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Counterintuitive Defocusing Technique Produces 3-D Microscope Images

By defocusing a microscope, biologists have developed a simple technique that produces three-dimensional images of red blood cells.

One of the great inventions in history was the development of the microscope. The ability to see the enormous variety of life and patterns on such a tiny scale changed our understanding of the world and our relationship to it.

Since then microscopists have dramatically improved their instruments so that they record not only with visible light but other wavelengths and even with other recording media entirely such as electrons and neutrons and so on.

One of the most difficult challenges, however, is determining the three-dimensional structure of small objects. Healthy red blood cells, for example, have a famous doughnut-shaped structure and changes to this structure are an important indicator of various diseases and conditions.

These shapes are straightforward to see but determining their entire 3-D structure with a resolution of a few hundred nanometers is tricky; not least because red blood cells are largely transparent and difficult to see with ordinary bright field microscopy.

Today, Paula Roma and pals at the Federal University of Minas Gerais in Brazil reveal a new and relatively simple technique for determining the entire 3-D structure of red blood cells based on the counterintuitive technique of defocusing.

Biologists have long recognized the limitations of bright field microscopy with biological samples because many are largely transparent. (Bright field microscopy is the ordinary microscope technique that most people come across in high school.)

Light shines onto or through the sample revealing anything that strongly absorbs or scatters light.

The trouble is that anything that is transparent is more or less invisible because light passes straight through it. And unfortunately, many biological samples fall into this category—including red blood cells which are largely transparent.

One way round this is to defocus the image slightly. Since the red blood cells have a refractive index, they bend the light that passes through it. This bending introduces a phase change in the light.

This phase change makes it easier to separate this light from light that has not passed through cell. And by doing this, the red blood cells become darker and easier to see.

That allows more detailed analysis of the cells. By measuring the changes in intensity that this process introduces, it is possible to work out the shape of the surface generating the phase changes. So that gives a three dimensional image of the upper surface of the cell.

Now Roma and co say it’s possible to go further. They show that by taking two images of the cell, both defocused by different amounts, it is possible to work out the shape of the cell’s bottom surface as well. In other words, this weird kind of defocused stereo image gives you the 3-D shape of the entire cell.

The results are impressive. The technique works with ordinary white light, although this has to be filtered to remove the red wavelengths that red blood cells might absorb.

To get two images of the same cell, Roma and co use a beam splitter to send light to two cameras that are both defocused by different amounts. The results can then be processed using a straightforward algorithm to produce 3-D images of the cells

To test the idea, Roma and co placed red blood cells in various concentrations of salt solutions to make them swell. They recorded images of 25 cells and processed the results, which are shown above.

They say the images have a resolution of within 300 nanometers, significantly better than is possible with similar techniques. In particular, they show how the cells adhere to the surface they are attached to.

The setup is also relatively straightforward. “The technique could be easily adopted by nonspecialists,” they say.

That’s an interesting result that shows how fascinating low cost advances can still be made in microscopy.

Ref: : Total 3D Imaging Of Phase Objects Using Defocusing Microscopy: Application o Red Blood Cells

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