Volumetric imaging of fast cellular dynamics with deep learning enhanced bioluminescence microscopy

Morales-Curiel, Luis Felipe; Gonzalez, Adriana Carolina; Castro-Olvera, Gustavo; Lin, Li-Chun; El-Quessny, Malak; Porta-de-la-Riva, Montserrat; Severino, Jacqueline; Morera, Laura Battle; Venturini, Valeria; Ruprecht, Verena; Ramallo, Diego; Loza-Álvarez, Pablo; Krieg, Michael

doi:10.1038/s42003-022-04292-x

Cited by 12 publications

(31 citation statements)

References 79 publications

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“…Other groups have employed custom built microscopes that we expect would be capable of achieving high frame rate bioluminescent light recordings. 43 – 45 A caveat to these designs is that they require calibration and a level of expertise in construction that many may not have the time or resources necessary to effectively utilize for the initial adoption of bioluminescence imaging. For this reason, we chose to use off-the-shelf components including a pre-assembled dissection microscope for this study and expect to improve on our results going forward.…”

Section: Discussionmentioning

confidence: 99%

Method for optimizing imaging parameters to record neuronal and cellular activity at depth with bioluminescence

Silvagnoli,

Taylor,

Slaviero

et al. 2024

Neurophoton.

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. Significance Optical imaging has accelerated neuroscience in recent years. Genetically encoded fluorescent activity sensors of calcium, neurotransmitters, and voltage are commonly used for optical recording of neuronal activity. However, fluorescence imaging is limited to superficial regions for in vivo activity imaging, due to photon scattering and absorbance. Bioluminescence imaging offers a promising alternative for achieving activity imaging in deeper brain regions without hardware implanted within the brain. Bioluminescent reporters can be genetically encoded and produce photons without external excitation. The use of enzymatic photon production also enables prolonged imaging sessions without the risk of photobleaching or phototoxicity, making bioluminescence suitable for non-invasive imaging of deep neuronal populations. Aim To facilitate the adoption of bioluminescent activity imaging, we sought to develop a low cost, simple in vitro method that simulates in vivo conditions to optimize imaging parameters for determining optimal exposure times and optical hardware configurations to determine what frame rates can be captured with an individual lab’s imaging hardware with sufficient signal-to-noise ratios without the use of animals prior to starting an in vivo experiment. Approach We developed an assay for modeling in vivo optical conditions with a brain tissue phantom paired with engineered cells that produce bioluminescence. We then used this assay to limit-test the detection depth versus maximum frame rate for bioluminescence imaging at experimentally relevant tissue depths using off-the-shelf imaging hardware. Results We developed an assay for modeling in vivo optical conditions with a brain tissue phantom paired with engineered cells that produce bioluminescence. With this method, we demonstrate an effective means for increasing the utility of bioluminescent tools and lowering the barrier to adoption of bioluminescence activity imaging. Conclusions We demonstrated an improved method for optimizing imaging parameters for activity imaging in vivo with bioluminescent sensors.

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Section: Discussionmentioning

confidence: 99%

Method for optimizing imaging parameters to record neuronal and cellular activity at depth with bioluminescence

Silvagnoli,

Taylor,

Slaviero

et al. 2024

Neurophoton.

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“…The eNL with mTurquoise and mNeongreen as acceptors worked particularly well and showed video-rate calcium dynamics in cultured cells and transgenic Caenorhabditis elegans animals. 12 , 22 Several variants with different calcium affinity were optimized to visualize calcium in subcellular organelles such as the endoplasmatic reticulum and mitochondria. 57 A ratiometric BRET sensor (termed CalfluxVTN) was developed in which the Nluc moiety and a Venus fluorophore were connected by a troponin-C calcium sensor with optimized linker residues to maximize dynamic range.…”

Section: Functional Imaging Of Physical and Physiological Properties ...mentioning

confidence: 99%

“… 3 Finally, PhAST has the advantage that the light output increases with presynaptic activity and, in principle, these photons can be imaged on a camera detector and can thus also be purposed as an indicator for synaptic activity. 3 , 12 …”

Section: Functional Control Over Neuronal Activity With Bioluminescencementioning

confidence: 99%

“…This was successfully achieved by fusing Syn-ATP to Synaptophysin 4 or TeNL to SNG-1 synaptogyrin 95 to enhance the localization of the luciferase at presynaptic varicosities in mouse neurons or C. elegans ASH neurons, respectively; but also the nucleus in mouse embryonic stem cells. 12 Codon optimization of Gaussia luciferase cDNA sequence has been shown to increase expression levels in the heterologous host compared with its wildtype form, 115 which became the standard in the field. 24 , 95 Likewise, the insertion of artificial introns is commonly known to increase transgene expression in C. elegans 95 , 116 and specific 3’UTRs might regulate expression through mRNA stabilization in a cell-type specific manner.…”

Section: Workhop: Optimizing Bioluminescent Signals For In ...mentioning

confidence: 99%

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Bioluminescence as a functional tool for visualizing and controlling neuronal activity in vivo

Porta-de-la-Riva,

Morales-Curiel,

Carolina Gonzalez

et al. 2024

Neurophoton.

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. The use of bioluminescence as a reporter for physiology in neuroscience is as old as the discovery of the calcium-dependent photon emission of aequorin. Over the years, luciferases have been largely replaced by fluorescent reporters, but recently, the field has seen a renaissance of bioluminescent probes, catalyzed by unique developments in imaging technology, bioengineering, and biochemistry to produce luciferases with previously unseen colors and intensity. This is not surprising as the advantages of bioluminescence make luciferases very attractive for noninvasive, longitudinal in vivo observations without the need of an excitation light source. Here, we review how the development of dedicated and specific sensor-luciferases afforded, among others, transcranial imaging of calcium and neurotransmitters, or cellular metabolites and physical quantities such as forces and membrane voltage. Further, the increased versatility and light output of luciferases have paved the way for a new field of functional bioluminescence optogenetics, in which the photon emission of the luciferase is coupled to the gating of a photosensor, e.g., a channelrhodopsin and we review how they have been successfully used to engineer synthetic neuronal connections. Finally, we provide a primer to consider important factors in setting up functional bioluminescence experiments, with a particular focus on the genetic model Caenorhabditis elegans , and discuss the leading challenges that the field needs to overcome to regain a competitive advantage over fluorescence modalities. Together, our paper caters to experienced users of bioluminescence as well as novices who would like to experience the advantages of luciferases in their own hand.

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“…In addition, when multiple markers are applied, the total exposure time increases with an increase in the number of objects, and the individual objects are observed at different times. To overcome this problem, a method was employed to simultaneously detect different wavelengths by separating the optical path [15][16][17] . Yao et al developed a phasor analysis method that is commonly used to distinguish spectrally similar luminophores 18 .…”

Section: Introductionmentioning

confidence: 99%

Creating coveted bioluminescence colors for simultaneous myriad-color bioimaging

Hattori,

Wazawa,

Orioka

et al. 2024

Preprint

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Bioluminescence, an optical marker that does not require excitation by light, allows researchers to simultaneously observe multiple targets, each exhibiting a different color. Notably, the colors of the bioluminescent proteins must sufficiently vary to enable simultaneous detection. Here, we aimed to introduce a method that can be used to expand the color variation by tuning dual-acceptor bioluminescence resonance energy transfer (BRET). Using this approach, we could visualize multiple targets with up to 20 colors through single-shot acquisition using a color CMOS camera. Overall, this method enables simple and simultaneous observation of numerous biological targets and phenomena.

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Volumetric imaging of fast cellular dynamics with deep learning enhanced bioluminescence microscopy

Cited by 12 publications

References 79 publications

Method for optimizing imaging parameters to record neuronal and cellular activity at depth with bioluminescence

Method for optimizing imaging parameters to record neuronal and cellular activity at depth with bioluminescence

Bioluminescence as a functional tool for visualizing and controlling neuronal activity in vivo

Creating coveted bioluminescence colors for simultaneous myriad-color bioimaging

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