A method for the depth-sensitive detection of fluorescent light is presented. It relies on a structured illumination restricting the excitation volume and on an interferometric detection of the wave front curvature. The illumination with two intersecting beams of a white-light laser separated in a Sagnac interferometer coupled to the microscope provides a coarse confinement in lateral and axial direction. The depth reconstruction is carried out by evaluating shearing interferograms produced with a Michelson interferometer. This setup can also be used with spatially and temporally incoherent light as emitted by fluorophores.
A simulation workflow of the method was developed using a combination of a solution of Maxwell's equations with the Monte Carlo method. These simulations showed the principal feasibility of the method.
The method is validated by measurements at reference samples with characterized material properties, locations and sizes of fluorescent regions. It is demonstrated that sufficient signal quality can be obtained for materials with scattering properties comparable to dental enamel while maintaining moderate illumination powers in the milliwatt range. The depth reconstruction is demonstrated for a range of distances and penetration depths of several hundred micrometers.
The propagation of different focused beams (e.g., Gaussian or quasi-Bessel beams) through scattering media is studied. The finite-difference time-domain method, a numerical solution of Maxwell’s equations, is applied to propagate the light beams in two dimensions. The focused beams are modeled by applying the angular spectrum of the plane waves method. The results show that weakly focused beams exhibit comparable performance to strongly focused beams in delivering focused light deep into scattering media.
A severe drawback to the scalar Monte Carlo (MC) method is the difficulty of introducing diffraction when simulating light propagation. This hinders, for instance, the accurate modeling of beams focused through microscope objectives, where the diffraction patterns in the focal plane are of great importance in various applications. Here, we propose to overcome this issue by means of a direct extinction method. In the MC simulations, the photon paths’ initial positions are sampled from probability distributions which are calculated with a modified angular spectrum of the plane waves technique. We restricted our study to the two-dimensional case, and investigated the feasibility of our approach for absorbing yet nonscattering materials. We simulated the focusing of collimated beams with uniform profiles through microscope objectives. Our results were compared with those yielded by independent simulations using the finite-difference time-domain method. Very good agreement was achieved between the results of both methods, not only for the power distributions around the focal region including diffraction patterns, but also for the distribution of the energy flow (Poynting vector).
This article [J. Biomed. Opt.. 19, , 071404 (2014)] was originally published online on 6 January 2014 with Figs. 2 and 3 reversed, though the captions were correct. The corrected figures and captions are reprinted below.
A highly efficient method based on Maxwell’s theory was developed, which enables the calculation of the scanning of a focused beam through scattering media. Maxwell’s equations were numerically solved in two dimensions using finite difference time domain simulations. The modeling of the focused beam was achieved by applying the angular spectrum of plane waves method. The scanning of the focused beam through the scattering medium was accomplished by saving the results of the near field obtained from one simulation set of plane waves incident at different angles and by an appropriate post processing of these data. Thus, an arbitrary number of focus positions could be simulated without the need to further solve Maxwell’s equations. The presented method can be used to efficiently study the light propagation of a focused beam through scattering media which is important, for example, for different kinds of scanning microscopes.
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