colony showing frames at various times during 48 hours of growth in a BioStation CT. Frames were taken at 7 minute intervals.
Figure 2: (A) Image of a hESC with a mask placed on the colony by the segmentation recipe. Areas of debris and background are also masked indicating that an enhancement recipe is needed to improve the accuracy of the evaluation. (B) Image of the same colony as shown in “A” after enhancement has been performed. The mask now selects only the colony and noise has been eliminated from the selection.
Figure 3: Graph showing increase in colony size (in pixels) over 48 hours as determined using CL-Quant software and Photoshop. This graph plots the raw data and shows that colonies start at different sizes. It also shows that both methods of measurement are in good agreement.
Figure 4: Graph showing the same data as in Figure 3 after normalization to show the percent increase in colony size over 48 hours. This shows that growth rates are similar regardless of starting colony size and that both measures of analysis give similar results.
Figure 5: Graph showing the means for the normalized data in Figure 4. This graph clearly shows good agreement between the analyses done using video bioinformatics (CL-Quant software) and Photoshop and establishes that the recipe is truthful. The slight downturn in area for the Photoshop data at frame 325 is due to several videos not being included in the Photoshop analysis.
Video bioinformatics tools are powerful for rapidly extracting data from video images. Our protocol for quantifying hESC colony growth demonstrates one application of video bioinformatics to a biological problem. This method is quantitative and has the interesting feature of revealing data from individual hESC colonies. Video bioinformatics recipes can be developed to monitor other cellular processes such as proliferation, migration, apoptosis, and cell attachment to the substrate, and cell attachment to adjacent cells. The accuracy of the recipes developed using CL-Quant software was validated using Photoshop and was found to be truthful. Once the appropriate recipes are developed, video data from any source can be analyzed very quickly. The time required to analyze video data using video bioinformatics tools is significantly less than the time needed for analysis performed by hand using Photoshop. Moreover, the analysis done by the computer is less prone to error, as the computer will analyze the data the same way each time, while a human performing an analysis may make errors or slightly different judgments each time an image is analyzed. Although not discussed as part of this protocol, videos can also be examined for morphological changes in the colony. This parameter would be useful in cases where treatment groups are included.
The development of this method was made possible by funding from the California Tobacco-Related Disease Research Program, the California Institute for Regenerative Medicine, and NSF IGERT grant on Video Bioinformatics (#0903667) at UCR (http://www.cris.ucr.edu/IGERT/index.php). Sabrina Lin is supported by a Dissertation Fellowship from the Graduate Division, Shawn Forteno is supported by an NIH MARC Fellowship, and Shruthi Satish is supported by a Graduate Division Fellowship. We are grateful to Anna Trtchounian for her help preparing the figures. We also thank Sam Alworth and Ned Jastromb for teaching us how to use the CL-Quant software.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
mTeSR1 Human Embryonic Stem Cell Maintenance Medium | Stem Cell Technologies | 05850 | or any suitable medium for hESC culture. | |
BD Matrigel | BD Bioscience | 356234 | or other suitable substrate. | |
DMEM/F12 Basal Medium | Invitrogen | 11330-032 | ||
Phosphate Buffered Saline without Ca2+ and Mg2+ | ||||
Accutase Enzyme Cell Detachment Medium | eBioscience | 00-4555-56 | or other suitable detachment enzyme. | |
3mm Glass beads | Fisher Scientific | 11-312A | optional. | |
12-well Tissue Culture Plates | BD Falcon | 353043 | or any other plate format. | |
Nikon BioStation CT/IM | or other incubator/microscope suitable for collecting video data. | |||
CL-Quant software (Nikon) and/or Photoshop (Adobe). |