The field will never be the same

A report of the use of the OMX microscope in high-speed live-cell imaging just came out in PNAS (Carlton et al. 2010 Fast live simultaneous multiwavelength four-dimensional optical microscopy.  Proc. Natl. Acad Sci. USA 107 16016-16022 PMID: 20705899).  In an accompanying commentary, Jason Swedlow offers the opinion that the field of live cell imaging will never be the same.

The OMX microscope made a big splash a couple of years ago by showing that one can go beyond the apparent physical limits of light microscopy, improving resolution beyond the 200-nm diffraction limit, by using a structured light source.  [A second method of breaking the diffraction limit, using focused spots of light, came out at almost the same time.]  By using multiple interfering beams of light, Sedat and co-workers were able to double the resolution of normal microscopy, and observe objects such as the nuclear pore in fixed cells.  Now Carlton et al. report a revamped and extensively tuned version of OMX, optimized for stability and speed, that offers a more flexible and general platform for microscopy.  And the first thing they did with it — though it appears that this was not their original plan — was to study the effect of the light you use to image a cell on the viability of the cell itself.

The results are kind of stunning.  We’ve known for many years that light is somewhat toxic, especially to cells containing large numbers of fluorophores — but not this toxic.  The assay Carlton et al. use to assess toxicity is to follow imaged cells for 5 complete divisions after the imaging experiment.  If they complete all 5 divisions successfully, then they are considered unperturbed.  They used cells that contain a lac repressor-GFP fusion, which binds to an amplified lac operator causing a spot on the DNA, and found that to get to the level where cells were reliably unperturbed, they had to cut the amount of light they were using to between 1/100 and 1/10,000 of the levels normally used for imaging.  Wow.  This is a new and powerful statement of Heisenberg’s Uncertainty Principle as it relates to cell biology.  You can only see it if you change it.

These results seem to offer a grim prospect for the future of live cell imaging. The images at 1/100x normal light levels are barely images — the signal to noise ratio is so low that the authors had to resort to time-averaging of the data even to be sure that they had any signal at all.  Fortunately Carlton et al. also show that image denoising algorithms can extract useful data even out of images collected at 1/10,000x normal light levels. As an added bonus, reducing the light intensity also reduces bleaching, allowing more frequent image collection and better time resolution. So let’s be adult and glass-half-full about this; although the truth (assuming that these observations hold up for other cell types, other localizations of fluorescent label, and other imaging modalities) that the levels of light we typically use to image may be seriously perturbing the processes we hope to study is certainly inconvenient, it is better to know it than not.  And the new microscopy platform allows us to increase both the spatial and the temporal resolution of light microscopy — if we do it right — which is potentially extraordinarily powerful.  I tend to agree with Jason that the field won’t — and shouldn’t — ever be the same.

Carlton PM, Boulanger J, Kervrann C, Sibarita JB, Salamero J, Gordon-Messer S, Bressan D, Haber JE, Haase S, Shao L, Winoto L, Matsuda A, Kner P, Uzawa S, Gustafsson M, Kam Z, Agard DA, & Sedat JW (2010). Inaugural Article: Fast live simultaneous multiwavelength four-dimensional optical microscopy. Proceedings of the National Academy of Sciences of the United States of America PMID: 20705899