The reviewer’s review reviewed.

We have just published a paper in Nature Methods (2006) 3: 35-40 in which we introduce a new caging chromophore to the scientific community. Our first application of the new chromophore is to "Caged Calcium". We have synthesized a photolabile EGTA derivative, and characterized this new probe chemically and biologically (viz. affinity before and after photolysis, rate of Ca release, 2-photon cross section, quantum yield, UV uncaging in muscle fibers, and 2-photon uncaging in droplets and living cells). This is the first paper in this field with such a comprehensive and complete characterization of a new probe. The properties of the chromophore are so superior to others that I regard the work as a landmark contribution to this important field.

Wen-Hong Li wrote an interesting News and Views to accompany our recent paper. In his review he mentioned “Several caged compounds useful for two-photon uncaging applications have been developed recently(refs. 3,5-8), and in some cases, rapid release of effector molecules has been demonstrated (ref.9).”

I feel that the way Dr. Li highlights these other caging chromophores is extremely unhelpful to a general reader of a News and Views. He references five other caged compounds: his ref. 5 Bhc-glu (Proc. Natl. Acad. Sci. USA (1999) 96, 1193-1200); ref. 6 azid-1 (Biophys. J. (1999) 96: 489-499; ref. 7 MNI-glu (J. Neuroscience Meth. (2001) 112: 29-42; fef. 8 Org. Lett. (2002) 4: 3419-3422; and ref. 9 DEACM-sulfate (caged protons) Angew. Chem. (2005) 44: 1195-1198, giving the impression in this context that there are plethora of caging chromophores out there that are also “useful” for 2-photon photolysis, just like NDBF-EGTA. Li does not mention how these are useful, and if one actually looks at the references one will discover why.

Of the 3 cages that have measured 2-photon cross sections (Bhc-glu, azid-1, BQH-Ac), only one has any accompanying 2-photon biology, Bhc-glu. However, the biology reported by Roger Tsien and co-workers using Bhc-glu is “odd” in that the rise-time for the AMPA receptor is reported as 200 milliseconds, rather than the normal 100 microseconds (Note added later in 2006: Sam Wang-who was the postdoc with Denk who did the work at that time- read this entry and told me this was the integrated current over the line scan, not from point uncaging. So I misunderstood this figure in his paper. I am not sure this correction makes the data or Bhc-glu any better?). Thus, it is hard to say that any real 2-photon biology was done using Bhc-glu. Furthermore, the rate of uncaging of Bhc-glu was not discussed, because it had been measured by David Trentham and colleagues in 1993 and is slow (140/s). (We mention this is our Supplementary Table.) Significantly, there has been no report of any 2-photon biology with azid-1 (the only caged calcium in Dr. Li's list, also made by Roger Tsien and co-workers) since its introduction in 1997 as this cage releases calcium too slowly for focal photolysis (rate=500/s). The MNI-glu reference used by Li has no report of any 2-photon measurements whatsoever, and the DEACM-sulfate reports a fast uncaging rate but also has no report of any 2-photon biology. In contrast, we measure the rate of calcium uncaging from NDBF-EGTA, and report a 2-photon cross section and use 2-photon uncaging to induce localised calcium release in cardiac myocytes.

Secondly, Dr. Li states that “further improvements of …its resistance to pH interference should broaden its applications in studying calcium signaling.” We feel this statement suggests that because NDBF-EGTA is based on EGTA it will not really be useful until there is no pH dependence of the calcium affinity. If this were actually the case then the two other cages we have made based on EGTA (NP-EGTA) and EDTA (DM-nitrophen) would and could not have been used in over 200 biology papers! Thus, again Dr. Li states something that a general reader of a News and Views will find misleading.

The status of N&V in the scientific community is very high. We read them so that we can understand work outside our own expertise. Thus, the factual accuracy of a N&V is paramount. We feel that Dr. Li is still reviewing our paper (the detailed comments show he was one of the reviewers of the paper), rather than communicating the great overall strengths of our work to the general reader of Nature Methods. Obviously a Errata for a N&V is ridiculous but I feel this entry goes some way to correcting Li's review.