Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Filters applied. Clear all
. Jan-Feb 2011;16(1):016014.
doi: 10.1117/1.3528656.

Optically Sectioned in Vivo Imaging With Speckle Illumination HiLo Microscopy

Affiliations
Free PMC article

Optically Sectioned in Vivo Imaging With Speckle Illumination HiLo Microscopy

Daryl Lim et al. J Biomed Opt. .
Free PMC article

Abstract

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.

Figures

Figure 1
Figure 1
A HiLo microscope setup is identical to a standard wide-field epi-fluorescence microscope except for the addition of a laser beam and diffuser in the illumination path, resulting in speckle illumination at the sample. The resulting fluorescence is captured by a digital camera. When the diffuser is static, the speckle illumination exhibits high contrast (top right panel). When the diffuser is rapidly oscillated by a galvanometric motor, the resulting speckle becomes blurred over the course of the camera exposure, effectively simulating uniform illumination (bottom right panel). Speckle and uniform illumination images are acquired pairwise, and processed into an optically sectioned HiLo image.
Figure 2
Figure 2
Volumetric imaging of a mouse brain hippocampus slice (100 μm thick) labeled by cytoplasmic EGFP by in utero electroporation.z-stacks comprising 200 slices with 0.5 μm step size were acquired with a HiLo microscope and a commercial confocal microscope (Olympus FluoView 1000), in both cases using a 20× water immersion objective (Olympus UApo∕340). Co-located z-slices (single frames) presented in the left column were taken with (a) standard wide-field microscopy; (b) HiLo microscopy; and (c) confocal microscopy. HiLo exposure time was 2×400 ms per slice (power limited with an illumination power at the sample of about 4 mW), with images acquired at 1200×1200 resolution. Confocal exposure time was 2880 ms per slice (scan speed limited). Scale bar is 50 μm.
Figure 3
Figure 3
(a) Confocal sectioning curve (solid red line) and HiLo sectioning curves processed with different σw ’s (solid blue and dashed lines) using the same data. (b) Bandpass filters used to generate HiLo sectioning curves (solid blue and dashed lines). Solid black line depicts the magnitude of the in-focus detection OTF for comparison. (c) FWHM (averaged over nine measurements) of HiLo sectioning curves as a function of σw1. Dashed cyan line indicates a linear fit (R2=0.998).
Video 1
Video 1
Videos made from same imaging data as Fig. 2. (a) Maximum intensity projection of HiLo z-stack. (b) Maximum intensity projection of confocal z-stack. (MPEG, 9.4MB)
Video 2
Video 2
Macroscopic HiLo videos of a live white planarian labeled by immersion with fluorescein diacetate (Sigma-Aldrich). All panels were generated from the same two raw images. (a) uniform illumination image. (b), (c), and (d) bottom left are HiLo images with decreasing depths of field, post-processed using filters with (b) σw1= 4, (d) 2, and (c) 1 pixels respectively. Images were acquired with a 4× macroscope objective (Olympus MVX PLAPO 2XC), a 600×600 pixel resolution, and an exposure time of 2×60 ms with an illumination power of about 4 mW at the sample. The scale bar is 0.4 mm. (MPEG, 6.9MB)
Video 3
Video 3
In vivo imaging video. (a) Uniform illumination and (b) HiLo microscopic images of live C. elegans genetically encoded to express the calcium indicator cameleon in a specific subset of locomotor motorneurons. Images were acquired with a 40× microscope objective (Olympus LCPlanFL N), 600×600 pixel resolution, and an exposure time of 2×60 ms with an illumination power of about 4 mW at the sample. The scale bar is 40 μm. (MPEG, 10MB)
Video 4
Video 4
In vivo imaging video. (a) Uniform illumination and (b) HiLo macroscopic images of live zebrafish labeled with EGFP driven by a CD41 promoter, and anaesthesized with Tricaine. Images were acquired with a 4× macroscope objective (Olympus MVX PLAPO 2XC), a 600×600 pixel resolution, and an exposure time of 2×40 ms with an illumination power of about 4 mW at the sample. The scale bar is 0.4 mm). (MPEG, 10MB)

Similar articles

See all similar articles

Cited by 19 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback