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New Microscope Watches Cells in 3D
Jamie found a story about a new 3D Microscope which creates 3D videos of cells in action. Traditionally scientists have had to choose between high resolution and animation, so no doubt this device will cure the common cold.
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Photo-damage to cells is indeed a concern, but the described technique actually has the advantage that this can minimized as much as physically possible. Many visualization techniques involve either (1) having the cell absorb light, so that you can differentiate different regions based on absorption (may require staining with something sufficiently absorptive), or (2) having something fluoresce, which requires that species to absorb and then re-emit light (typically requires staining or genetic engineering so a target protein is fluorescent). Obviously both (1) and (2) require the sample to actively absorb photos, which means that some amount of photo-heating is unavoidable. Moreover fluorescent molecules often lead to undesired side-reactions and degrade over time (so-called "photo-bleaching"). With fluorescence imaging, you can select an excitation wavelength outside of the absorption bands of everything in solution (especially water!), and thereby minimize photo-heating and photo-damage.
The article says that they are actually imaging the refracted light. Since this technique doesn't require any amount of sample absorption at all, they can use a minimally absorbing wavelength, thereby keeping sample damage to an absolute minimum. In fact since they are measuring refracted light, the technique works best at wavelengths where absorption is as low as possible (but refractive index contrast is as high as possible).
From the description, it doesn't sound like the illumination would be much more intense than what a normal microscope generates. Most cells don't experience significant photo-damage under such illumination conditions.
Some current imaging systems use a raster-scanned focused-laser spot to generate the images. By using high-quality detectors the light-levels can be kept low enough that cell damage is prevented. Thus the technique from the article probably induces less cell damage than currently used techniques. Not to mention that the fact that you don't have to stain or modify the cells eliminates the toxicity (or perturbing effect) or those staining agents.
"Cellular Organization and Substructure Measured Using Angle-Resolved Low-Coherence Interferometry", Wax A, Yang C, Backman V, Badizadegan K, Boone C, Dasari RR, Feld MS. Biophysical Journal 82: 2256-2264 (2002).
In the experimental section of that article they say: This appears to be one of their more recent publications:
"Quantitative phase imaging of live cells using fast Fourier phase microscopy", Niyom Lue, Wonshik Choi, Gabriel Popescu, Takahiro Ikeda, Ramachandra R. Dasari, Kamran Badizadegan, and Michael S. Feld. Applied Optics, Vol. 46, Issue 10, pp. 1836-1842.
In that paper they say: The illumination sources are not very intense, but are powerful enough to cause cell damage if they were highly focused. From looking over the papers it doesn't seem that this is the case. For what it's worth, the papers do not mention cell damage as being a concern.
Overall the technique seems to have serious promise. It essentially involves doing laser interferometry on the sample at multiple angles, and reconstructing the 3D image. As they mention in their papers, it has the advantage of interfacing with conventional confocal microscope designs. Thus it could be added as an option on existing setups. It appears to have some exacting requirements (like all holography/interferometry it will be sensitive to vibrations, etc.), but overall seems like the type of thing that could be rapidly built into existing labs and commercial instruments.
Until June, I had been working in a live-cell imaging laboratory for nearly four years. There's a whole list of criteria that will cell proliferation while being grown on a microscope. My lab had (before I arrived) already proven they could grow cells on a microscope stage that would match cells grown in an incubator (ie, number of mitotic events). These like this are important.
A few people have mentioned bits about imaging and I thought I'd kind of list the important ones:
'Normal' brightfield microscopes are the kind that you'll find in a high school classroom. They work because the sample absorbs light. When they talk about fixing and staining cells, you can use these. Usually cells are transparent and won't attenuate light as it passes through the cell.
Confocal works by exciting a fluorophor with a laser and measuring the emitted photons. Its neat. But you're pointing a laser at a cell.
DIC, AFM and others work on varied principles. AFM is atomic force. They basically poke a cell and measure how it pushes back. But you're poking a cell. DIC are light based but as far as I have seen not extremely popular in the field of live cell imaging for one reason or another.
Phase-Contrast I left till last as its really the only microscope that can be used for live-cell imaging. It works by measuring not how much the light is attenutated, but how much its slowed as it passes through the cell. Basically, a small fraction of light is slowed and difracted as it passes through the sample. The light that passes through unaffected is attenuated after the sample so it and the two groups have approximately the same intensity. Then normal interference will give an image on the detector.
As usual for science articles, it got most of the details wrong. We've had phase-contrast microscopes for over 50 years now. Zernike got the Nobel Prize for inventing them in 1953 http://nobelprize.org/educational_games/physics/mi croscopes/phase/index.html We can use these to measure living cells. We could do drug screening. Nothing the article said was really new and frankly it was rather irritating.
What is exciting though is the fact that this might allow the machine vision guys to be able to reliably segment live-cell image data. Currently this is a problem with no *acceptable* solution. [By acceptable I mean to say, something that has an accuracy over 90-95% for any cell line I give it as well as not using anything like a nuclear stain] Once we get this level of segmentation there is an unlimited number of things we can do:
"Where's the any key?" - Homer Simpson
The presented technique does indeed have limitations--sample thickness being a major one. However the fact that it requires no sample prep (e.g. staining) seems like a big advantage. For many studies, having video of the 3D structure of a cell will be irrelevant compared to what more traditional techniques can tell you (e.g. labeling a protein and using fluorescent to monitor its localization). However for other studies, realtime 3D visualization may be very useful (e.g. cellular dynamics). I agree that it's not the cure-all that the article hypes it to be... but I can see it becoming useful for a number of research topics.
As to how difficult it is to get working... The papers indicate that it can be fitted onto a conventional confocal microscope. However because it is an interferometry technique, things like vibrations must be minimized. So it's probably a bit finnicky, but I any research lab with experience in optics could build one if they really wanted to. The technique uses off-the-shelf technology, so commercial instruments (probably sold as add-ons to existing microscopes) could easily be built. I'm not an expert in the field, so I can't predict whether there would be a strong demand for such an instrument.
(Note: I've used various microscopies in my research, but not on biological samples, so please correct any mistakes I've made in that regard.)
I have to admit that the results obtained with this new kind of microscope are spectacular. You'll find additional references and images of a cervical cancer cell taken using this new imaging technique on this ZDNet post.
This looks a LOT like digital inline holography, but I didn't see in the article what technique they're useing. I did some minor DIH work at Dalhousie University, back in 2004. Last I heard, a couple of the profs there are developing a commercial product.