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.
Despite the growing popularity of SPIM, the fundamental imaging depth limit of this microscopy technique has yet to characterized either theoretically or experimentally. @cite_38 reported that in their experiments the signal strength and the noise strength are equal at 10 MFPs, but did not characterize the factors that affect this maximum imaging depth. Here, using the openSPIM system, @cite_16 we also show a depth limit close to 10 MFPs, and take this result further to identify the major factors that influence this imaging depth.
@cite_38 @cite_16
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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. A compact real-time fluorescence lifetime imaging microscopy (FLIM) system based on an array of low dark count 0.13?m CMOS singlephoton avalanche diodes (SPADs) is demonstrated. Fast background-insensitive fluorescence lifetime determination is achieved by use of a recently proposed algorithm called ‘Integration for Extraction Method’ (IEM) [J. Opt. Soc. Am. A 25, 1190 (2008)]. Here, IEM is modified for a wider resolvability range and implemented on the FPGA of the new SPAD array imager. We experimentally demonstrate that the dynamic range and accuracy of calculated lifetimes of this new camera is suitable for widefield FLIM applications by imaging a variety of test samples, including various standard fluorophores covering a lifetime range from 1.6ns to 16ns, microfluidic mixing of fluorophore solutions, and living fungal spores of Neurospora Crassa. The calculated lifetimes are in a good agreement with literature values. Real-time fluorescence lifetime imaging is also achieved, by performing parallel 32 × 16 lifetime calculations, realizing a compact and low-cost FLIM camera and promising for bigger detector arrays.