This data set contains the infrared spectra of C57Bl/6J female mice subjected to conventional dose rate irradiations and to FLASH radiotherapy. Spectra were measured via Fourier transform infrared microspectroscopy.

1. Description of methods used for collection-generation of data:

Animals: All animal procedures were conducted in accordance with the Swiss ethics committee (VD3603) for animal experimentation. 10 weeks old female C57Bl/6J mice (purchased from Charles River Laboratories, France) were divided into two controls (labeled as Ctrl_1 and Ctrl_2), two animals irradiated with conventional dose rates (Conv_1 and Conv_2) and two animals irradiated with FLASH-RT (FLASH_1 and FLASH_2) (n = 6).

Irradiations: Whole brain irradiations at 10 Gy were performed on a prototype Oriatron 6e (PMB Alcen, France), a 6-MeV electron beam linear accelerator (LINAC). FLASHRT was delivered in 1 pulse of 1.8 μs and conventional irradiations at 0.1 Gy/s.

Sample preparation: At 24 h post-RT, the whole brain was snap-frozen in isopentane cooled in liquid nitrogen. Prior to the FTIRM measurements, 4 μm-thick brain cryosections were deposited onto low-e microscope slides (Kevley Technologies). Sections were immersed in zinc formalin solution (Sigma-Aldrich) and then, rinsed with Millipore ultrapure water and dried.

FTIRM Measurements: FTIRM was performed at the MIRAS beamline of ALBA Synchrotron using the BRUKER 3000 Hyperion microscope coupled to a Vertex 70 spectrometer. For the measurements, a conventional infrared source was employed. Each infrared spectrum was acquired in the mid-infrared range after 4 co-added scans with a 4 cm−1 spectral resolution. Several regions of interest (ROIs) were selected in the brain sections: • ROI 1: Hip SO (Hippocampus stratum oriens), • ROI 2: Hip SP (Hippocampus pyramidal layer), • ROI 3: Hip SR (Hippocampus stratum radiatum), • ROI 4: Hip DG (Hippocampus dentate gyrus), • ROI 5: Cortex FR/MO (Frontal and somatomor areas of the Cortex), • ROI 6: Cb W (Cerebellum white matter), • ROI 7: Cb G (Cerebellum gray matter).

1. Methods for processing the data: Spectral data was cut into two distinct spectral ranges: the Higher Wavenumber (HW, 3000-2800 cm-1) or Amides and Fingerprint (AFP, 1800-950 cm-1). In both spectral regions, data was processed using a Savitzky-Golay filter was applied (2nd derivative order; 5 points filter, 3rd polynomial order), followed by unit vector normalization. Data processing was performed with the Quasar software (version 1.7).

2. Instrument- or software- specific information needed to interpret the data: Any software able to read csv files.

3. Instruments, calibration and standards information: Hyperion 3000 microscope, Vertex 70 Spectrometer (Bruker Optics GmbH).

4. Environmental or experimental conditions: SR-FTIRM measurements were carried out on dried tissues at room temperature.