Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

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.

Improving high resolution retinal image quality using speckle illumination HiLo imaging

“…In addition, the background shot noise rejection in HiLo is imperfect, since only a bias from the shot noise can be corrected [24]. It is therefore important to have good knowledge of the specifications of the imaging and illumination setup to produce the optimal HiLo image.…”

Cell Biology of Mitotic Recombination

“…We can look forward to a myriad of technical advances in protein tagging as well as in microscopy (e.g., stochastic optical reconstruction microscopy [STORM], structured illumination microscopy [SIM], HiLo, fluorescence resonance energy transfer [FRET], fluorescence redistribution after photobleaching [FRAP], and single-particle tracking) (Fischer et al 2011;Lim et al 2011;Ball et al 2012;Ishikawa-Ankerhold et al 2012). These advances when combined with genetic studies will allow a more in-depth look at the architecture of foci as well as provide further insight into the regulation of focus assembly and disassembly.…”

Super-resolution two-photon microscopy via scanning patterned illumination

“…Inventions of stimulated emission depletion (STED) microscopy, photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM) have reached ~10 nm resolution by manipulating the activation and depletion (on-off) of fluorescent tags. Other techniques have achieved more modest resolution enhancement by increasing the effective numerical aperture [5-7], confocal spatial phase modulation [10], anti-bunching properties of photons from single molecules [13], structure of the illuminated or emitted light [8, 11, 12, 14], or a combination of the aforementioned techniques [9, 15]. …”