Title: Data from: Video-rate raman-based metabolic imaging by airy light-sheet illumination and photon-sparse detection Dataset DOI: 10.11578/1908656 Data, code and/or products within this dataset support the follwoing manuscript: Manuscript Title: Video-rate raman-based metabolic imaging by airy light-sheet illumination and photon-sparse detection Journal: PNAS DOI: 10.1073/pnas.2210037120 Description/Abstract: Data supporting manuscript submitted to PNAS: Video-Rate Raman-based Metabolic Imaging by Airy Light-Sheet Illumination and Photon-Sparse Detection. The data set includes: [1] raw data and [2] related images used in the analyses described within the manuscript. Despite its massive potential, Raman imaging represents just a modest fraction of all research and clinical microscopy to date. This is due to the ultralow Raman scattering cross-sections of most biomolecules that impose low-light or photon-sparse conditions. Bioimaging under such conditions is suboptimal, as it either results in ultralow frame rates or requires increased levels of irradiance. Here, we overcome this tradeoff by introducing Raman imaging that operates at both video rates and 1,000-fold lower irradiance than state-of-the-art methods. To accomplish this, we deployed a judicially designed Airy light-sheet microscope to efficiently image large specimen regions. Further, we implemented subphoton per pixel image acquisition and reconstruction to confront issues arising from photon sparsity at just millisecond integrations. We demonstrate the versatility of our approach by imaging a variety of samples, including the three-dimensional (3D) metabolic activity of single microbial cells and the underlying cell-to-cell variability. To image such small-scale targets, we again harnessed photon sparsity to increase magnification without a field-of-view penalty, thus, overcoming another key limitation in modern light-sheet microscopy. **Data Use**: *License*: [CC-BY 4.0](https://creativecommons.org/licenses/by/4.0/) *Recommended Citation*: Vasdekis AE (2023) Data from: Video-rate raman-based metabolic imaging by airy light-sheet illumination and photon-sparse detection [Dataset]. University of Idaho. https://doi.org/10.11578/1908656 Resource URL: https://data.nkn.uidaho.edu/dataset/data-video-rate-raman-based-metabolic-imaging-airy-light-sheet-illumination-and-photon Creator(s): 1. Full Name: Andreas E. Vasdekis Unique identifier: https://orcid.org/0000-0003-4315-1047 Affiliation(s): University of Idaho Other Contributor(s): NULL Publisher: University of Idaho Publication Year: 2023 Language(s): American English Subject(s): 1. NATURAL SCIENCES 1.3 Physical Science 1.6 Biological Science Keywords/Tags: Raman imaging, photon-sparse imaging, light-sheet microscopy Resource Type General: Dataset Dates: NULL Date available for the public: 2023-02-22 Sizes: 273.9 MB Format(s): Compressed data deirectory (zip) with txt, csv and tiff files. Version: NULL Funding References: U.S. Department of Energy Award: DE-SC0022282 URL: NULL Title: Integrative Imaging of Plant Roots during Symbiosis with Mycorrhizal Fungi Spatial/Geographical Coverage Location: NULL Temporal Coverage: NULL Granularity of the Data: NULL Contact Info: Contact Name: Andreas Vasdekis Contact Email: andreasv@uidaho.edu Related Content: Peer-reviewed manuscript-PNAS | https://doi.org/10.1073/pnas.2210037120 Project associated code-GitHub | https://github.com/aevasdekis/photon-sparse#photon-sparse Data Files: readme.txt Figure 4: file directory containing data files necessary to compile figure 4 of associated manuscript Figure_4a.csv: data for compiling the histogram presented in figure 4a cell: ID of the cell observed sig_D2O: intensity values of a single cell grown in D2O bg_D2O: intensity values of background in D2O sig-bg_D2O: intensity difference between signal and background for a single cell grown in D2O sig_H2O: intensity values of a single cell grown in H2O [data only available for 1st 10 rows of data (cells h1-h10)] bg_H2O: intensity values of background in H2O [data only available for 1st 10 rows of data (cells h1-h10)] sig-bg_H2O: intensity difference between signal and background for a single cell grown in H2O [data only available for 1st 10 rows of data (cells h1-h10)] Figure_4b.csv: data for compiling the time-trace presented in figure 4b time_min: temporal axis values (in sec) cell: intensity values for a single cell grown in D2O cell_norm: intensity values for a single cell grown in D2O, normalized at t = 1 min Figure 5: file directory containing data files necessary to compile figure 5 of associated manuscript 220121_cloud_40x_90pct_focus.tif: 3D tif stack (xyt) representing single photon clouds as a function of time (t) of a single cell at focus Figure_5_error_analysis.csv: data of cell area error value represented in the insets of figure 5 photons-per-voxel: number of photons per unit voxel xy-area: cell area in the x-y plane in pixel units xy-area-per_norm: cell area in the x-y plane in pixel units normalized at 10 photons per voxel xz-area: cell area in the x-z plane in pixel units xz-area-per_norm: cell area in the x-z plane in pixel units normalized at 10 photons per voxel Figure 6: file directory containing data files necessary to compile figure 6 of associated manuscript cell_1x1_figure_6a.tif: 2D tif image of a Y. lipolytica cell (as per Fig. 5) under pixel-level projection cell_2x2_figure_6b.tif: 2D tif image of a Y. lipolytica cell (as per Fig. 5) under sub-pixel projection Figure_6_traces.csv: data for compiling the traces presented in the insets of figure 6a and 6b Header Key: distance-pixel: x-axis distance in pixel units distance-μm: x-axis distance in micron units trace_1x1: trace of the Y. lipolytica cell under pixel-level projection trace_2x2: trace of the Y. lipolytica cell under sub-pixel projection