Quantitative third-harmonic generation imaging of mouse visual cortex areas reveals correlations between functional maps and structural substrates

Abstract

The structure of brain regions is assumed to correlate with their function, but there are very few instances in which the relationship has been demonstrated in the live brain. This is due to the difficulty of simultaneously measuring functional and structural properties of brain areas, particularly at cellular resolution. Here, we performed label-free, third-harmonic generation (THG) microscopy to obtain a key structural signature of cortical areas, their effective attenuation lengths (EAL), in the vertical columns of functionally defined primary visual cortex and five adjacent visual areas in awake mice. EALs measured by THG microscopy in the cortex and white matter showed remarkable correspondence with the functional retinotopic sign map of each area. Structural features such as cytoarchitecture, myeloarchitecture and blood vessel architecture were correlated with areal EAL values, suggesting that EAL is a function of these structural features as an optical property of these areas. These results demonstrate for the first time a strong relationship between structural substrates of visual cortical areas and their functional representation maps in vivo. This study may also help in understanding the coupling between structure and function in other animal models as well as in humans.

Label-free three-photon imaging of intact human cerebral organoids: tracking early events in brain development and deficits in Rett Syndrome

“…First, we characterized the extinction (combined scattering and absorption) lengths of WT and MT organoids. Our label-free THG imaging characterization shows that the extinction lengths of a fixed WT and a MT organoid are 162.5 µm and 148.2 µm, respectively which are approximately half of the attenuation length of a primary visual cortex of an awake mouse brain[42, 43] (Fig. 3C-D).…”

“…Optimized designs using high power lasers and high sensitivity photomultipliers have enabled the application of THG microscopy to 3D tissue microscopy by improving the imaging depth in tissues with varying scattering coefficients [35][36][37][38]. THG microscopy has been applied recently to the non-invasive monitoring of human adipose tissue [39], cell nuclei and cytoplasm in liver tissue [40] and subcortical structures within an intact mouse brain [41][42][43]. THG imaging provides the advantages of micron scale resolution, label free imaging, deep tissue penetration depth, and non-destructiveness.…”

Long-termin vivothree-photon imaging reveals region-specific differences in healthy and regenerative oligodendrogenesis

“…2d). Therefore, we measured mouse-specific effective attenuation lengths (EALs) in the adult posterior parietal cortex to estimate the pulse energy delivered at each focal plane as described previously 21,28,34 (Extended Data Figure 5; Methods). The pulse energy at the focal plane was calculated using the equation below:…”

DEEP2: Deep Learning Powered De-scattering with Excitation Patterning

“…In addition to the experimentally acquired beads instances, we also acquired the blood vessel instances at increasing depths using the DEEP-TFM. Hence, to train the network, we used an equivalent vascular structural image volume acquired using a three-photon Fluorescent microscope 30 . The vascular image stack consists of 800 z-stacks, each had an increment of 1.5 um and each sized 512 pixels * 512 pixels with a spatial resolution of 0.6um corresponding to a field of view of 300um.…”