oreste acuto : institut pasteur , france mark m. davis : stanford university , ca christopher c. goodnow : the australian national university , australia claire hivroz : institut national de la sante et de la recherche medicale unite 520 , france bernard malissen : centre d'immunologie inserm - cnrs de marseille - luminy , france anjana rao : harvard medical school , ma michael reth : max planck institute of immunobiology , germany lawrence e. samelson : national cancer institute , national institutes of health , md alexander tarakhovsky : rockefeller university , ny salvatore valitutti : institut national de la sante et de la recherche medicale unite 563 , france arthur weiss : university of california san francisco , ca rose zamoyska : national institute for medical research , uk mark m. davis : if you ask ,  what 's the value of imaging ? one important thing is that it allows us to escape the tyranny of population biochemistry . we 've been forced to indulge in the fantasy that if you have a million cells in a tube , and you hit them with something , that they 're all going to do exactly the same thing at the same time . however , we know that a lot of stochastic processes are going on . if you just grind the cells up at different time points , you 're averaging over all those processes . it 's only when you can see an individual cell that you can see it was stimulated , and then went on a particular pathway . we 're getting much more precise answers to questions like the amount of calcium that 's being released during t cell activation . we can see that one peptide gives you x amount of calcium , and two give you 2x , and so forth ( 3 ) . lawrence e. samelson : with imaging , it 's not just that you 're able to overcome the heterogeneity of the cell population ; you 're able to overcome the heterogeneity of the intracellular molecular populations . many of the molecules that we tag and look at , in terms of intracellular signaling proteins , exist in different pools in the cell : some are cytoplasmic , some are recruited to the membrane , others are associated with the cytoskeleton . it would be impossible to study these different populations without imaging the individual cells and individual molecules . bernard malissen : we are also feeling out the speed at which signaling processes are working , and it 's very fast . we are just putting our finger on the speed at which a t cell is responding to an antigen . but in addition , frap technology allows us to see things that we never imagined , in terms of the turnover and the dynamics of structures that we think are static . because you see an immune synapse , you think everything is just sitting there and immobile with no turnover . mmd : there are a lot of technical challenges involved in asking questions of a detailed molecular nature , of molecules that are within particular cells , at particular times , and in particular places . but there are already some that are specifically designed as indicators of a particular kind of phosphorylation . we need many more of those for all the different signals that might be going on . rose zamoyska : single molecule imaging is coming , and it 's not that far off . that really is going to add another dimension to the way we 're going to be able to analyze signaling events . but we should never forget that biochemistry gives us the fastest way to establish protein bm : ultimately , genetics should allow you to order a pathway without having help of biophysics . if you have powerful genetics , like in the yeast , you can do very nice ordering and epistatic analyses of pathways , and you do n't need imaging . rz : one of the problems with biochemistry is that you can never look at the consequences of the signals , because the cells are lysed . and one of the advantages of imaging is that we 're developing techniques where you 'll be able to look at the differentiation consequences , downstream of particular signals . christopher c. goodnow : whenever i hear the biochemistry angle , i always think about arthur kornberg and how proud he was of dna polymerase in the tube , until the geneticists knocked it out in bacteria , and they replicated just fine . it 's very hard to order pathways and i think the triangulation between biochemistry , genetics , and imaging is going to be essential . you need to know where things are in the cell , and their kinetics and the dynamics , which is what the imaging tells you . you need to know what could happen , which is what the test tube experiment tells you . and then the genetics tells you what is actually needed for a given process . as a community , we do n't put enough emphasis , in our publication and referee processes , on when the genetics and the biochemistry are disconnected . for example , we have a zap-70 catalytic domain point mutant mouse , which has a severe problem with thymic positive selection ( unpublished data ) . but when we look at tyrosine phosphorylation and do old - fashioned calcium analysis , we ca n't see any problem . the question is if you did high speed imaging , would we suddenly be able to connect the genetics with the biochemistry ? michael reth : the immune synapse is a nice example of how difficult it can be to correlate image analysis data with the biochemical behavior of molecules inside a cell . when the synapse was first discovered , everybody thought that one had finally found the molecular organization of signaling proteins at the activated t cell antigen receptor . yet when people later on analyzed synapse formation at different time points and used antiphosphotyrosine antibodies ( 4 ) , it became clear that tcr signaling happens much earlier than synapse formation , and that the synapse may have more to do with the secretion of cytokines than with the initiation of the tcr signal . mmd : absolutely , when we first see things we 're invariably going to misinterpret them . mr : another source of artifacts is the use of gfp - tagged proteins to follow the behavior of molecules in a living cell . this is a powerful tool , but it is important to first test whether the fusion to gfp is not drastically changing the localization or biochemical behavior of the fusion proteins inside the cell . another problem is the use of fluorescent reagents to visualize structures in living cells like lipid rafts . in this respect , it was interesting to see that when fitc - coupled cholera toxin was used to detect raft structures it resulted in raft aggregation ( 5 ) . mr : one problem we have to solve is how to quantify our image analysis . when i see a publication with a beautiful picture of a cell showing the localization of the gfp - tagged protein to a particular membrane structure , i wonder how representative this image is of the behavior of these proteins in the cell population . to overcome the bias generated by our eyes and our brain , we need to apply computer programs for image analysis which , similar to a facscan , can quantify images and order cells according to the image phenotype . you look at a picture in a journal , and you say ,  how do i know this is representative ?  that gets into the question of honesty , and ethics , and basically good science . but programs for quantitation exist ; you can track movement ; you can quantitate velocity ; you can quantitate dynamics . but it is possible to ask authors for quantification , or even to show the originals from which the quantitation was made . alexander tarakhovsky : do you think that there is an absolute necessity to introduce ways to quantify images ? sv : i think it will be in the interests of people doing morphology to somehow enforce measurements of intensity , for instance , at the immune synapse . mmd : the referees always ask us for quantitation : what does the cell population do ? when the referees have been skeptical about our interpretation of the images , they ask for all the images , and we send all the images on a cd . so the vetting is actually easier than it used to be , in the sense that you can transmit vast amounts of information very quickly , so that people can be sure that you 're not being subjective . as to a program that bill gates would sell us to do this i can imagine a very simple kind of software being good for some kinds of imaging analysis , but completely useless for other kinds . claire hivroz : one of the problems that we have to face is that while these new imaging techniques are really nice , they are sources of artifacts , also . we really need to do the analysis carefully . biologists are not physicists , and sometimes we do n't know how to analyze the image , or we do n't know exactly how to use the software . i think we have to make great efforts to work with physicists , to really understand what we are doing because being sure , for example , that what we are looking at by fret is really an interaction between two proteins is not that easy . mr : by using gfp - tagged proteins we can employ imaging to localize proteins to certain subcellular structures , but what is really lacking are more techniques to not only measure the localization , but also the activity of a protein . of course , we can measure the intracellular increase of calcium by fluorescent dyes , but these dyes can not be localized to certain places inside the cell . what we need are more gfp variants that change their fluorescence upon modification , like calcium binding , phosphorylation , or oxidation . with such techniques , you can then see in real time , not only the localization of molecules in the cell , but also at what time point these proteins become active or modified . anjana rao : i 'm dealing with a program of transcriptional regulation that is sensitive to very low intracellular calcium levels  lower than can be measured reliably by the available technology . so i think , in the case of calcium imaging , we 're clearly going to need some new technology in the picture . mmd : there 's still a bag of genetic tricks that hasn't really been applied to imaging but which could be used . there is development of photo - activatable gfp , for example , and temperature - sensitive mutations . so it might be possible to give cells a burst of a laser , for example , to alter the function of molecules that you had modified . at ccg : the strength of genetics is in the specificity of the dissection . but the limitation is that you just have no temporal control or spatial control . the ideal scenario would be to replace genetics with pharmacology . to have small molecules that you can add in to inhibit signaling pathways , which are as specific as genetic manipulations . but , of course , where we get into trouble is in knowing how specific a compound is . this is where the neurobiologists , and the cardiologists , have it over us as immunologists . they 've got a panoply of quite specific agents to dissect processes such as cardiac contractility . at : do you think that genetic manipulation could allow conditional protein inactivation at a given time point ? . the only thing would be some adaptation of photo - inactivatable or temperature - sensitive alleles , which as mark [ mmd ] has pointed out , could allow you to eliminate a wild - type signaling protein both rapidly and in a specific region of a cell . mr : new in vivo imaging studies can follow the dynamic behavior of single lymphocytes in the tissue of a living mouse . this , in combination with genetics , will allow us to understand better the dynamics of cellular interactions inside the immune system . to follow the behavior and activity of single molecules inside a lymphocyte is much more demanding technically , as these are rapid and very dynamic processes . one needs machines with a very fast kinetic image acquisition if one wants to follow these events in real time . furthermore , if one wants to track single fluorescent molecules , one has to use a lot of laser light to detect them , and this could be another potential source of artifacts . laser light not only results in photobleaching , but can also generate reactive oxygen species . as many signaling processes inside the cell are highly sensitive to redox changes , it is possible that shining a lot of laser light on the molecules can change their regulation and behavior . chris [ ccg ] talked about a point mutation which does n't show any biochemical difference . we might not be able to validate that our gfp constructs are behaving normally using simple overexpression systems . i would argue that maybe we can use genetic approaches to test the constructs we use to validate the fact that we 're expressing the right amount of protein , in the right place , at the right time , and that those proteins are regulated properly . ar : in terms of technology development , there are two things that i would focus on . microinjection of mutant proteins into cells that do n't have that protein would be a very good thing to develop . the protein can be tagged in various ways , with small fluorescent probes for instance . think about the work that gunther blobel did , for instance , with the nuclear pore and with nuclear import and export . and then he used em to localize them in the basket of the nuclear pore . the stable immune synapse is such a structure that could benefit very greatly from em analysis . so , without discussing limitations , tell me how do you think in ten years your dreams will be fulfilled ? mr : my dream is a very powerful computer program that is able to generate a virtual cell where we can test our hypothesis of protein signals inside cells . using the primary sequence data , this program should be able to generate the tertiary structure of the protein not only in its frozen ( crystal ) stage , but also its normal dynamic stage in solution . with such a program , one could follow the conformational changes of proteins and better understand the dynamic processes of protein  protein interactions during cell signaling . so that is a dream , but i think one has to wait quite a while for such a program . mmd : i do n't think a computer would be able to know how evolution built a cell . but in my childhood , there was a movie called  fantastic voyage ,  where there was a tiny , little ship that was built , and miniaturized humans were in it . they cruised around the bloodstream , and they looked at the lymphocytes and other things from this point of view . so a good dream would be one where you miniaturize the ship even more , such that you could go into a cell , through a channel or something , and cruise around the cell and watch different bits of the machinery moving up and down , or doing whatever it does . ar : i think you 'd find what schrdinger predicted in 1944 ( 6 ) . i think the whole workings of the cell are governed by probability and stochastic movements that , in turn , are governed by the affinity of interaction . my dream would be to apply the imaging techniques we 're talking about at the membrane , today , to find out what happens in the nucleus . les : mine is another cautionary tale from early twentieth - century physics , and that is the heisenberg uncertainty principle . we have to watch out for what we do to these systems . certainly by manipulating systems we create artifacts . we create a big center and attack very fundamental , basic paradigms in a few systems . it is not strictly necessary to know everything , with everybody working in their own little corner . why not combine genetics , imaging , and biochemistry in a dedicated center where scientists can perform challenging experiments , like physicists do ?
during a recent roundtable discussion , we captured some personal perspectives on the new insight that advanced imaging techniques promise to bring to the study of lymphocyte signaling . 
 the experts present their views on the power of imaging , the problems that need to be overcome , and the potential of the technology .