[Cytometry] Compensation, Cy5PE, and APC

Howard Shapiro hms at shapirolab.com
Sun Feb 9 12:13:49 EST 2014

Mario wrote- 

> I'd like to correct a few things here.

So would I.

> First, the Calibur was not "way ahead of its time when it came to time delay exclusion." This was already being done in the early 1970s with the first two laser machines in the Herzenberg lab, and all of the predecessors to the Calibur (FACS II, FACS IV, etc)  did this as well.

In the *early* 1970s? 

The laser-based FACS was first described in March, 1972 (Bonner, Hulett, Sweet, & Herzenberg, Rev Sci Inst 43:404); the first immunofluorescence results were published in July, 1972 (Julius, Masuda & Herzenberg, PNAS 69:1934). An August, 1973 paper (Hulett, Bonner, Sweet, & Herzenberg, Clin Chem 19:813) described a two-laser instrument; a 633 nm He-Ne beam detected scatter (forward only; Los Alamos didn’t come up with side scatter until 1974 or 1975), and the more powerful 488 nm beam downstream could be used to excite fluorescence at two wavelengths.  

Delay exclusion would not have been an issue in 1973. The first description of *two-color* immunofluorescence from Stanford (Loken, Parks, & Herzenberg, J Histochem Cytochem 25:899) appeared in July, 1977. I don’t think anybody else published earlier.  The instrument used only one laser. The 515 nm argon line was used to excite both fluorescein- and rhodamine-labeled antibodies (polyclonal; usable monoclonals were a few years in the future), necessitating that the authors *invent* compensation, which was done in analog electronics (log amps were not used, making the process relatively straightforward).

Tomas Hirschfeld at Block had worked out the details of both analog and digital compensation by then, but, since the Block flow cytometers had several fluorescence excitation beams from 1974 on, it would have been unnecessary to use compensation with them. It was definitely necessary to keep track of signals from multiple beams; the electronics ahead of Block's dedicated computer could track 33,000 cells/second when the first and last beams were separated by 1.6 centimeters! But Bernie Shoor and Mack Fulwyler, then running BD’s FACS program, told Block that nobody would ever need three beams or four colors. The Herzenbergs’ early recognition of what could be done with monoclonals was almost certainly even more important for cytometry than was the initial development and commercialization of sorting. 

These days, however, there do seem to be times when we get to the “Fifty-Seven Channels and Nothin' On” stage.


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