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.
As an alternative approach to estimate the depth limit of 2PM, @cite_21 used probabilistic model to estimate the contrast between in-focus and out-of-focus fluorescence. They reported a depth limit between 15-22 MFPs at which the image contrast falls to one reported the depth limit as 10-15 MFP, calculated at excitation wavelength, which translates to 15-22 MFPs at emission wavelength of 560 nm. .
@cite_21
2013297745 2113245224 2161844736 1969128393
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. Endogenous fluorescence provides morphological, spectral, and lifetime contrast that can indicate disease states in tissues. Previous studies have demonstrated that two-photon autofluorescence microscopy (2PAM) can be used for noninvasive, three-dimensional imaging of epithelial tissues down to approximately 150 μm beneath the skin surface. We report ex-vivo 2PAM images of epithelial tissue from a human tongue biopsy down to 370 μm below the surface. At greater than 320 μm deep, the fluorescence generated outside the focal volume degrades the image contrast to below one. We demonstrate that these imaging depths can be reached with 160 mW of laser power (2-nJ per pulse) from a conventional 80-MHz repetition rate ultrafast laser oscillator. To better understand the maximum imaging depths that we can achieve in epithelial tissues, we studied image contrast as a function of depth in tissue phantoms with a range of relevant optical properties. The phantom data agree well with the estimated contrast decays from time-resolved Monte Carlo simulations and show maximum imaging depths similar to that found in human biopsy results. This work demonstrates that the low staining inhomogeneity (∼20) and large scattering coefficient (∼10 mm−1) associated with conventional 2PAM limit the maximum imaging depth to 3 to 5 mean free scattering lengths deep in epithelial tissue. 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. A novel architecture for performing hue-saturation-value (HSV) domain enhancement of digital color images with non-uniform lighting conditions is proposed in this paper for video streaming applications. The approach promotes log-domain computation to eliminate all multiplications, divisions and exponentiations utilizing the effective logarithmic estimation modules. An optimized quadrant symmetric architecture is incorporated into the design of homomorphic filter for the enhancement of intensity value. Efficient modules are also presented for conversion between RGB and HSV color spaces. The design is able to bring out details hidden in shadow regions of the image. It is capable of producing 187.86 million outputs per second (MOPs) on Xilinx's Virtex II XC2V2000-4ff896 field programmable gate array (FPGA) at a clock frequency of 187.86 MHz. It can process over 179.1 (1024 X 1024) frames per second and consumes approximately 70.7 and 76.8 less hardware resource with 127 and 280 performance boost when compared to the designs with machine learning algorithm [10], and with separated dynamic and contrast enhancements [11], respectively.