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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. We have developed a technique, called HiLo microscopy, to reject the out-of-focus contributions.

To begin, 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. We have therefore achieved half of our goal. That is, we have extracted the high-frequency in-focus content of our standard image. The remaining task is to extract the low-frequency in-focus content.

To achieve the remaining task, we acquire a second image of our object, this time using “structured” illumination rather than uniform illumination. By structured illumination, we mean any 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. Our second image is thus spatially modulated by this illumination structure. Importantly, the contrast of the imaged modulation becomes vanishingly small for object signals that arise from out of focus. That is, a measure of the local contrast of the imaged modulation provides of measure of the degree to which the object is in focus, or contains in-focus contributions. Several techniques may be used to measure the local modulation contrast (e.g. by performing local variance measurements (1), single sideband demodulation (2), double sideband demodulation, etc.). In general, these are coarse grained, meaning they provide local modulation contrast measurements with only low resolution. That is, they enable an extraction of only the low-frequency in-focus content of our image. However, by proper adjustment of the low-pass filtering provided by such coarse-grained contrast measurements, the low-frequency in-focus content derived from the structured illumination image can be made to exactly complement the high-frequency in-focus content derived from the uniform illumination image (described above). A seamless fusion of the 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.

Publications Related to this Research Area

Fast optically sectioned fluorescence HiLo endomicroscopy

Tim N. Ford, Daryl Lim, Jerome Mertz,

Journal of Biomedical Optics

We describe a nonscanning, fiber bundle endomicroscope that performs optically sectioned fluorescence imaging with fast frame rates and real-time processing. Our sectioning technique is based on HiLo imaging, wherein two widefield images are acquired under uniform and structured illumination and numerically processed to reject out-of-focus background. This work is an improvement upon an earlier demonstration of widefield optical sectioning through a flexible fiber bundle. The improved device features lateral and axial resolutions of 2.6 and 17 μm, respectively, a net frame rate of 9.5 Hz obtained by real-time image processing with a graphics processing unit (GPU) and significantly reduced motion artifacts obtained by the use of a double-shutter camera. We demonstrate the performance of our system with optically sectioned images and videos of a fluorescently labeled chorioallantoic membrane (CAM) in the developing G. gallus embryo. HiLo endomicroscopy is a candidate technique for low-cost, high-speed clinical optical biopsies.

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Optical sectioning microscopy with planar or structured illumination

Jerome Mertz,

Nature Methods

A key requirement for performing three-dimensional (3D) imaging using optical microscopes is that they be capable of optical sectioning by distinguishing in-focus signal from out-of-focus background. Common techniques for fluorescence optical sectioning are confocal laser scanning microscopy and two-photon microscopy. But there is increasing interest in alternative optical sectioning techniques, particularly for applications involving high speeds, large fields of view or long-term imaging. In this Review, I examine two such techniques, based on planar illumination or structured illumination. The goal is to describe the advantages and disadvantages of these techniques.

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Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

Daryl Lim, Tim N. Ford, Kengyeh K. Chu, Jerome Mertz,

Journal of Biomedical Optics

We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish.

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Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection

J. Mertz and J. Kim,

Journal of Biomedical Optics

t is well known that light-sheet illumination can enable optically sectioned wide-field imaging of macroscopic samples. However, the optical sectioning capacity of a light-sheet macroscope is undermined by sample-induced scattering or aberrations that broaden the thickness of the sheet illumination. We present a technique to enhance the optical sectioning capacity of a scanning light-sheet microscope by out-of-focus background rejection. The technique, called HiLo microscopy, makes use of two images sequentially acquired with uniform and structured sheet illumination. An optically sectioned image is then synthesized by fusing high and low spatial frequency information from both images. The benefits of combining light-sheet macroscopy and HiLo background rejection are demonstrated in optically cleared whole mouse brain samples, using both green fluorescent protein (GFP)-fluorescence and dark-field scattered light contrast.

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Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle

Silvia Santos, Kengyeh K. Chu, Daryl Lim, Nenad Bozinovic, Tim N. Ford, Claire Hourtoule, Aaron C. Bartoo, Satish K. Singh, Jerome Mertz,

Journal of Biomedical Optics

We present an endomicroscope apparatus that exhibits out-of-focus background rejection based on wide-field illumination through a flexible imaging fiber bundle. Our technique, called HiLo microscopy, involves acquiring two images, one with grid-pattern illumination and another with standard uniform illumination. An evaluation of the image contrast with grid-pattern illumination provides an optically sectioned image with low resolution. This is complemented with high-resolution information from the uniform illumination image, leading to a full-resolution image that is optically sectioned. HiLo endomicroscope movies are presented of fluorescently labeled rat colonic mucosa.

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Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy

D. Lim, K. K. Chu and J. Mertz,

Optics Letters

We describe a method of obtaining optical sectioning with a standard wide-field fluorescence microscope. The method involves acquiring two images, one with nonuniform illumination (in our case, speckle) and another with uniform illumination (in our case, randomized speckle). An evaluation of the local contrast in the speckle-illumination image provides an optically sectioned image with low resolution. This is complemented with high-resolution information obtained from the uniform-illumination image. A fusion of both images leads to a full resolution image that is optically sectioned across all spatial frequencies. This hybrid illumination method is fast, robust, and generalizable to a variety of illumination and imaging configurations.

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HiLo principle

GFP-labeled brain slice

HiLo (left) and confocal (right) images of GFP-labeled mouse brain slice. HiLo was taken with speckle (non-uniform) and randomized-speckle (uniform) illumination. Net acquisition time with HiLo was about 10 times less than with confocal.