In recent years, tremendous efforts have been spent on deep tissue imaging using phase conjugation, a technique used to undo the effects of light scattering in a thick tissue. Despite the early debates between Yariv and Wolf, it is still not well understood physically how deep can a field propagate into biological tissue and still be phase conjugated. In order to answer this question, we developed a light scattering theory to describe the evolution of the phase associated with a field scattered by a thick tissue block. The multiple scattering through the sample is simplified to a series of single scattering through consecutive thin tissue slices. With this theory, we identify the limits of the phase conjugation operation and recover the previous results by Yariv and Wolf, which asserts that phase conjugation is rooted in small angle approximation. Importantly, we discover the fundamental principle that rules phase conjugation: the mean axial wavenumber of a field progressively decreases to zero as it scatters multiple times. At this point, phase becomes a spatially random variable and phase conjugation becomes impossible. This result describes a fundamental phenomenon: the interaction between a deterministic object and a deterministic field can result in a random scattered field. We show that this phenomenon is rooted into Heisenberg’s uncertainty principle.
White-light diffraction tomography (WDT) is a recently developed 3D imaging technique based on a quantitative phase imaging system called spatial light interference microscopy (SLIM). The technique has achieved a sub-micron resolution in all three directions with high sensitivity granted by the low-coherence of a white-light source. Demonstrations of the technique on single cell imaging have been presented previously; however, imaging on any larger sample, including a cluster of cells, has not been demonstrated using the technique.
Neurons in an animal body form a highly complex and spatially organized 3D structure, which can be characterized by neuronal networks or circuits. Currently, the most common method of studying the 3D structure of neuron networks is by using a confocal fluorescence microscope, which requires fluorescence tagging with either transient membrane dyes or after fixation of the cells. Therefore, studies on neurons are often limited to samples that are chemically treated and/or dead.
WDT presents a solution for imaging live neuron networks with a high spatial and temporal resolution, because it is a 3D imaging method that is label-free and non-invasive. Using this method, a mouse or rat hippocampal neuron culture and a mouse dorsal root ganglion (DRG) neuron culture have been imaged in order to see the extension of processes between the cells in 3D. Furthermore, the tomogram is compared with a confocal fluorescence image in order to investigate the 3D structure at synapses.
Even with the recent rapid advances in the field of microscopy, non-laser light sources used for light microscopy have not been developing significantly. Most current optical microscopy systems use halogen bulbs as their light sources to provide a white-light illumination. Due to the confined shapes and finite filament size of the bulbs, little room is available for modification in the light source, which prevents further advances in microscopy.
By contrast, commercial projectors provide a high power output that is comparable to the halogen lamps while allowing for great flexibility in patterning the illumination. In addition to their high brightness, the illumination can be patterned to have arbitrary spatial and spectral distributions. Therefore, commercial projectors can be adopted as a flexible light source to an optical microscope by careful alignment to the existing optical path.
In this study, we employed a commercial projector source to a quantitative phase imaging system called spatial light interference microscopy (SLIM), which is an outside module for an existing phase contrast (PC) microscope. By replacing the ring illumination of PC with a ring-shaped pattern projected onto the condenser plane, we were able to recover the same result as the original SLIM. Furthermore, the ring illumination is replaced with multiple dots aligned along the same ring to minimize the overlap between the scattered and unscattered fields. This new method minimizes the halo artifact of the imaging system, which allows for a halo-free high-resolution quantitative phase microscopy system.