@article {69994, title = {A multimodal adaptive optical microscope for in vivo imaging from molecules to organisms}, journal = {bioRxiv}, year = {2025}, month = {2025 Jun 16}, abstract = {

Understanding biological systems requires observing features and processes across vast spatial and temporal scales, spanning nanometers to centimeters and milliseconds to days, often using multiple imaging modalities within complex native microenvironments. Yet, achieving this comprehensive view is challenging because microscopes optimized for specific tasks typically lack versatility due to inherent optical and sample handling trade-offs, and frequently suffer performance degradation from sample-induced optical aberrations in multicellular contexts. Here, we present MOSAIC, a reconfigurable microscope that integrates multiple advanced imaging techniques including light-sheet, label-free, super-resolution, and multi-photon, all equipped with adaptive optics. MOSAIC enables non-invasive imaging of subcellular dynamics in both cultured cells and live multicellular organisms, nanoscale mapping of molecular architectures across millimeter-scale expanded tissues, and structural/functional neural imaging within live mice. MOSAIC facilitates correlative studies across biological scales within the same specimen, providing an integrated platform for broad biological investigation.

Preprint:\ https://www.biorxiv.org/content/early/2025/06/13/2025.06.02.657494

}, doi = {10.1101/2025.06.02.657494}, url = {https://www.biorxiv.org/content/early/2025/06/13/2025.06.02.657494}, author = {Fu, Tian-Ming and Liu, Gaoxiang and Milkie, Daniel E. and Ruan, Xiongtao and G{\"o}rlitz, Frederik and Shi, Yu and Ferro, Valentina and Divekar, Nikita S. and Wang, Wei and York, Harrison M. and Kilic, Velat and Mueller, Matthew and Liang, Yajie and Daugird, Timothy A. and Gacha-Garay, Maria J. and Larkin, Kathryn A. and Adikes, Rebecca C. and Harrison, Nathaniel and Shirazinejad, Cyna and Williams, Samara and Nourse, Jamison L. and Sheu, Shu-Hsien and Gao, Liang and Li, Tongchao and Mondal, Chandrani and Achour, Kemal and Hercule, Wilmene and Stabley, Daniel and Emmerich, Kevin and Dong, Peng and Drubin, David and Liu, Zhe J. and Clapham, David and Mumm, Jeff S. and Koyama, Minoru and Killilea, Alison and Bravo-Cordero, Jose Javier and Keene, C. Dirk and Luo, Liqun and Kirchhausen, Tom and Pathak, Medha M. and Arumugam, Senthil and Nunez, James K. and Gao, Ruixuan and Matus, David Q. and Martin, Benjamin L. and Swinburne, Ian A. and Betzig, Eric and Legant, Wesley R. and Upadhyayula, Srigokul} } @article {69622, title = {A modular chemigenetic calcium indicator for multiplexed in vivo functional imaging.}, journal = {Nat Methods}, year = {2024}, month = {2024 Sep 20}, abstract = {

Genetically encoded fluorescent calcium indicators allow cellular-resolution recording of physiology. However, bright, genetically targetable indicators that can be multiplexed with existing tools in vivo are needed for simultaneous imaging of multiple signals. Here we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several that efficiently label the brain in animals. When bound to a near-infrared dye-ligand, WHaloCaMP shows a 7{\texttimes} increase in fluorescence intensity and a 2.1-ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a to image Ca responses in vivo in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae and to quantify Ca concentration using fluorescence lifetime imaging microscopy (FLIM).

}, issn = {1548-7105}, doi = {10.1038/s41592-024-02411-6}, url = {https://doi.org/10.1038/s41592-024-02411-6}, author = {Farrants, Helen and Shuai, Yichun and Lemon, William C and Monroy Hernandez, Christian and Zhang, Deng and Yang, Shang and Patel, Ronak and Qiao, Guanda and Frei, Michelle S and Plutkis, Sarah E and Grimm, Jonathan B and Hanson, Timothy L and Tomaska, Filip and Turner, Glenn C and Stringer, Carsen and Keller, Philipp J and Beyene, Abraham G and Chen, Yao and Liang, Yajie and Lavis, Luke D and Schreiter, Eric R} } @article {68665, title = {Fast and sensitive GCaMP calcium indicators for imaging neural populations.}, journal = {Nature}, year = {2023}, month = {2023 Mar 15}, abstract = {

Calcium imaging with protein-based indicators is widely used to follow neural activity in intact nervous systems, but current protein sensors report neural activity at timescales much slower than electrical signalling and are limited by trade-offs between sensitivity and kinetics. Here we used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators. The resulting {\textquoteright}jGCaMP8{\textquoteright} sensors, based on the calcium-binding protein calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (half-rise times of 2 ms) and the highest sensitivity for neural activity reported for a protein-based calcium sensor. jGCaMP8 sensors will allow tracking of large populations of neurons on timescales relevant to neural computation.

}, issn = {1476-4687}, doi = {10.1038/s41586-023-05828-9}, author = {Zhang, Yan and Rozsa, Marton and Liang, Yajie and Bushey, Daniel and Wei, Ziqiang and Zheng, Jihong and Reep, Daniel and Broussard, Gerard Joey and Tsang, Arthur and Tsegaye, Getahun and Narayan, Sujatha and Obara, Christopher J and Lim, Jing-Xuan and Patel, Ronak and Zhang, Rongwei and Ahrens, Misha B and Turner, Glenn C and Wang, Samuel S-H and Korff, Wyatt L and Schreiter, Eric R and Svoboda, Karel and Hasseman, Jeremy P and Kolb, Ilya and Looger, Loren L} } @article {68930, title = {Stimulus edges induce orientation tuning in superior colliculus.}, journal = {Nature Communications}, volume = {14}, year = {2023}, month = {2023 Aug 08}, pages = {4756}, abstract = {

Orientation columns exist in the primary visual cortex (V1) of cat and primates but not mouse. Intriguingly, some recent studies reported the presence of orientation and direction columns in the mouse superficial superior colliculus (sSC), while others reported a lack of columnar organization therein. Using in vivo calcium imaging of sSC in the awake mouse brain, we found that the presence of columns is highly stimulus dependent. Specifically, we observed orientation and direction columns formed by sSC neurons retinotopically mapped to the edge of grating stimuli. For both excitatory and inhibitory neurons in sSC, orientation selectivity can be induced by the edge with their preferred orientation perpendicular to the edge orientation. Furthermore, we found that this edge-induced orientation selectivity is associated with saliency encoding. These findings indicate that the tuning properties of sSC neurons are not fixed by circuit architecture but rather dependent on the spatiotemporal properties of the stimulus.

}, keywords = {Animals, Calcium, Neurons, Photic Stimulation, Superior Colliculi, Visual Cortex}, issn = {2041-1723}, doi = {10.1038/s41467-023-40444-1}, author = {Liang, Yajie and Lu, Rongwen and Borges, Katharine and Ji, Na} } @article {67763, title = {An adaptive optics module for deep tissue multiphoton imaging in vivo.}, journal = {Nature Methods}, volume = {18}, year = {2021}, month = {2021 Oct 01}, pages = {1259-1264}, abstract = {

Understanding complex biological systems requires visualizing structures and processes deep within living organisms. We developed a compact adaptive optics module and incorporated it into two- and three-photon fluorescence microscopes, to measure and correct tissue-induced aberrations. We resolved synaptic structures in deep cortical and subcortical areas of the mouse brain, and demonstrated high-resolution imaging of neuronal structures and somatosensory-evoked calcium responses in the mouse spinal cord at great depths in vivo.

}, issn = {1548-7105}, doi = {10.1038/s41592-021-01279-0}, author = {Rodriguez, Cristina and Chen, Anderson and Rivera, Jos{\'e} A and Mohr, Manuel A and Liang, Yajie and Natan, Ryan G and Sun, Wenzhi and Milkie, Daniel E and Bifano, Thomas G and Chen, Xiaoke and Ji, Na} } @article {67273, title = {A Distinct Population of L6 Neurons in Mouse V1 Mediate Cross-Callosal Communication.}, journal = {Cerebral Cortex}, year = {2021}, month = {2021 May 14}, abstract = {

Through the corpus callosum, interhemispheric communication is mediated by callosal projection (CP) neurons. Using retrograde labeling, we identified a population of layer 6 (L6) excitatory neurons as the main conveyer of transcallosal information in the monocular zone of the mouse primary visual cortex (V1). Distinct from L6 corticothalamic (CT) population, V1 L6 CP neurons contribute to an extensive reciprocal network across multiple sensory cortices over two hemispheres. Receiving both local and long-range cortical inputs, they encode orientation, direction, and receptive field information, while are also highly spontaneous active. The spontaneous activity of L6 CP neurons exhibits complex relationships with brain states and stimulus presentation, distinct from the spontaneous activity patterns of the CT population. The anatomical and functional properties of these L6 CP neurons enable them to broadcast visual and nonvisual information across two hemispheres, and thus may play a role in regulating and coordinating brain-wide activity events.

}, issn = {1460-2199}, doi = {10.1093/cercor/bhab084}, author = {Liang, Yajie and Fan, Jiang Lan and Sun, Wenzhi and Lu, Rongwen and Chen, Ming and Ji, Na} } @article {65618, title = {jYCaMP: an optimized calcium indicator for two-photon imaging at fiber laser wavelengths.}, journal = {Nature Methods}, volume = {17}, year = {2020}, month = {2020 May 25}, pages = {694-97}, abstract = {

Femtosecond lasers at fixed wavelengths above 1,000 nm are powerful, stable and inexpensive, making them promising sources for two-photon microscopy. Biosensors optimized for these wavelengths are needed for both next-generation microscopes and affordable turn-key systems. Here we report jYCaMP1, a yellow variant of the calcium indicator jGCaMP7 that outperforms its parent in mice and flies at excitation wavelengths above 1,000 nm and enables improved two-color calcium imaging with red fluorescent protein-based indicators.

}, issn = {1548-7105}, doi = {10.1038/s41592-020-0835-7}, author = {Mohr, Manuel Alexander and Bushey, Daniel and Abhi Aggarwal and Marvin, Jonathan S and Kim, Jeong Jun and Marquez, Emiliano Jimenez and Liang, Yajie and Patel, Ronak and Macklin, John J and Lee, Chi-Yu and Tsang, Arthur and Tsegaye, Getahun and Ahrens, Allison M and Chen, Jerry L and Kim, Douglas S and Wong, Allan M and Looger, Loren L and Schreiter, Eric R and Podgorski, Kaspar} } @article {65322, title = {Kilohertz two-photon fluorescence microscopy imaging of neural activity in vivo.}, journal = {Nature Methods}, volume = {17}, year = {2020}, month = {2020 Mar 02}, pages = {287-290}, abstract = {

Understanding information processing in the brain requires monitoring neuronal activity at high spatiotemporal resolution. Using an ultrafast two-photon fluorescence microscope empowered by all-optical laser scanning, we imaged neuronal activity in vivo at up to 3,000 frames per second and submicrometer spatial resolution. This imaging method enabled monitoring of both supra- and subthreshold electrical activity down to 345 μm below the brain surface in head-fixed awake mice.

}, issn = {1548-7105}, doi = {10.1038/s41592-020-0762-7}, author = {Wu, Jianglai and Liang, Yajie and Chen, Shuo and Hsu, Ching-Lung and Chavarha, Mariya and Evans, Stephen W and Shi, Dongqing and Lin, Michael Z and Tsia, Kevin K and Ji, Na} } @article {65321, title = {Rapid mesoscale volumetric imaging of neural activity with synaptic resolution.}, journal = {Nature Methods}, volume = {17}, year = {2020}, month = {2020 Mar 02}, pages = {291-4}, abstract = {

Imaging neurons and neural circuits over large volumes at high speed and subcellular resolution is a difficult task. Incorporating a Bessel focus module into a two-photon fluorescence mesoscope, we achieved rapid volumetric imaging of neural activity over the mesoscale with synaptic resolution. We applied the technology to calcium imaging of entire dendritic spans of neurons as well as neural ensembles within multiple cortical regions over two hemispheres of the awake mouse brain.

}, issn = {1548-7105}, doi = {10.1038/s41592-020-0760-9}, author = {Lu, Rongwen and Liang, Yajie and Meng, Guanghan and Zhou, Pengcheng and Svoboda, Karel and Paninski, Liam and Ji, Na} } @article {64889, title = {Dynamic super-resolution structured illumination imaging in the living brain.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, year = {2019}, month = {2019 Apr 26}, pages = {9586-91}, abstract = {

Cells in the brain act as components of extended networks. Therefore, to understand neurobiological processes in a physiological context, it is essential to study them in vivo. Super-resolution microscopy has spatial resolution beyond the diffraction limit, thus promising to provide structural and functional insights that are not accessible with conventional microscopy. However, to apply it to in vivo brain imaging, we must address the challenges of 3D imaging in an optically heterogeneous tissue that is constantly in motion. We optimized image acquisition and reconstruction to combat sample motion and applied adaptive optics to correcting sample-induced optical aberrations in super-resolution structured illumination microscopy (SIM) in vivo. We imaged the brains of live zebrafish larvae and mice and observed the dynamics of dendrites and dendritic spines at nanoscale resolution.

}, issn = {1091-6490}, doi = {10.1073/pnas.1819965116}, author = {Turcotte, Raphael and Liang, Yajie and Tanimoto, Masashi and Zhang, Qinrong and Li, Ziwei and Koyama, Minoru and Betzig, Eric and Ji, Na} } @article {49597, title = {High-throughput synapse-resolving two-photon fluorescence microendoscopy for deep-brain volumetric imaging .}, journal = {eLife}, volume = {8}, year = {2019}, month = {2019 Jan 03}, abstract = {

Optical imaging has become a powerful tool for studying brains . The opacity of adult brains makes microendoscopy, with an optical probe such as a gradient index (GRIN) lens embedded into brain tissue to provide optical relay, the method of choice for imaging neurons and neural activity in deeply buried brain structures. Incorporating a Bessel focus scanning module into two-photon fluorescence microendoscopy, we extended the excitation focus axially and improved its lateral resolution. Scanning the Bessel focus in 2D, we imaged volumes of neurons at high-throughput while resolving fine structures such as synaptic terminals. We applied this approach to the volumetric anatomical imaging of dendritic spines and axonal boutons in the mouse hippocampus, and functional imaging of GABAergic neurons in the mouse lateral hypothalamus .

}, issn = {2050-084X}, doi = {10.7554/eLife.40805}, author = {Meng, Guanghan and Liang, Yajie and Sarsfield, Sarah and Jiang, Wan-Chen and Lu, Rongwen and Dudman, Joshua Tate and Aponte, Yeka and Ji, Na} } @article {49367, title = {In vivo measurement of afferent activity with axon-specific calcium imaging.}, journal = {Nature Neuroscience}, year = {2018}, month = {2018 Aug 20}, abstract = {

In vivo calcium imaging from axons provides direct interrogation of afferent neural activity, informing the neural representations that a local circuit receives. Unlike in somata and dendrites, axonal recording of neural activity-both electrically and optically-has been difficult to achieve, thus preventing comprehensive understanding of neuronal circuit function. Here we developed an active transportation strategy to enrich GCaMP6, a genetically encoded calcium indicator, uniformly in axons with sufficient brightness, signal-to-noise ratio, and photostability to allow robust, structure-specific imaging of presynaptic activity in awake mice. Axon-targeted GCaMP6 enables frame-to-frame correlation for motion correction in axons and permits subcellular-resolution recording of axonal activity in previously inaccessible deep-brain areas. We used axon-targeted GCaMP6 to record layer-specific local afferents without contamination from somata or from intermingled dendrites in the cortex. We expect that axon-targeted GCaMP6 will facilitate new applications in investigating afferent signals relayed by genetically defined neuronal populations within and across specific brain regions.

}, issn = {1546-1726}, doi = {10.1038/s41593-018-0211-4}, author = {Broussard, Gerard Joey and Liang, Yajie and Fridman, Marina and Unger, Elizabeth K and Meng, Guanghan and Xiao, Xian and Ji, Na and Petreanu, Leopoldo and Tian, Lin} } @article {49181, title = {Three-photon fluorescence microscopy with an axially elongated Bessel focus.}, journal = {Optics Letters}, volume = {43}, year = {2018}, month = {2018 Apr 15}, pages = {1914-1917}, abstract = {

Volumetric imaging tools that are simple to adopt, flexible, and robust are in high demand in the field of neuroscience, where the ability to image neurons and their networks with high spatiotemporal resolution is essential. Using an axially elongated focus approximating a Bessel beam, in combination with two-photon fluorescence microscopy, has proven successful at such an endeavor. Here, we demonstrate three-photon fluorescence imaging with an axially extended Bessel focus. We use an axicon-based module that allowed for the generation of Bessel foci of varying numerical apertures and axial lengths, and apply this volumetric imaging tool to image mouse brain slices and for in vivo imaging of the mouse brain.

}, issn = {1539-4794}, doi = {10.1364/OL.43.001914}, author = {Rodriguez, Cristina and Liang, Yajie and Lu, Rongwen and Ji, Na} } @article {48891, title = {A general method to fine-tune fluorophores for live-cell and in vivo imaging.}, journal = {Nature Methods}, volume = {14}, year = {2017}, month = {2017 Oct}, pages = {987-994}, abstract = {

Pushing the frontier of fluorescence microscopy requires the design of enhanced fluorophores with finely tuned properties. We recently discovered that incorporation of four-membered azetidine rings into classic fluorophore structures elicits substantial increases in brightness and photostability, resulting in the Janelia Fluor (JF) series of dyes. We refined and extended this strategy, finding that incorporation of 3-substituted azetidine groups allows rational tuning of the spectral and chemical properties of rhodamine dyes with unprecedented precision. This strategy allowed us to establish principles for fine-tuning the properties of fluorophores and to develop a palette of new fluorescent and fluorogenic labels with excitation ranging from blue to the far-red. Our results demonstrate the versatility of these new dyes in cells, tissues and animals.

}, keywords = {Animals, Brain, Cell Line, Coloring Agents, Drosophila, Image Processing, Computer-Assisted, Larva, Mice, Microscopy, Fluorescence, Photochemical Processes, Staining and Labeling}, issn = {1548-7105}, doi = {10.1038/nmeth.4403}, author = {Grimm, Jonathan B and Muthusamy, Anand K and Liang, Yajie and Brown, Timothy A and Lemon, William C and Patel, Ronak and Lu, Rongwen and Macklin, John J and Keller, Philipp J and Ji, Na and Lavis, Luke D} } @article {48304, title = {Video-rate volumetric functional imaging of the brain at synaptic resolution.}, journal = {Nature Neuroscience}, volume = {20}, year = {2017}, month = {2017 Feb 27}, pages = {620-8}, abstract = {

Neurons and neural networks often extend hundreds of micrometers in three dimensions. Capturing the calcium transients associated with their activity requires volume imaging methods with subsecond temporal resolution. Such speed is a challenge for conventional two-photon laser-scanning microscopy, because it depends on serial focal scanning in 3D and indicators with limited brightness. Here we present an optical module that is easily integrated into standard two-photon laser-scanning microscopes to generate an axially elongated Bessel focus, which when scanned in 2D turns frame rate into volume rate. We demonstrated the power of this approach in enabling discoveries for neurobiology by imaging the calcium dynamics of volumes of neurons and synapses in fruit flies, zebrafish larvae, mice and ferrets in vivo. Calcium signals in objects as small as dendritic spines could be resolved at video rates, provided that the samples were sparsely labeled to limit overlap in their axially projected images.

}, issn = {1546-1726}, doi = {10.1038/nn.4516}, author = {Lu, Rongwen and Sun, Wenzhi and Liang, Yajie and Kerlin, Aaron and Bierfeld, Jens and Seelig, Johannes D and Wilson, Daniel E and Scholl, Benjamin and Mohar, Boaz and Tanimoto, Masashi and Koyama, Minoru and Fitzpatrick, David and Orger, Michael B and Ji, Na} } @article {6892, title = {Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes.}, journal = {Nature Methods}, volume = {11}, year = {2014}, month = {2014 Feb}, pages = {175-82}, abstract = {

The quality of genetically encoded calcium indicators (GECIs) has improved dramatically in recent years, but high-performing ratiometric indicators are still rare. Here we describe a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors with a reduced number of calcium binding sites per sensor. These {\textquoteright}Twitch{\textquoteright} sensors are based on the C-terminal domain of Opsanus troponin C. Their FRET responses were optimized by a large-scale functional screen in bacterial colonies, refined by a secondary screen in rat hippocampal neuron cultures. We tested the in vivo performance of the most sensitive variants in the brain and lymph nodes of mice. The sensitivity of the Twitch sensors matched that of synthetic calcium dyes and allowed visualization of tonic action potential firing in neurons and high resolution functional tracking of T lymphocytes. Given their ratiometric readout, their brightness, large dynamic range and linear response properties, Twitch sensors represent versatile tools for neuroscience and immunology.

}, issn = {1548-7105}, doi = {10.1038/nmeth.2773}, author = {Thestrup, Thomas and Litzlbauer, Julia and Bartholom{\"a}us, Ingo and Mues, Marsilius and Russo, Luigi and Dana, Hod and Kovalchuk, Yuri and Liang, Yajie and Kalamakis, Georgios and Laukat, Yvonne and Becker, Stefan and Witte, Gregor and Geiger, Anselm and Allen, Taylor and Rome, Lawrence C and Chen, Tsai-Wen and Kim, Douglas S. and Garaschuk, Olga and Griesinger, Christian and Griesbeck, Oliver} }