As might be expected of living things, cells don’t sit still. They fidget, changing shape and volume in response to their environment, for instance. And because cells are small, the changes are neither large nor slow. Red blood cell membranes, for example, move tens of nanometers over tens of milliseconds. For researchers, the problem has been how to track such movement and relate it back to cellular function. Now a team from MIT in Cambridge, Mass., and from Harvard Medical School in Boston has combined simultaneous quantitative phase imaging with nanometer-scale resolution and fluorescence imaging to develop a diffraction phase and fluorescence microscope that reveals functional details. The researchers demonstrated the microscope in experiments on red blood cells and on mitotic kidney cells. According to Michael S. Feld, director of the George R. Harrison Spectroscopy Laboratory at MIT, the microscope is an important step in a decade-long effort to probe biological structures with light. Rather than use intensity changes arising from absorption differences, the techniques developed at MIT employ interferometry. In this approach, a probe beam traverses a sample and is combined with a reference beam to create an interference pattern. Refractive index variations in the sample create contrast, illuminating internal cellular structures without a contrast agent and enabling detection of optical path length changes that are much smaller than a wavelength. Both of these are advantages over conventional microscopy, where the resolution is limited to, at best, about a half wavelength, and where contrast can be minimal without an external contrast agent. A DNA-stained kidney cell is shown in fluorescence mode (A). The phase map is measured in diffraction phase and fluorescence microscopy mode (B), and the overlaid image is presented (C). However, interferometers can be too sensitive. The phase difference between sample and reference beams can fluctuate so much that it drowns out any signal from a cell. To circumvent this problem, the team developed a common-path approach with the sample and reference beams traveling in proximity so that they fluctuate in tandem. Then the difference between the two is stable despite the noise in the individual signals. The researchers began with an Olympus microscope equipped with an ultraviolet lamp for epifluorescence imaging. They added an argon laser operating at 514 nm for transmission phase imaging. For diffraction phase and fluorescence imaging, they took the light from the sample out through the microscope’s video port and ran it through a series of lenses and mirrors. Along the way, they used a diffraction grating with a periodicity of 30 μm to separate the beam into multiple diffraction orders, each of which carried the frequency information. They employed a spatial filter and a pinhole to block everything but the zeroth and first order, with the first-order beam containing the entire frequency information while the zeroth contained only low-frequency information. The effect was to transform the zeroth beam into the reference beam and the first-order one into the sample beam, with both traveling along a common path. The fluorescence generated by the sample traveled along the same path, but, because of the optics, the zeroth order was almost entirely blocked. As a result, the fluorescence, shifted in wavelength from the excitation by an amount dependent on the fluorophore, arrived in the first-order beam. For detection, the researchers used a Nikon CCD camera with a resolution of 3000 x 2000 pixels that measured 7.8 μm square. As described in the Sept. 4 issue of Optics Express, tests by the researchers with no sample in the field of view indicated that the optical path length of the setup was stable. The mean standard deviation of the path length was 0.23 nm, with a variation of a tenth of that. The largest source of noise, the researchers found, was caused by mechanical stability. They demonstrated the ability of the setup by imaging the membrane fluctuations of living red blood cells, which took milliseconds, and white cell activity, which took minutes. For the first, they placed droplets of whole blood between coverslips and imaged a red blood cell. They captured thermal fluctuations, disturbing the cell’s membrane and creating flickering, a phenomenon known for more than a century that has been the subject of much research. For the second, they placed a whole blood smear containing a red blood cell and a white blood cell. Over many seconds, they captured the process, whereby the white blood cell attacked the foreigner, eventually breaking through its membrane and destroying it. To show that the instrument could combine diffraction phase with fluorescence imaging, they experimented with kidney cells in culture. After treating the cells with a fluorescent dye that binds to DNA to reveal the cell nucleus, they imaged the cells directly in culture dishes. They found that, for example, they could overlay the fluorescent image of a cell undergoing division with the phase image of the same cell. The fluorescence indicated two separated nuclei, which also appeared, although not as distinctly, in the phase image. Although providing the stability needed for interferometry, the grating does cause light loss. That deficit could be an issue for weak fluorophores, which don’t produce many photons to start with. The problem could be minimized, the researchers noted, by using sinusoidal amplitude grating, which maximizes the diffraction of the positive and negative first orders. That technique would produce the two beams needed for phase imaging while keeping light loss to only half. Applications of the method include studies of the effects of various membrane-binding molecules on the mechanical properties of cell membranes. Red blood cells could be a convenient model for such studies. As an example of what can be done, Gabriel Popescu, a postdoctoral researcher who headed up the diffraction phase and fluorescence microscope development efforts at MIT, said recent findings, now in press, have quantified red blood cell flickering, indicating positive tension in the cell membrane.