Deep imaging in scattering media with selective plane illumination microscopy

Abstract

In most biological tissues, light scattering due to small differences in refractive index limits the depth of optical imaging systems. Two-photon microscopy (2PM), which significantly reduces the scattering of the excitation light, has emerged as the most common method to image deep within scattering biological tissue. This technique, however, requires high-power pulsed lasers that are both expensive and difficult to integrate into compact portable systems. Using a combination of theoretical and experimental techniques, we show that if the excitation path length can be minimized, selective plane illumination microscopy (SPIM) can image nearly as deep as 2PM without the need for a high-powered pulsed laser. Compared to other single-photon imaging techniques like epifluorescence and confocal microscopy, SPIM can image more than twice as deep in scattering media ( ? 10 times the mean scattering length). These results suggest that SPIM has the potential to provide deep imaging in scattering media in situations in which 2PM systems would be too large or costly.

Determining the Depth Limit of Bioluminescent Sources in Scattering Media

“…The imaging depth in biological tissue is typically limited by scattering; on average, a 2 photon scatters more than ten times within the first millimeter of most biological 3 tissues [19,20]. This scattering results in blurring of the image and reduced image 4 contrast.…”

“…Together these effects limit the maximum depth at which cellular-resolution 5 imaging is possible [19]. 6 To improve image quality within scattering tissue, researchers often perform in vivo imaging contrast does not suffer from autofluorescence and excitation scattering, which 27 are the primary factors that limit deep fluorescent imaging in scattering media [20]. 28 While bioluminescence imaging still suffers from attenuation and blurring due to 29 emission scattering, with a bright enough source, one would expect to achieve a greater 30 imaging depth than epifluorescence [7,26,34].…”

“…To sufficiently illuminate deeper portions of tissue in standard 51 epifluorescence microscopy, increased excitation energy is used to obtain a larger 52 emitted signal [5]. However, out-of-plane autofluorescence and excitation scattering also 53 increase and reduce contrast [30], limiting the depth limit to 3 MFPs for epifluorescence 54 imaging [20].…”

“…Light propagation through scattering media can be accurately modeled using the 109 radiative transfer equation (RTE). The standard integro-differential equation for a 110 plane-parallel medium is defined as [20]:…”

“…σ t corresponds to the extinction cross section, made 114 up of σ s , scattering cross section, and σ a , absorption cross section (not shown). For 115 situations in which there is little to no absorption, the extinction cross-section can be 116 represented with just the scattering cross-section (σ t = σ s ), which is the case for brain 117 imaging [20], as most brain tissue is predominantly scattering. The change in the 118 intensity of light at a certain (θ, φ) with respect to depth in the medium (left-hand side) 119 depends on two processes (if there is no intensity generation at this z): loss of intensity 120 that is either absorbed or scattered (right hand side, term 1) or light from another 121 direction (θ , φ ) scattering into the (θ, φ) direction (right hand side, term 2).…”