Large field-of-view metabolic profiling of murine brain tissue following morphine incubation using label-free multiphoton microscopy

Abstract

Background: Although the effects on neural activation and glucose consumption caused by opiates such as morphine are known, the metabolic machinery underlying opioid use and misuse is not fully explored. Multiphoton microscopy (MPM) techniques have been developed for optical imaging at high spatial resolution. Despite the increased use of MPM for neural imaging, the use of intrinsic optical contrast has seen minimal use in neuroscience.

New method: We present a label-free, multimodal microscopy technique for metabolic profiling of murine brain tissue following incubation with morphine sulfate (MSO₄). We evaluate two- and three-photon excited autofluorescence, and second and third harmonic generation to determine meaningful intrinsic contrast mechanisms in brain tissue using simultaneous label-free, autofluorescence multi-harmonic (SLAM) microscopy.

Results: Regional differences quantified in the cortex, caudate, and thalamus of the brain demonstrate region-specific changes to metabolic profiles measured from FAD intensity, along with brain-wide quantification. While the overall intensity of FAD signal significantly decreased after morphine incubation, this metabolic molecule accumulated near the nucleus accumbens.

Comparison with existing methods: Histopathology requires tissue fixation and staining to determine cell type and morphology, lacking information about cellular metabolism. Tools such as fMRI or PET imaging have been widely used, but lack cellular resolution. SLAM microscopy obviates the need for tissue preparation, permitting immediate use and imaging of tissue with subcellular resolution in its native environment.

Conclusions: This study demonstrates the utility of SLAM microscopy for label-free investigations of neural metabolism, especially the intensity changes in FAD autofluorescence and structural morphology from third-harmonic generation.

Keywords: Label-free imaging; Metabolism; Morphine; Multiphoton microscopy; Nonlinear optics.

Fig. 1.

Label-free images of brain tissue acquired using SLAM microscopy. (A) Mosaicked image of all autofluorescence channels from an untreated murine brain slice. (B-E) 2PAF, 3PAF, SHG, and THG channels, respectively, for the brain slice in (A). (F) Adjacent brain slice treated with 20 μM MSO₄. (G-J) Corresponding 2PAF, 3PAF, SHG, and THG images, respectively. Red arrows point to bright-yellow structures, which are likely to be neurons in this channel.

Fig. 2.

SLAM images of brain tissue for all channels, and quantification of dynamic range for all data used in this investigation. (A) Brain-slice and data from SLAM microscopy datasets. (B-E) Whole-slice mosaics from 2PAF (B), 3PAF (C), SHG (D), and THG (E) channels. (F-I) Montage with individual 2PAF (F), 3PAF (G), SHG (H), and THG (I) for individual mosaics and for whole-brain tissue. (J) Histogram for all SLAM microscopy data captured from each individual channel from numerous brain tissue. (K) Box-and-whisker plot illustrating the distribution of individual SLAM channels for brain tissue collected in this investigation. Statistics generated from n = 9 slices from N = 2 mice.

Fig. 3.

Brain-wide quantification of FAD and NAD(P)H. Isolated FAD channels from tissue incubated in (A) control aCSF, (B) 20 μM morphine sulfate, and (C) 40 μM morphine sulfate. (D-F) NAD(P)H channels that correspond to the FAD channels between (A-C). (G) Histograms for the FAD and NAD(P)H channels for the data in (AF), and (H) the box and whisker plots for the FAD data in each condition. * denotes p < 0.01 and Cohen’s D below −1 or above 1. Statistics from n = 3 slices from N = 1 mouse.

Fig. 4.

Results for data from Fig. 3 separated by cortex (A, D), caudate (B, E) and thalamus (C, F). Notably, histograms of the 2PAF and 3PAF data (A-C) and corresponding box and whisker plot (D-F). * denotes p < 0.01 and Cohen’s D below −1 or above 1. Statistics generated from n = 3 slices (one control, one 20 μM, and one 40 μM slice) from N=1 mouse.

Fig. 5.

Whole-brain statistics for treated and untreated brain tissue. The results in this figure summarize data from all datasets captured in this study. (A) Histogram comparing 2PAF of FAD between control and morphine-treated samples. (B) Bar plots illustrating mean 2PAF of untreated (n = 210,915 pixels) and treated (n = 284,744 pixels) conditions, and (C) corresponding box-and whisker plot. (D) Histogram, (E) bar graph, and (F) box-and-whisker plot separated by control (n = 210,915 pixels), 20 μM treatment (n = 181,510 pixels), and 40 μM (n = 67,576 pixels) morphine treatment. Error bars +/− standard mean error (S.E.M). * denotes p < 0.01 and Cohen’s D below −1 or above 1. Statistics generated from n = 4 control slices, n = 4 slices exposed to 20 μM MSO₄, and n = 1 slice exposed to 40 μM MSO₄, from N = 2 mice.