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
2014644083 2083575776 2013297745 2141354231
We examine the performance of confocal microscopes designed for probing structures embedded in turbid media. A heuristic scheme is described that combines a numerical Monte Carlo simulation of photon transport in a turbid medium with a geometrical ray trace through the confocal optics. To show the effects of multiple scattering on depth discrimination, we compare results from the Monte Carlo simulations and scalar diffraction theory. Experimental results showing the effects of the pinhole diameter and other variables on imaging performance at various optical depths in suspensions of polystyrene microspheres were found to correspond well with the Monte Carlo simulations. The major conclusion of the paper is that the trade-off between signal level and background scattered-light rejection places a fundamental limit on the sectioning capability of the microscope. 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. 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. 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.