Abstract
Optical microscopy remains a fundamental tool for modern biological discovery owing to its excellent spatial resolution and versatile contrast in visualizing cellular and sub-cellular structures. Yet, the time domain is paramount for the observation of biological dynamics in living systems. Commonly, acquisition of microscopy data involves scanning of a spherically- or cylindrically-focused light beam across the imaged volume, which significantly limits temporal resolution in 3D. Additional complications arise from intense light scattering of biological tissues, further restraining the effective penetration depth and field of view of optical microscopy techniques. To overcome these limitations, we devised a fast optoacoustic micro-tomography (OMT) approach based on simultaneous acquisition of 3D image data with a high-density hemispherical ultrasound array having effective detection bandwidth beyond 25 MHz. We demonstrate fast three-dimensional imaging of freely-swimming zebrafish larvae, achieving 3D imaging speed of 100 volumes per second with isotropic spatial resolution approaching the dimensions of large cells across a field of view exceeding 50mm3. As opposed to other microscopy techniques based on optical contrast, OMT resolves optical absorption acoustically using unfocused light excitation. Thus, no penetration barriers are imposed by light scattering in deep tissues, suggesting it as a powerful approach for multi-scale functional and molecular imaging applications.
Highlights
Microscopy refers to a myriad of methods aimed at resolving structures invisible to the naked eye
Optoacoustic imaging has emerged as a promising modality capable of high-resolution imaging beyond the penetration limits of optical microscopy[8]
optoacoustic micro-tomography (OMT) uses unfocused light illumination to image optical absorption contrast with acoustic resolution. This enables the acquisition of 3D tomographic data from the entire imaged volume for each laser pulse, resulting in large fields of view exceeding 50 mm[3] and volumetric imaging frame rates of 100 Hz, an unprecedented performance among other microscopy methods
Summary
Microscopy refers to a myriad of methods aimed at resolving structures invisible to the naked eye. The angular resolution of the human eye is estimated to be approximately 1 arcminute, i.e., points separated by less than ~75 μm cannot be distinguished via normal vision at the closest focusing distance (~25 cm)[1] This resolution barrier can be readily overcome with different microscopic techniques based on electromagnetic radiation[2], electron beams[3] or ultrasound[4]. Numerous implementations have been reported that use point-by-point scanning of focused transducers with detection bandwidths from several MHz up to the GHz range, corresponding to acoustic spatial resolution down to micrometer scales[15, 16]. We introduce a novel tomographic design that enables accurate real-time 3D imaging of optical absorption with microscopic resolution, approaching single cell dimensions at depths beyond the limits imposed by light scattering
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