Photoacoustic and fluorescence imaging methods combined

Researchers have demonstrated a new endoscope that combines photoacoustic and fluorescent imaging in a device about the thickness of a human hair. Described in the journal Biomedical Optics Express, the device could one day provide new insights into the brain by enabling blood dynamics to be measured at the same time as neuronal activity.

Acquiring fluorescent and photoacoustic images with the same device provides automatically co-registered images with complementary information. Fluorescent signals, which are created when a fluorescent marker absorbs light and re-emits it with a different wavelength, are most useful for labelling specific regions of tissue. On the other hand, photoacoustic images, which capture an acoustic wave generated after the absorption of light, do not require labels and thus can be used to image blood dynamics, for example.

A research team led by Emmanuel Bossy, from France’s Centre national de la recherche scientifique (CNRS), in collaboration with Paul C Beard’s team from University College London, has now developed a multimodality endoscope that can acquire photoacoustic and fluorescent images of red blood cells and fluorescent beads. The endoscope uses a technique called optical wavefront shaping to create a focused spot of light at the imaging tip of a very small multimode optical fibre.

“Light propagating into a multimode fibre is scrambled, making it impossible to see through the fibre,” Bossy said. “However, this type of fibre is advantageous for endoscopy because it is extremely small compared to the bundles of imaging fibres used for many medical endoscopic devices.”

To see through the multimode optical fibre, the researchers used the spatial light modulator to send specific light patterns through the fibre and create a focus spot at the imaging end. When the focus spot hits the sample, it creates a signal that can be used to build up an image point by point by raster scanning the spot over the sample. Although other researchers have used multimode fibres for fluorescence endoscopy, the new work represents the first time that photoacoustic imaging has been incorporated into this type of endoscope design.

The researchers added photoacoustic imaging by incorporating an additional, very thin optical fibre with a special sensor tip that is sensitive to sound. Because commercially available fibre-optic acoustic sensors are not sensitive or small enough for this application, the researchers used a very sensitive fibre-optic sensor recently developed by Beard’s research team.

“The focused spot of light allows us to build the image pixel by pixel while also increasing the strength of fluorescence and photoacoustic signals because it concentrates the light at the focal spot,” Bossy explained. “This concentrated light combined with a sensitive detector made it possible to obtain images using only one laser pulse per pixel, whereas commercial fibre-optic acoustic sensors would have required many laser pulses.”

The researchers fabricated a prototype microendoscope that measured just 250 x 125 µm² and used it to image fluorescent beads and blood cells using both imaging modalities. They successfully detected multiple 1 µm fluorescent beads and individual 6 µm red blood cells.

“Combining these imaging modalities could improve our understanding of the brain’s structure and behaviour in specific conditions such as after treatment with a targeted drug,” Bossy said. “The endoscope’s small size helps minimise damage to tissue when inserting it into the brains of small animals for imaging.”

Because fluorescence endoscopy in a rodent’s brain has been performed by other scientists, the researchers are confident that their dual modality device will work in similar conditions. They are now continuing work to increase the device’s acquisition speed, with a goal of acquiring a few images per second.

Image caption: The researchers demonstrated their new device by using it to image fluorescent beads (green) and red blood cells (red). The field of view is the size of a hair. Image credit: Emmanuel Bossy, CNRS/Université Grenobe Alpes Laboratoire Interdisciplinaire de Physique.

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