Flow scanning structured illumination

We apply the principle of structured illumination microscopy to a microfluidic device. The necessary phase shifts are no longer obtained by controlled displacement of the illumination pattern but by flowing the sample itself through the microfluidic channel. We demonstrate the technique experimentally by reconstructing superresolved images of yeast cells.

The resolution of an optical imaging system is subject to the diffraction limit, which for a fixed wavelength is governed by the numerical aperture (NA) of the system. A popular technique to go beyond these limits is structured illumination (SI) microscopy, in which spectral beating of object modes introduced by a given illumination pattern, usually periodic, folds high-resolution information into lower spatial frequencies (Moire patterns) which can be detected by the imaging device. The resolution can be improved by a factor of two in the linear case, and greater improvements can be obtained using nonlinearity. This imaging technique, however, requires the acquisition of several raw images (at least three) with a series of precise displacements of the illumination pattern in order to remove phase ambiguity. Previous SI microscopes relied on mechanical moving parts (e.g. piezoelectric actuators) or on a spatial light modulator (SLM). These methods add complexity to the imaging system and displacing the illumination pattern significantly reduces the image acquisition speed. Further, mechanical movement is subject to vibration error and artifacts, while SLMs are limited by their pixel size.

Microfluidic microscopy adds a new degree of freedom to imaging by flowing the object of interest through a microfluidic channel. It has received renewed attention with the development of integrated optofluidic devices, which are lensless imagers that place flowing samples directly over a detector. To date, this flow has been used to enhance resolution using small holes before the detector in a scanning imaging device or to introduce sub-pixel displacements and digitally increase the resolution, but always using a uniform illumination. Here, we add the element of structured illumination, using the flow to provide the necessary scanning for SI microscopy. We also re-introduce an objective lens into the system, effectively decoupling the resolution from pixel size and injecting another degree of freedom for imaging, e.g. for optimization of recording. The resulting scheme retains all the benefits of microfluidics, including high sample throughput and object sorting, while enabling easy integration with existing microscopes and flow cytometers.

Experimental Setup

The experimental setup is shown in Figure. 1. A 500 μm wide, 50 μm deep microfluidic channel is etched on a glass slide and is located at the focal plane of a 20X optical microscope.

The objective is part of a 4f imaging configuration with an aperture located at the confocal plane. The resulting value of the numerical aperture (NA = 0.1) corresponds to a resolution limit of approximately 4μm. To generate the structured light, a 532nm continuous laser is patterned using a transmission grating and then demagnified to reduce fringe spacing. This device illuminates the microfluidic channel with a steady sinusoidal pattern: 2.8 μm stripes orientated orthogonally to the fluid flow direction.

A suspension of yeast particles in glycerol is flowed through the microfluidic channel at a constant flow velocity. Multiple images are recorded by a CCD camera (pixel size 9.9 μm) at a constant frame rate (15 fps). Figure. 2(a-c) show three consecutive frames. It is clear that different features of the object are revealed as it flows past the stationary illumination pattern.

Figure 1 - A 500μm wide, 50 μm deep fluidic channel is located near the focal plane of a 20\times objective lens. The designed imaging system has low numerical aperture (NA = 0.1). The structured illumination source is a steady sinusoidal-profile (2.78 μm stripes) orthogonal to the flow direction. Images are recorded on a CCD camera at frame rate 15 fps.

Figure 2 - Three consecutive frames of two yeast particles under structured illumination. The constant phase shift (0.14 pi) of the illumination pattern between consecutive frames shows the evolution of intensity.

The yeast image without structured illumination is shown in Fig.3(a), and numerical reconstruction of the yeast particles using Eq. (4) is shown in Fig. 3(b). It is clear that SI provides greater visibility and reveals more details than the uniform illumination image, even for a 1D illumination pattern. The amount of improvement can be quantified using the visibility V= (I_max-I_min)/(I_max +I_min) , where V = 0.15 corresponds to the Rayleigh resolution criterion. Fig \ref{Fig3}(e) shows cross-sections of the intensity along the line connecting the two particles. For uniform illumination, the left yeast particle is well below the Rayleigh limit (V_left = 0.06) while the right particle is barely visible (V_right = 0.17). In contrast, structured illumination improves the visibility to 0.32 and 0.34, respectively.

Related Publications

Flow-based Structured Illumination
C-H. Lu, N. C. Pegard and J. W. Fleischer, 2013, Applied Physics Letters, 102 161115 (2013)

Microfluidic Structured Illumination Microscopy
C. H. Lu, N. C. Pegard and J. W. Fleischer, Frontiers in Optics, (2012)

Structured illumination optofluidic microscope
J. W. Fleischer,and N. C. Pegard
Provisional Patent Application 61-609,991 - 2011