Outer edge to center intensity measurement of irregular 2D objects. For annulus method as described on the web in 2004, click here.

Description:

In 1998 and 1999 two papers were published that included a novel image analysis method for fluorescent microscopy of cultured cells developed as a NIH-Image macro set (later converted to ImageJ). The software measured from the perimeter of a closed 2D shape towards the interior using thin bands referred to as the annulus method. The power of this was that the shape could be irregular. chan1998.pdf and bailly1999.pdf In addition, subsequent papers relied on this software, at least one with the added functionality of Pearson's correlation for quantification of colocalization in each annulus.

The second paper, Bailly 1999 is about the arp2/3 complex and its role in f-actin protrusions and measurements relied on the annulus method. The biology being interrogated was f-actin remodeling at the outer edge of a cell plated on a coverlip, in this case f-actin network expansion reliant on the arp2/3 complex.

Sixteen years later Barry et al publish a paper in which they describe a novel annulus method and use it to describe f-actin remodeling relying on the arp2/3 complex. They added functionality of measuring through time (although, possibly, at the expense of precision spatial resolution at the leading edge). Sadly, they did not cite the earlier work.

It was common practice of the Analytical Imaging Facility at the Albert Einstein College of Medicine to provide both the macro sets and training for proper use from the late 1990s through September 2008 (perhaps later; I took a new position elsewhere in September 2008). The macros resided on the public website for at least one more year. The macros also resided, and continue to reside, on a password protected web site and were made available to requestors through 2015. In 2015, following Barry 2015, the macros and explanation of development and testing were copied to a public website.

But there appears to be more demand for radial or concentric from center out measurements.

Bibliography

Barry DJ, Durkin CH, Abella JV, Way M. Open source software for quantification of cell migration, protrusions, and fluorescence intensities. J Cell Biol. 2015 Apr 13;209(1):163-80. doi: 10.1083/jcb.201501081. Epub 2015 Apr 6. PMID: 25847537

Bailly M, Macaluso F, Cammer M, Chan A, Segall JE, Condeelis JS. Relationship between Arp2/3 complex and the barbed ends of actin filaments at the leading edge of carcinoma cells after epidermal growth factor stimulation. J Cell Biol. 1999 Apr 19;145(2):331-45. PMID: 10209028

Chan AY, Raft S, Bailly M, Wyckoff JB, Segall JE, Condeelis JS. EGF stimulates an increase in actin nucleation and filament number at the leading edge of the lamellipod in mammary adenocarcinoma cells. J Cell Sci. 1998 Jan;111 ( Pt 2):199-211. PMID: 9405304

Personal note:
I must acknowledge the then contributors of the NIH-Image listserv and the author of NIH-Image, Wayne Rasband, for their invaluable help developing the methods and code. Wayne Rasband deserves further acknowledgement for including in ImageJ many of the functions we needed to rewrite and further develop the methods, thus making our task much faster and robust and extending to other researchers a larger library of excellent image analysis routines.

Website by Michael Cammer, mcammer@gmail.com. Last updated 3 July 2018.

Earlier personal email to a colleague in 2015:

Packaging various analytical methods for general use is laudable.  However, origins of the methods need to be noted.

Two papers which defined the methods in Barry’s Figure 4 C & E are completely absent in his citations. 

Figure 1 of Bailly, 1999 (1) is essentially Figure 4E Barry’s paper except that we chose to represent the profile from the lamellipod in towards the center without the complete intensity profile and to report results in absolute distance, not normalized along radii completing the path to the center.  This was essential both to correlate with TEM the location and function of Arp2/3 and f-actin and to simultaneously confirm the method of analysis.

The method was previously published in Chan 1998 in which Figure 5 is clearly a model for Barry’s Figure 4C.  In fact, the method we initially coded was an iterative erode as Barry implements.  At the time NIH-Image did not have a function to measure bands, pixels along an edge, or recast a perimeter as a segmented line, so the major part of the coding was managing masking to accurately report intensities at this edge.  The method was coded by Michael Cammer (with invaluable consultation of the NIH-Image listserv and Wayne Rasband) as macros within NIH-Image to meet specifications defined by John Condeelis.

Once these papers were published the methods, and additional variations, were available to anybody who asked.  Within Einstein College of Medicine researchers using the Analytical Imaging Facility were encouraged to adopt this method and variations.  The code was also publicly available on the facility website. 

Barry’s paper focuses on both f-actin and Arp2/3 as a validation for his methods.  Given that these papers defined the method both to measure f-actin and Arp2/3, it is difficult to understand the complete omission from Barry’s paper .