Framing next-generation imaging questions: Outstanding problems that must be addressed if we are to understand cell biology

摘要

It is clear that the enormous variety of imaging technologies particularly those directed towards single cells have transformed our understanding of how living cells function. It is also evident that this field of study has matured to the extent that bioimaging is itself almost a self-standing discipline. There remains, however, quite profound problems both in terms of the imaging technologies themselves and also those that are presented to us by cell biology. We must become more aware that imaging protocols may modify the processes we are observing, there is growing body of evidence for example, that large photon dosages associated with eg stimulated emission depletion imaging or PALM/STORM imaging to a region of a single living cells can lead to cellular damage or modification of molecular behavior in the vicinity of the laser irradiation. This potential problem is related to both high intensity and long exposure times. Similarly, some techniques that operate towards the edge of present detection sensitivities (eg single molecule techniques) requiring extended illumination times necessary to acquire an image are also subject to some concerns that the measurement is modifying the process under scrutiny. Another problem that is becoming the subject of greater awareness is that the attachment of a photoprotein to a protein of interest (PoI) may lead to modified behaviour of the PoI or even other protein that are affected by the augmented exposure of the photoprotein-PoI construct. It should be emphasised, however, that these foregoing comments in no way represent any criticism of the profound contribution the originators of these techniques have made, they are made simply to allow us to formulate a ‘wish-list’ for our next generation imaging needs.