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.
The depth limits of confocal and multi-photon microscopy is a well-studied property @cite_36 @cite_11 @cite_37 @cite_23 @cite_30 @cite_28 . Light scattering by tissue is the primary factor that limits the imaging depth: the stronger the scattering, the shallower the depth limit. To meaningfully account for the effect of tissue scattering while comparing the imaging capabilities of various microscopy techniques, depth limits of imaging systems are typically expressed as a multiple of the number of mean free paths (MFPs), or scattering length, within the sample tissue @cite_36 @cite_37 . The mean free path is defined as the average distance a photon travels before a scattering event.
@cite_30 @cite_37 @cite_36 @cite_28 @cite_23 @cite_11
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It is highly desirable to be able to optically probe biological activities deep inside live organisms. By employing a spatially confined excitation via a nonlinear transition, multiphoton fluorescence microscopy has become indispensable for imaging scattering samples. However, as the incident laser power drops exponentially with imaging depth due to scattering loss, the out-of-focus fluorescence eventually overwhelms the in-focal signal. The resulting loss of imaging contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation intensity. Herein we propose to significantly extend this depth limit by multiphoton activation and imaging (MPAI) of photo-activatable fluorophores. The imaging contrast is drastically improved due to the created disparity of bright-dark quantum states in space. We demonstrate this new principle by both analytical theory and experiments on tissue phantoms labeled with synthetic caged fluorescein dye or genetically encodable photoactivatable GFP. A model for the propagation of a focused light beam in a strongly scattering medium is used to analyse various factors that limit the capability of two-photon fluorescence microscopy (TPFM) to image deep sections of optically thick biological specimens. The TPFM imaging depth is shown to be limited by three main factors: (1) beam broadening as a result of multiple small-angle scattering, leading to a loss of submicron lateral resolution; (2) strong near-surface fluorescence at large imaging depths as a result of the increase in average source power in order to compensate for scattering losses; (3) reduction in useful two-photon fluorescence signal level because of the exponential attenuation of the excitation power. The influence of these factors is examined in a small-angle diffusion approximation of radiative transfer theory. The first two of them are shown to set a fundamental TPFM limit, whereas the last is an instrumental limitation and appears to be the most critical to state-of-the-art commercial two-photon laser scanning microscopy systems for the vast majority of fluorophores in current use. We have analyzed how the maximal imaging depth of two-photon microscopy in scattering samples depends on properties of the sample and the imaging system. We find that the imaging depth increases with increasing numerical aperture and staining inhomogeneity and with decreasing excitation-pulse duration and scattering anisotropy factor, but is ultimately limited by near-surface fluorescence with slight improvements possible using special detection strategies. We investigate limits of the confocal microscope when applied to imaging through scattering layers and compare its performance with that of a correlation (heterodyne) microscope. Confocal laser scanning microscopy is shown to make possible imaging through scattering media owing to the spatial filtering of the signal back-reflected from the sample. Its performance is limited by the noise of the detection system and or insufficient rejection of scattered light, depending on the sample under investigation. Correlation microscopy with narrow or broad bandwidth light can extend these limits by selective, optical amplification of the image information.