Faster Fluorescent Holography

Spatiotemporal modulations of illumination intensity in the CHIRPT microscope are achieved by imaging a spinning modulation mask to the focal plane of the microscope. A spatial filter placed in the pupil plane of the objective lens allows illumination intensity to form by the interference of two beams in the object plane. The microscope and illumination intensity are shown here at a snapshot in time. (Source: J. Field / CSU)

Spatiotemporal modulations of illumination intensity in the CHIRPT microscope are achieved by imaging a spinning modulation mask to the focal plane of the microscope. A spatial filter placed in the pupil plane of the objective lens allows illumination intensity to form by the interference of two beams in the object plane. The microscope and illumination intensity are shown here at a snapshot in time. (Source: J. Field / CSU)

Optical micro­scopy experts at Colorado State University are pushing the envelope of biological imaging. Jeffrey Field, a research scientist in electrical engineering and director of CSU’s Micro­scope Imaging Network, has designed and built a fluores­cence-detection microscope that combines three-dimensional and high-resolution image processing that’s also faster than compa­rable techniques. He named his new micro­scope CHIRPT: Coherent Holographic Image Recon­struction by Phase Transfer.

Field and other optics scientists work in a world of tradeoffs. For example: an advanced deep-tissue imaging technique called multiphoton fluores­cence micro­scopy employs a short, bright laser pulse focused tight to one spot, and the fluores­cence intensity from that one spot is recorded. Then, the laser moves to the next spot, then the next, to build up high-reso­lution 3D images. The technique offers sub­cellular detail, but it’s relatively slow because it illuminates only one tiny spot at a time. Other techniques, like spinning disk confocal micro­scopy, are faster because they shine light on multiple spots, not just one, and they scan simul­taneously over a larger area. But unlike multi­photon, these techniques require col­lecting an image with a camera. As a result, fluores­cent light emitted from the specimen is blurred on the camera, leading to loss in resolution, and with it, subcellular detail.

The goal of Field and colleagues is working around each of these limi­tations – speed, resolution, size of field – to break through established boundaries in light micro­scopy. Field and Bartels’ new micro­scope builds upon a previously published technique, and permits digital re-focus of fluores­cent light. It il­luminates not one point, but multiple points by harnessing delocalized illu­mination spread over a large area. The physical principles they are using are similar to holo­graphy, in which scattered light is used to build a 3-D image.

CHIRPT and confocal imaging of fluorescently labelled mouse intestine slices. (a) and (b) are digitally refocused CHIRPT images. (c) and (d) are conventional confocal images. In all four, features in focus are denoted by arrows. (Source: J. Field / CSU)

CHIRPT and confocal imaging of fluorescently labelled mouse intestine slices. (a) and (b) are digitally refocused CHIRPT images. (c) and (d) are conventional confocal images. In all four, features in focus are denoted by arrows. (Source: J. Field / CSU)

Using a large illu­mination field, followed by back-end signal processing, the microscope can define distinct light modu­lation patterns of many points within the field of view. It builds up a 3-D image by combining the signals from all those distinct patterns. “The idea is that you have a fluoro­phore at any point in the specimen, and the temporal structure of its fluores­cence will be distin­guishable from all others,” Field said. “So you can have this huge array of fluoro­phores, and just with this single-pixel detector, you can tell where every one of them is in that 2D field.”

So what does this new technique allow? Deep-tissue images in three dimensions, with better depth of field than comparable techniques. Depth of field, like in photography, means background images are in sharp focus along with the main image. And the CSU researchers can work at 600 frames per second, which is many times faster than esta­blished techniques. With their new micro­scope, images can also be post-processed to remove aber­rations that obscure the object of interest. It’s akin to being able to focus a picture after it’s been taken.

The CHIRPT micro­scope could allow biomedical researchers to produce sharp, 3-D images of cells or tissue over a much larger volume than conven­tional fluorescence micro­scopy methods allow. It could lead to things like imaging multi­cellular processes in real time that, with a conven­tional light micro­scope, could only be seen one cell at a time. (Source: CSU)

Reference: J. J. Field et al.: Single-pixel fluorescent imaging with temporally labeled illumination patterns, Optica 3, 971 (2016); DOI: 10.1364/OPTICA.3.000971

Link: Microscope Imaging Network Foundational Core Facility, Colorado State University, Fort Collins, USA

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