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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.