While two-photon microscopy has ushered in a revolution in brain imaging over the past 15 years, several limitations remain. Firstly, penetration depths remain limited to a few hundred microns, and typically, either exogenous dyes or transgenic animals are required. Here we present a different strategy for deep tissue brain imaging using intrinsic scattering contrast, i.e. contrast arising from endogenous properties of biological tissues. We demonstrate imaging depths of 2.5-3x higher than standard two-photon microscopy in the living brain. This penetration depth was achieved with a continuous wave infrared superluminescent diode light source, with optical powers at the sample of approximately 4 mW.
Our data show that OCM enables visualization of cell bodies through a negative contrast mechanism. Neurites (axons, dendrites) exhibit high backscatter due to their size (on the order of a wavelength) and refractive index contrast [27
]. Even so, due to the high refractive index of myelin, myelinated axons in the en face
plane appear as highly scattering processes against an already highly scattering background, and can thus be enhanced through performing a maximum intensity projection.
By comparison, neuronal cell bodies appear as low scattering regions. However, the cell body is known to consist of multiple organelles of higher refractive index than the cytosol. Therefore, one possible explanation for the relatively low scattering from neuronal cell bodies is that the organelles and subcellular structures are packed tightly enough to present negligible refractive index variations on a sufficiently large spatial scale that would lead to significant backscatter. An alternative explanation is that the large-scale refractive index variations between cytosol and organelles within a cell are much smaller than refractive index variations between neurites and extracellular fluid. Therefore, in viable tissue, intracellular organelles in the cell body are tightly packed or refractive index variations are not significant; therefore backscattering from organelles is low compared to backscattering from neurites. In models of light scattering designed to detect pre-cancerous changes, cell nuclei were assumed to account for the periodic fine structure in reflectance from biological tissue [28
]. However, recent experimental measurements of refractive index have shown relatively small variations in refractive index within the cell itself and in particular, less refractive index contrast between the nucleus and other cellular regions than was previously thought [29
]. This recent work is in agreement with our observation that neuronal cell bodies appeared as relatively lower scattering regions against a highly scattering background of neurites. A similar phenomenon occurs in the retina, where OCT images show highly backscattering plexiform layers and less backscattering nuclear layers [30
Our data show that anoxic depolarization is accompanied by cell swelling and increased scattering from subcellular compartments, resulting in a loss of contrast. A number of physiological processes occur upon energy failure. Firstly, channels open and the cell swells as water invades the intracellular space. At first glance, entrance of the water into the intracellular space would seem to lead to a reduction in backscatter as cell size increases [31
] and the average cellular refractive index is reduced. However, the entrance of water into the cell may also reduce the organelle packing density and increase inhomgeneities in the intracellular refractive index, thereby leading to wavelength-scale intracellular refractive index variations that result in high scattering. Intracellular organelle swelling [34
] and shape changes, as well as membrane failure, nuclear condensation and fragmentation may also lead to increased scattering. The neurite environment also increases scattering after anoxic depolarization due to processes such as dendritic beading [35
]. Diffusion weighted MRI [37
] measures changes in the apparent diffusion coefficient are caused by intercompartmental water shifts associated with cytotoxic edema. Our results suggest that analogous optical scattering markers can be defined to measure cell viability using intrinsic contrast.
We anticipate that these results will enable a number of novel in vivo
and ex vivo
optical imaging paradigms. Volumetric OCM data may provide information about cell density, volume, myelination, and capillary density at cortical depths exceeding 1 mm. These in vivo
techniques have potential advantages over conventional histological methods, since measurements can be performed repeatedly without animal sacrifice, and do not require fixation, slicing, or staining. The capability to image myelinated axons using only intensity-based information, as opposed to birefringence [40
], will enable all-optical tractography and studies of brain connectivity. As myelin presents the main barrier that causes anisotropic diffusion in white matter, our measurements of microscopic fiber directionality could be used to directly validate diffusion MRI [41
]. While current imaging depths are already sufficient to image the entire cortex in mice, alternative imaging geometries may enable greater depth penetration, and improve sensitivity to non-transverse fiber orientations. Further studies will be required to investigate changes in scattering contrast after fixation, dehydration, and optical clearing procedures.