Methods & Software
I. Single Live Cell Tracking Method
Single Live Cell Tracking
for tracing dynamic properties and detailed genealogy of individual yeast or bacteria cells over many generations
Single cell approach can reveal surprising behaviors that are often hidden in more traditional ensemble measurements. I have developed a long-term single cell imaging technique together with analytical software that can accurately trace dynamic properties and detailed genealogical information of each single cell over many generations. Because of its simplicity and reliability, this method has also been applied to a large scale study in Rhodococcus (up to 96-well culture plate) in Sinskey Lab, to simultaneously monitor the cell metabolism and lipid formation dynamics under different conditions. If you may find this method useful for you, please feel free to email qiongy@mit.edu for more information.
Example 2 : Three videos of tracking single cells dynamically in a growing colony.
I. Experimental setup
"Everything should be made as simple as possible, but not simpler."
I like to search for the simplest solutions to any problems. After different trials, I found the following protocol to be the simplest and most reliable for our purpose.
The play-list below contains three videos that show the automatic segmentation and detailed genealogical tracing of a growing Cyanobacteria family. Two of them have the segmented images overlayed with the phase contrast images.
Just two steps:
Lay a small 2% low melting agarose pad made with the corresponding media on top of 1ul cell culture. --> The pad is critical for keeping the cells grow in two dimension.
Drop the same 2% liquid agarose media on top. --> The 2nd step has three key roles: to keep the pad in a fixed position once solidified; to prevent the pad from drying out up to 1 week; using the same agarose media is to keep the refractive index consistent.
II. Analytical software
This software is based on the marker-controlled watershed algorithm and performs on phase contrast images to segment and track the history(such as genealogy) of each cell. The obtained information will be used to get dynamic properties from other interesting channels (such as GFP,YFP, etc).
Several features:
automatically detect markers and perform segmentation on phase contrast images.
automatically identify cell genealogies/relationships and generate a family tree.
report errors and allow interactive corrections, such as adding or deleting markers by simple clicks, or renaming cells based on its correct history.
Notes:
Interactive correction are only necessary for images with bad quality or with cell morphologies that tend to be over-segmented or under-segmented.
Example 1 : Interactive correction of the automatic segmentation.
Example 3 : Segmentation for cells with special morphology
Very long cells tend to cause over segmentation. In this case, the interactive marker correction is a easy and reliable way to generate correct segmentation.
The above figures:Left: The program automatically detected all makers (green dots), and reported one unexpected marker (in white circle).Middle: User deleted the wrong marker by clicking on it (blue star).Right: User added two correct markers by clicking on the figure (blue star).This last figure is the resulting segmentation based on the corrected markers.
II. Single Molecule FISH Method
Single Molecule FISH
for detecting individual endogenous gene transcripts, applicable to a variety of biological specimens
Examples
The following figure is one example of simultaneously imaging three endogenous gene transcripts in a single A549 cell,
with single molecule resolution:
Fig.A. Merged DIC and DAPI image of a single A549 cell.
Fig.B. Single mRNA molecules of three endogenous genes from the same cell shown in Fig.A.
The individual transcripts are shown as distinct dots in pseudo-colors (COX-2 in red, FLJ1112 in green and FKBP5 in blue).
Fig.C. Zoom in of the yellow squared region in Fig.B.
Description of method
Raj A., van den Bogaard P., Rifkin S., van Oudenaarden A., Tyagi S. Imaging individual mRNA molecules using multiple singly labeled probes. Nature Methods, 2008 5(10) pp. 877-9.