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. 2018 Nov 21;8(1):17178.
doi: 10.1038/s41598-018-35006-1.

Cancellation of Bessel beam side lobes for high-contrast light sheet microscopy

Giuseppe Di Domenico  1   2 , Giancarlo Ruocco  3 , Cristina Colosi  3 , Eugenio DelRe  4   5 , Giuseppe Antonacci  3   6
Affiliations

Cancellation of Bessel beam side lobes for high-contrast light sheet microscopy

Giuseppe Di Domenico et al. Sci Rep. .

Abstract

An ideal illumination for light sheet fluorescence microscopy entails both a localized and a propagation invariant optical field. Bessel beams and Airy beams satisfy these conditions, but their non-diffracting feature comes at the cost of the presence of high-energy side lobes that notably degrade the imaging contrast and induce photobleaching. Here, we demonstrate the use of a light droplet illumination whose side lobes are suppressed by interfering Bessel beams of specific k-vectors. Our droplet illumination readily achieves more than 50% extinction of the light distributed across the Bessel side lobes, providing a more efficient energy localization without loss in transverse resolution. In a standard light sheet fluorescence microscope, we demonstrate a two-fold contrast enhancement imaging micron-scale fluorescent beads. Results pave the way to new opportunities for rapid and deep in vivo observations of large-scale biological systems.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
(a) Generation of the droplet illumination in a light sheet imaging system. The amplitude of an expanded laser beam was modulated by an SLM and focused by an objective lens (Oill) of NA = 0.25. Fluorescence light from a test sample was collected by a second orthogonal objective lens (Ocol) of NA = 0.35 and a tube lens L3. A CCD camera was used to acquire the image by integrating the fluorescence signal emitted during a yˆ scan. Li lenses; λ/2 half-wave plate; P linear polarizer; F bandpass filter. (b,c) To generate the droplet illumination, a set of amplitude binary masks composed of two concentric rings of inner and outer radii, r2 and r1 respectively, and thickness dr were displayed by the SLM.
Figure 2
Figure 2
(a) Plot of the ratio between the optical power (for z = 0) distributed across the droplet side lobes with respect to the Bessel lobe power as a function of the ratio r2/r1. The solid line is the theoretical prediction while red dots are experimental data. A plot of the ratio between the axial FWHM of the droplet beam and the Gaussian beam as a function of the ratio r2/r1 is reported in the same graph. Top-view (xz plane) (b) and radial (c) intensity profiles of a Gaussian, an ideal Bessel beam (ϵ=1) and a short Bessel beam (ϵ=0.78) of comparable axial FWHM to that of the droplet beam as a function of the number of Rayleigh lengths and for an equal NA. For r2/r1 ~ 0.57, the light distributed across the droplet side lobes is about 62% and 48% lower than that of an ideal and a short Bessel beam respectively. Associated beam amplitude masks (inset).
Figure 3
Figure 3
(a) Representative Bessel beam obtained with a single ring of radius r1 (top) and droplet beam with a ratio of r2/r1 = 0.57 (bottom). (b) Measured radial intensity profile of the two beams along the dashed line together with the theoretical prediction (green line). The power distributed across the side lobes of the Bessel beam is suppressed by more than ~50% in the case of the droplet beam.
Figure 4
Figure 4
Fluorescence images in both log (a,b) and linear (c,d) scales of embedded micron-sized beads obtained with a Bessel beam (a,c) and a droplet beam illumination (b,d) in a light sheet microscope. The intensity profiles (e) along the dashed line show a two-fold reduction in the background light given by the droplet illumination as a result of the suppression of the out-of-focus side lobes. Scale bar is 10 µm. (f) Map the intensity ratio between the images obtained with Bessel and droplet illumination.
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