Lensless microscopes have simple and compact optics that, together with associated computational algorithms, allow for large fields of view and the refocusing of the captured images. One limitation of existing lensless techniques has been the accurate reconstruction of images of optically dense biological tissue.

The latest iteration of lensless microscopy is the Bio-FlatScope. The device looks inward to image micron-scale targets like cells and blood vessels inside the body—even through the skin. Bio-FlatScope captures images that no lensed camera can see. For example, showing dynamic changes in the fluorescent-tagged neurons in running mice.

One advantage over other microscopes is that light captured by Bio-FlatScope can be refocused after the fact to reveal 3D details. And without lenses, the scope’s field of view is the size of the sensor (at close range to the target) or wider, without distortion.

Jacob Robinson, PhD, associate professor in electrical & computer engineering and bioengineering at Rice University, led the recent effort to test Bio-FlatScope in living creatures. The team’s proof-of-concept study also imaged plants, hydra and, to a limited degree, a human.

The small, low-cost Bio-FlatScope may open up to clinical uses, especially for imaging difficult-to-reach areas of the body.

The results appear in Nature Biomedical Engineering in the paper, “In vivo lensless microscopy via a phase mask generating diffraction patterns with high-contrast contours.

The Bio-FlatScope mechanism combines a sophisticated phase mask to generate patterns of light that fall directly onto the chip, according to the researchers. The phase mask looks more like the random map of a natural landscape, with no straight lines. “We had to start from scratch and think about how to make it function in a realistic biological setting,” Robinson said.

“Being random allows the mask to be pretty diverse in gathering light from all directions,” said Vivek Boominathan, PhD, a postdoctoral research associate in the Computational Imaging Group headed by Ashok Veeraraghavan, PhD, professor of electrical and computer engineering at Rice University and co-developer of FlatCam—a thin sensor chip with a mask that replaces lenses in a traditional camera. “And then we take the random input, which is called Perlin noise, and do some processing to get these high-contrast contours.”

At the sensor, light that comes through the mask appears as a point spread function—a pair of blurry blobs that seems useless but is actually key to acquiring details about objects below the diffraction limit that are too small for many microscopes to see. The blobs’ size, shape, and distance from each other indicate how far the subject is from the focal plane. Software reinterprets the data into an image that can be refocused at will.

The researchers started small, first capturing cellular structures in a lily of the valley, then calcium activity in tiny hydra. They moved on to monitoring a running rodent, attaching the Bio-FlatScope to a rodent’s skull and setting it down on a wheel. The data showed fluorescent-tagged neurons in a region of the animal’s brain, connecting activity in the motor cortex with motion and resolving blood vessels as small as 10 microns in diameter.

The team identified vascular imaging as a potential clinical application of the Bio-FlatScope. Jimin Wu, a graduate student, used her lower lip to see if light passing through to the camera could deliver structural details of the blood vessels within.

“It was kind of an engineering challenge because it’s difficult to position the Bio-FlatScope at the correct position and keep it there,” Wu said. “But it showed us it could be a good tool for seeing signs of sepsis, because pre-sepsis changes the density of the vasculature. Cancer also alters the morphology of the microvasculature.”

Long term, the team sees potential for a camera that could curve around its subject, like brain tissue, “so it could match the morphology of what you’re looking at,” Robinson said. “Or maybe you could fold it up, stick it in place, and have it unfold and deploy.

“You could also do really interesting things by bending it for a fisheye effect, or you could curve it inward and have very high light-collection efficiency,” he said.

A comparison of images from Rice University’s Bio-FlatScope, left, and original FlatScope, right, show how much detail the new device captures in images of cells in a lily of the valley. Bio-FlatScope’s random phase mask proved better at imaging curved structures. [Robinson Lab/Rice University]
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