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Abstract
A non-axial-scanning multi-plane microscopic system incorporating multiplexed volume holographic gratings and slit array detection to simultaneously acquire optically sectioned images from different depths is presented. The proposed microscopic system is configured such that multiplexed volume holographic gratings are utilized to selectively produce axial focal points in two or more planes inside the sample, and then to use confocal slit apertures to simultaneously image these multiple planes onto corresponding detection areas of a CCD. We describe the design, implementation, and experimental data demonstrating this microscopic system’s ability to obtain optically sectioned multi-plane images of fluorescently labeled standard micro-spheres and tissue samples without scanning in axial directions.
© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Optical sectioning techniques offer the ability to acquire three-dimensional information from various samples by removal of out-of-focus background noise. The most commonly used optical sectioning imaging method with good background rejection is based on confocal approach [1–7]. Although confocal imaging offers fine optical sectioning capabilities to reject out-of-focus background, the price to pay for improved image quality is typically point-by-point scanning time. Efforts to improve scanning efficiency by developing high-speed lateral scanning mechanisms or increasing the number of detection and focal points [8–12] are ongoing. To reduce need for scanning, slit confocal techniques have been developed by utilizing a line aperture, rather than a single point pinhole, for a variety of biological imaging applications [6, 7, 13–17].
Typically, in slit confocal microscopy, multiple detectors are required along the length of the slit; hence, a one-dimensional or two-dimensional camera has also been adapted for high-speed operation [18, 19]. To further enhance acquisition speed, a single oscillating mirror [14], or a double-sided mirror [15] was used in slit confocal microscopy for imaging applications. Recently, an acousto-optic deflector [20] has been employed for line scanning in slit confocal over tissue samples, but this approach does not eliminate depth scanning. Theta slit confocal [21], based on oblique illumination, has been developed to simplify system configuration for lateral scanning. Spectrally encoded slit confocal [18], based on chromatic dispersion, is capable of obtaining images of label-free samples, but it does not resolve volumetric fluorescence samples. Dual slit configuration has also been reported [22] such that two focused beams are used to scan objects in opposite directions and are synchronized with corresponding pixel rows, acting as digital slits, on a CCD camera. Hence efforts in slit confocal techniques have been made toward developing high-speed lateral scanning mechanisms; however, the existing slit confocal systems still require scanning in axial direction.
Most recent multifocal confocal includes high-speed confocal fluorescence microscopy in Ref [25], which adapt Dammann grating to generate 3 X 3 points at a specific depth. Although it speed-up acquisition time, it still needs depth scanning. Parallel confocal approach [8] speeds up m ulti-plane image formation process through the use of double computer generated distorted gratings onto a spatial light modulator (SLM), producing two axial focal points for excitation at one wavelength using one distorted grating, and observing emission at a different emission wavelength with the other grating. Unfortunately, the separation between two planes is very limited, and it still requires scanning at two directions. In addition, to observe fluorescence images using a SLM is very stringent due to polarization selectivity.
Here, we present a new non-axial-scanning slit confocal imaging, which incorporates multiplexed volume holographic gratings (MVHGs), volume holographic gratings recording materials include PQ-PMMA [24], polymer-dispersed liquid crystals (PDLCs) [26], and thick photochromic polymer [27] and the recording strategy of multiplexed volume hologram can be found in [28–30].The presented system produces line focal points in two or more planes inside the sample, and then to use slit apertures to simultaneously image these multiple depths, which are projected laterally onto a CCD. Compared with previously described slit confocal systems, our approach offers parallelism for simultaneously acquiring optically sectioned images of volumetric tissue samples from multiple depths without axial scanning. Unlike the system described in [8] using computer generated gratings on a SLM, our approach does not rely on polarization selectivity to probe images, and an arbitrary arrangement of longitudinal line focal points can be achieved using appropriately MVHGs. Compared to our previous effort in [9], our approach, using MVHGs, produces multi-plane, slit confocal. Rather then laterally scanning along both X and Y direction, our approach acquires depths at the same time, and only scans along either X- or Y-direction. In contrast to the system described in [18] through chromatic dispersion techniques, our approach acquires images of label-free samples, as well as fluorescently labeled tissue samples. In this paper, we experimentally demonstrate realization of a multi-plane, non-axial-scanning slit confocal system to image standard microspheres and ex vivo biological tissue samples at multiple depths.
2. Method
Figure 1 shows a schematic diagram of the proposed slit confocal microscopy. The geometry is configured such that two focused lines, generated using MVHGs under Bragg-matched condition, occur inside a specimen at different depths, and also serve as the input focal lines for the subsequent slit apertures. Thus, optically sectioned images of a sample within different depths are simultaneously obtained using a CCD without scanning along axial direction.
Fig. 1 Schematic diagram of the proposed multi-plane slit confocal approach. Focused lines at two different depths are simultaneously generated by diffraction of the Bragg-matched MVHGs, and probed by corresponding confocal slit apertures. Optically sectioned images at multiple depths are acquired using a CCD in detection without axial scanning.
The MVHGs are formed by multiple exposures of a thick Phenanthrenquinone doped Polymethylmethacrylate (PQ-PMMA) material, based on shift-angular multiplexing [23], which can be illustrated using a k-sphere diagram in Fig. 2. Figure 2(a) shows the recording process, and and denote signal and reference wave vectors, respectively. The grating vector of the i th MVHG is given by [23]
Here, , is the recording wavelength in free space, is the grating vector of the i th hologram, and n is the refractive index of the recording material, and M is the number of multiplexed gratings. Figure 2(b) shows the reconstruction process, and propagation vectors of the probe and diffracted beams, based on Bragg condition [24], can be expressed as is the probe wavelength in free space. Because the Bragg degeneracy property, a MVHGs formed at a certain wavelength can be probed at a different wavelength . In the drawings of Fig. 2, M = 2. The numerical aperture of L1 and L2 are 0.65 M-40X (Newport Inc.) and 0.55 MLWD-50X (Newport Inc.). The nominal inter beam angle (φ) between signal and reference beams is 68°, multiplexing angle (Δφ) between reference beams is 14.5°. ∆z = 10 μm in Fig. 2 is determined by moving the lens of L1while MVHGs are recorded.Fig. 2 (a) Recording design based on K-sphere diagram. ksig,i = 1,2 and kref,i = 1,2 are respective signal and reference beams to form MVHGs. (b) Reconstruction under Bragg-matched condition. Probe beams satisfying K-sphere diagram to simultaneously produce two diffracted beams, kdiff,1 and kdiff,2, which produce different foci at different planes at the sample space through an objective lens.
3. Experimental results and discussion
In our experimental setup, two MVHGs with average diffraction efficiency of ~35% at 488nm, was located in the illumination arm. Two slit apertures with the pupil size of 5 µm were utilized simultaneously to reject out-of-focus light during imaging. An Olympus objective lens (ULWDMSPlan50X), a tube lens with a focal length of 200 mm, and a CCD (iXon897, Andor) were also used to build our proposed MVHG-based multi-plane slit confocal microscope. Lateral resolution of the proposed confocal system was evaluated by imaging an Air Force Resolution Chart. Figure 3(a) shows the smallest lines with a width of 0.78 μm are well resolved when the Chart is brought into focus at different depths. It is worth mentioning that the contrast of smallest line pairs in vertical direction is slightly higher than that along horizontal direction since asymmetrical slit aperture shape causes anisotropic confocal effect [24]. In addition, the system axial resolution was experimentally evaluated by scanning a fluorescently labeled microsphere (1 µm in diameter, Polyscience) in depth with a step of 0.1 µm. Figure 3(b) shows experimental results of the point spread function along axial direction (PSFz) using an excitation wavelength of 488 nm, and the full width of half maximum (FWHM) is respective 3 µm at depth 1, and 3.5 µm at Depth 2. The PSFz measurements are in agreement with simulation results under incoherent condition, using the following relation [24], which provides 3 µm.
where x, y and z presents object coordinate, , , belong to Fourier coordinate, is the amplitude PSF depended on objective lens and can be obtained as in cylindrical coordinate.Fig. 3 (a,i) Images of a 1951 USAF bar chart through the system. The line width of the smallest element in group 9 is 0.78 μm, and (a,ii) profile plots of smallest line pairs (a,i) in both vertical and horizontal direction. (b) The normalized axial resolution of the MVHG-based multi-plane slit confocal system, measured by scanning a 1 µm fluorescent microsphere. The full width of half maximum (FWHM) at different depths is ~3µm (Depth 1) and 3.5µm (Depth 2), respectively; the distance between the two depths is ~10 µm.
To verify the ability of our proposed multi-plane slit confocal system, a volumetric sample of fluorescently labeled green microspheres with sparsely distributed condition (5 µm in diameter, Polyscience) and embedded in a 1 mm thick slab of agarose (Invitrogen) was first prepared for imaging. The sparsely distributed beads were excited using a blue tunable laser source (Innova 304C, Coherent Inc.) at λ = 488 nm. With no need for axial scanning, Fig. 4(a) shows in-focus fluorescent beads obtained by CCD, and the out-of-focus light at the second depth is significantly suppressed. In Fig. 4(b), optically sectioned images of the same microspheres brought to in-focus at different depth while out-of-focus background is also significantly rejected. Further, we prepared more densely distributed fluorescently labeled green microspheres of 25 μm in diameter (Polyscience).
Fig. 4 (a,b) Optically sectioned images of sparsely distributed 5 µm fluorescent microspheres simultaneously acquired from two planes separated axially by 10 µm: (a) fluorescent microspheres located at Depth 1, and (b) microspheres located at Depth 2.
Figure 5 provides comparisons of standard wide-field and optical sectioning images captured using our MVHG-based multi-plane slit confocal approach. In Fig. 5(a), fluorescence images of microspheres with both in-focus and out-of-focus light are simultaneously captured from the CCD with uniform illumination. Figure 5(b) shows images of fluorescently labeled microspheres acquired at the same time at both depths using the MVHG-based multi-plane slit confocal, with a lateral scanning step of 0.1 µm along x axis, and no need of scanning in axial direction. Figure 5(c) compares the wide view and confocal out-of-focus background rejection, by plotting an intensity profile along a line, between the different techniques. The image degrades with imaging depth in two different depths is shown in Fig. 5(d). It shows solid evidence that the MVHG-based multi-plane slit confocal suppresses the out-of-focus background noise from the desired in-focus signal.
Fig. 5 Images of densely distributed 25 μm microspheres embedded in a agarose-gel. (a) The images of the beads are taken using standard wide-field condition. (b) Optically sectioned images are simultaneously acquired from two planes separated axially by 10 μm, using the MVHG-based multi-plane slit confocal system. (c) Comparison of intensity profiles between standard wide-view and our approach for out-of-focus background rejection.
To demonstrate the capability of MVHG-based multi-plane slit confocal microscopy to image a highly scattering tissue sample, we performed an in vitro imaging experiment to image rabbit corneal epithelium. Figure 6 shows images of fluorescently labeled rabbit corneal samples, taken with our MVHG-based multi-plane slit confocal microscope. The samples were stained with a fluorescent dye (PKH67) on the cell membranes and illuminated with the same excitation laser (488nm). As in the case of the microsphere experiment described earlier, the MVHG-based multi-plane slit confocal succeeds in removing background at multiple fluorescent planes in a highly scattering tissue sample.
Fig. 6 Resultant images of fluorescently labeled rabbit corneal samples acquired from two axially separated planes (Depths 1and 2), using the MVHG-based multi-plane slit confocal imaging.
4. Conclusion
In conclusion, we have developed MVHG based multi-plane slit confocal microscopy to simultaneously observe optically sectioned in vitro 3D images from different depths. In previous work [9], combination of MVHGs in illumination and pinhole confocal detection principle is significantly faster than alternative axial scanning mechanisms in imaging multiple depths within a volumetric tissue sample while effectively rejecting out-of-focus background. In this approach, the need of scanning direction is dramatically decreased to only one axis scanning.
The system is simple, and robust. In addition, it promises to increase throughput significantly because it captures multiple depths simultaneously. Our MVHGs may further record multiplexed Bessel beams [31,32], to penetrate deeper position. Furthermore, MVHGs, with corresponding confocal apertures, may suppress side lobes of multiplexed Bessel beams to provide multi-plane confocal imaging. Our approach can be extended to obtain more planes simultaneously with more MVHGs within a volume hologram using PQ-PMMA [24]. The acquisition speed of our approach can be further enhanced by a higher speed scanning mirror for lateral scanning.
Funding
Taiwan Ministry of Science and Technology (105-2628-E-002-008-MY3, 106-2221-E-002-157-MY3); National Taiwan University (NTU-106M103, NTU-106R7807).
Acknowledgements
The authors gratefully acknowledge Shu-Chi Chiang and Ying-Hou Chen for sample preparations, Yu-Hsin Chia for material preparation of volume holographic gratings, and also thank Kung-Bin Sung and Yi-You Huang for valuable comments.
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