HiLo Microscopy

In a standard optical imaging system, an object is illuminated by a source (lamp, laser, diodes, etc.) and the resulting object signal (reflectance, fluorescence, etc.) is imaged onto a detector array (CCD camera, CMOS camera, etc.). In most cases, the illumination is uniform. An image acquired in such a manner possesses both in-focus and out-of-focus contributions. In contrast, HiLo microscopy (not to be confused with HILO: highly inclined and laminated optical sheet microscopy) can be used to effectively discriminate out-of-focus information. 

HiLo is similar to SIM, but only requires two images: one structured illumination image and one conventional widefield image. Optically sectioned images are then digitally generated thanks to a fusion algorithm. The structured illumination image is used to identify low-frequency out-of-focus information, while the conventional widefield image contains high spatial frequency details that may have been lost via application of the illumination pattern. 

Principle of HiLo microscopy

HiLo involves acquiring a standard image with uniform illumination. As noted above, such an image contains both in-focus and out-of-focus content. By definition, out-of-focus content is blurred and hence contains only low spatial frequency components. To reject such out-of-focus content, it suffices, therefore, to apply a simple high-pass filter to the image, thereby selecting only high spatial frequency components and rejecting low-frequency components. Such high-frequency components are thus inherently in focus.

A second image of the object is acquired, this time using “structured” illumination rather than uniform illumination. Structured illumination in this context is an illumination pattern that imparts spatial variations to the object signal that can be recorded by the detector array. For example, the structured illumination may be a spatially varying intensity distribution such as produced by laser speckle, fringes, a grid pattern, a checkerboard pattern, etc. The second image is thus spatially modulated by this illumination structure. A seamless fusion of both the high and low frequency image contents then leads to a full resolution in-focus image that contains all frequency contents within the frequency bandwidth of the imaging system.

Reference:

Proc. of SPIE Vol. 8589 85890H-2