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.
@cite_35 measured the depth limit of confocal microscopy experimentally using a reflecting grating structure suspended in a solution of latex spheres and water. To simulate the effect of imaging deeper, increased the concentration of the latex spheres thereby increasing the number of mean free paths in their sample. When the concentration of the latex sphere was increased to 2
@cite_35
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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.