HiLo Microscopy Research Articles

Optically-sectioned two-shot structured illumination microscopy with Hilbert-Huang processing.

We introduce a fast, simple, adaptive and experimentally robust method for reconstructing background-rejected optically-sectioned images using two-shot structured illumination microscopy. Our innovative data demodulation method needs two grid-illumination images mutually phase shifted by π (half a grid period) but precise phase displacement between two frames is not required. Upon frames subtraction the input pattern with increased grid modulation is obtained. The first demodulation stage comprises two-dimensional data processing based on the empirical mode decomposition for the object spatial frequency selection (noise reduction and bias term removal). The second stage consists in calculating high contrast image using the two-dimensional spiral Hilbert transform. Our algorithm effectiveness is compared with the results calculated for the same input data using structured-illumination (SIM) and HiLo microscopy methods. The input data were collected for studying highly scattering tissue samples in reflectance mode. Results of our approach compare very favorably with SIM and HiLo techniques.

Depth-resolved cellular microrheology using HiLo microscopy

It is increasingly important to measure cell mechanical properties in three-dimensional environments. Particle tracking microrheology (PTM) can measure cellular viscoelastic properties; however, out-of-plane data can introduce artifacts into these measurements. We developed a technique that employs HiLo microscopy to reduce out-of-plane contributions. This method eliminated signals from 90% of probes 0.5 μm or further from the focal plane, while retaining all in-plane probes. We used this technique to characterize live-cell bilayers and found that there were significant, frequency-dependent changes to the extracted cell moduli when compared to conventional analysis. Our results indicate that removal of out-of-plane information is vital for accurate assessments of cell mechanical properties.

Analyzing speckle contrast for HiLo microscopy optimization

HiLo microscopy is a recently developed technique that provides both optical sectioning and fast imaging with a simple implementation and at a very low cost. The methodology combines widefield and speckled illumination images to obtain one optically sectioned image. Hence, the characteristics of such speckle illumination ultimately determine the quality of HiLo images and the overall performance of the method. In this work, we study how speckle contrast influence local variations of fluorescence intensity and brightness profiles of thick samples. We present this article as a guide to adjust the parameters of the system for optimizing the capabilities of this novel technology.

Single-Shot Optical Sectioning Using Two-Color Probes in HiLo Fluorescence Microscopy

We describe a wide-field fluorescence microscope setup which combines HiLo microscopy technique with the use of a two-color fluorescent probe. It allows one-shot fluorescence optical sectioning of thick biological moving sample which is illuminated simultaneously with a flat and a structured pattern at two different wavelengths. Both homogenous and structured fluorescence images are spectrally separated at detection and combined similarly with the HiLo microscopy technique. We present optically sectioned full-field images of Xenopus laevis embryos acquired at 25 images/s frame rate.

Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

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.

Holographic photolysis for multiple cell stimulation in mouse hippocampal slices.

BackgroundAdvanced light microscopy offers sensitive and non-invasive means to image neural activity and to control signaling with photolysable molecules and, recently, light-gated channels. These approaches require precise and yet flexible light excitation patterns. For synchronous stimulation of subsets of cells, they also require large excitation areas with millisecond and micrometric resolution. We have recently developed a new method for such optical control using a phase holographic modulation of optical wave-fronts, which minimizes power loss, enables rapid switching between excitation patterns, and allows a true 3D sculpting of the excitation volumes. In previous studies we have used holographic photololysis to control glutamate uncaging on single neuronal cells. Here, we extend the use of holographic photolysis for the excitation of multiple neurons and of glial cells.Methods/Principal FindingsThe system combines a liquid crystal device for holographic patterned photostimulation, high-resolution optical imaging, the HiLo microscopy, to define the stimulated regions and a conventional Ca2+ imaging system to detect neural activity. By means of electrophysiological recordings and calcium imaging in acute hippocampal slices, we show that the use of excitation patterns precisely tailored to the shape of multiple neuronal somata represents a very efficient way for the simultaneous excitation of a group of neurons. In addition, we demonstrate that fast shaped illumination patterns also induce reliable responses in single glial cells.Conclusions/SignificanceWe show that the main advantage of holographic illumination is that it allows for an efficient excitation of multiple cells with a spatiotemporal resolution unachievable with other existing approaches. Although this paper focuses on the photoactivation of caged molecules, our approach will surely prove very efficient for other probes, such as light-gated channels, genetically encoded photoactivatable proteins, photoactivatable fluorescent proteins, and voltage-sensitive dyes.

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

It 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.

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

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.