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GENERAL INFORMATION
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Title of Dataset: In vivo pump-probe and multiphoton fluorescence microscopy of melanoma and pigmented lesions in a mouse model

Authors: Wilson, Jesse; Degan, Simone; Gainey, Christina; Mitropoulos, Tanya; Simpson, Mary Jane; Zhang, Jennifer; Warren, Warren

Funder: NIH (R01-CA166555)

Associated Publications: Wilson, J. W., Degan, S., Gainey, C. S., Mitropoulos, T., Simpson, M. J., Zhang, J. Y., & Warren, W.S. (2014). Comparing in vivo pump–probe and multiphoton fluorescence microscopy of melanoma and pigmented lesions. Journal of Biomedical Optics, 20(5), 51012. https://doi.org/10.1117/1.jbo.20.5.051012

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DATA & FILE OVERVIEW
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This dataset contains over 20,000 microscopy images in TIFF stacks (approx ~35GB uncompressed). Each microscopy session folder contains text files with additional information about the mouse and the database session. Due to the large number of files, HTML index files are available to ease navigation. To use these navigation files, download the zipped data files and the zipped "Data_Index" folder. You will want to unzip and save all of the html files in the same location that the data files are stored. The HTML index files have to be co-located with the uncompressed data folder(s) you wish to browse or the index file will not work. The master index file is the home.html file. It has a list of all the mice in the study. Start with this file to use the index. 

A smaller sample dataset labelled Exploratory_Data has been created to allow for end users to more easily understand the data contents prior to downloading the entire collection given its large size.

File Format Note: The TIFF images are currently labelled with an .ijt file extension (this an arbitrary extension assignment, not a J Software labs file). The .tiff file extension was changed due to issues with the automatic thumbnail generation for the index files within Windows. Because of these issues, it is not recommended to change the extension back to .tiff; rather, the files should be opened in software that recognizes the TIF tags inside the file (e.g. ImageJ/FIJI, or in MATLAB using the imread function and specifying the format). More information on downloading and using ImageJ/FIJI is available at https://imagej.nih.gov/ij/ or at https://fiji.sc/

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METHODOLOGICAL INFORMATION
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We demonstrate a multimodal approach that combines a pump–probe with confocal reflectance and multiphoton autofluorescence microscopy. Pump–probe microscopy has been proven to be of great value in analyzing thin tissue sections of pigmented lesions, as it produces molecular contrast which is inaccessible by other means. However, the higher optical intensity required to overcome scattering in thick tissue leads to higher-order nonlinearities in the optical response of melanin (e.g., two-photon pump and one-photon probe) that present additional challenges for interpreting the data. We show that analysis of pigment composition in vivo must carefully account for signal terms that are nonlinear with respect to the pump and probe intensities. We find that pump–probe imaging gives useful contrast for pigmented structures over a large range of spatial scales (100 µm to 1 cm), making it a potentially useful tool for tracking the progression of pigmented lesions without the need to introduce exogenous contrast agents. 

Data format:
There are two types of image stacks: time-delay scans (delay-stacks) and depth scans (Z-stacks). Both types are saved as multi-page tiff files (each page is a single time delay or depth, respectively). Each acquisition channel (reflectance confocal, pump-probe) is its own tiff file and the ending of the file name denotes the channel number (..._CHx.ijt, where x is the channel number). Each stack is accompanied by a text file that summarizes the imaging parameters, such as image size, power levels, averages, time- or z-stack values, etc.)

Data Layout:
The data files are arranged hierarchically by subject (mouse), imaging session (date), and scan type (delay scan or Z-scan). Each folder contains a text file with information on the subject, imaging session, or image acquisition parameters, respectively. The information on subject and imaging sessions are also contained in the html index files to ease navigation.
Most keys for the subject/imaging session/image text or index files are self-explanatory, with some exceptions:
Scan Range: x/y (V) -> Scan range of the galvo scanner in x/y direction. This corresponds to a field-of-view of approximately 740 µm/V for the 10x objective, 370 µm/V for the 20x objective, and 185 µm/V for the 40x objective.
ms/line -> Time in milliseconds per scan line
z (um) or t (ps) -> Array of time delay or Z-values for the image stack
Note: If these parameters were unavailable, they are given as ‘null’.