We present results for a new type of fiber-coupled stimulated emission depletion (STED) microscope which uses a single fiber to transport STED and excitation light, as well as collect the fluorescence signal. Our method utilizes two higher-order eigenmodes of polarization maintaining (PM) fiber to generate the doughnut-shaped STED beam. The modes are excited with separate beams that share no temporal coherence, yielding output that is independent of fiber bending. We measured the resolution using 45 nm fluorescent beads and found a median bead image size of 116 nm. This resolution does not change as function of fiber bending radius, demonstrating robust operation. We report, for the first time, STED images of fixed biological samples collected in the epi-direction through fiber. Our microscope design shows promise for future use in super-resolution micro-endoscopes and
Two-photon Fiber STED Microscope Using Polarization Maintaining Fiber
We demonstrate a two photon (2P) fiber STED microscope in which the excitation and STED light are delivered to the sample in polarization maintaining (PM) fiber.
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- NSF-PAR ID:
- 10295312
- Date Published:
- Journal Name:
- Conference on Lasers and Electro-Optics
- Page Range / eLocation ID:
- SW2D.2
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract in vivo neural imaging in awake and freely-behaving animals. -
Vertebrate brains have a dual structure, composed of ( i ) axons that can be well-captured with graph-theoretical methods and ( ii ) axons that form a dense matrix in which neurons with precise connections operate. A core part of this matrix is formed by axons (fibers) that store and release 5-hydroxytryptamine (5-HT, serotonin), an ancient neurotransmitter that supports neuroplasticity and has profound implications for mental health. The self-organization of the serotonergic matrix is not well understood, despite recent advances in experimental and theoretical approaches. In particular, individual serotonergic axons produce highly stochastic trajectories, fundamental to the construction of regional fiber densities, but further advances in predictive computer simulations require more accurate experimental information. This study examined single serotonergic axons in culture systems (co-cultures and monolayers), by using a set of complementary high-resolution methods: confocal microscopy, holotomography (refractive index-based live imaging), and super-resolution (STED) microscopy. It shows that serotonergic axon walks in neural tissue may strongly reflect the stochastic geometry of this tissue and it also provides new insights into the morphology and branching properties of serotonergic axons. The proposed experimental platform can support next-generation analyses of the serotonergic matrix, including seamless integration with supercomputing approaches.more » « less
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The neighborhood of virtually every brain neuron contains thin, meandering axons that release serotonin (5-HT). These axons, also referred to as serotonergic fibers, are present in all vertebrate species (from fish to mammals) and are an essential component of biological neural networks. In the mammalian brain, they create dense meshworks that are macroscopically described by densities. It is not known how these densities arise from the trajectories of individual fibers, each of which resembles a unique random-walk path. Solving this problem will advance our understanding of the fundamental structure of neural tissue, including its plasticity and regeneration. Our interdisciplinary program investigates the stochastic structure of serotonergic fibers, by employing a range of experimental, computational, and theoretical methods. Transgenic mouse models (e.g., Brainbow) and brainstem cell cultures are used with advanced microscopy (3D-confocal imaging, STED super-resolution microscopy, holotomography) to visualize individual serotonergic fibers and their trajectories. Serotonergic fibers are modeled as paths of a superdiffusive stochastic process, with a focus on fractional Brownian motion (FBM). The formation of regional fiber densities is tested with supercomputer modeling in neuroanatomically accurate 2D- and 3D-brain-like shapes. Within the same framework, we are developing the mathematical theory of the reflected, branching, and spatially heterogenous FBM.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Conference Paper:
- https://doi.org/10.1364/CLEO_SI.2021.SW2D.2
