Single-frame 3D fluorescence microscopy with ultraminiature lensless FlatScope - PubMed (original) (raw)

Single-frame 3D fluorescence microscopy with ultraminiature lensless FlatScope

Jesse K Adams et al. Sci Adv. 2017.

Abstract

Modern biology increasingly relies on fluorescence microscopy, which is driving demand for smaller, lighter, and cheaper microscopes. However, traditional microscope architectures suffer from a fundamental trade-off: As lenses become smaller, they must either collect less light or image a smaller field of view. To break this fundamental trade-off between device size and performance, we present a new concept for three-dimensional (3D) fluorescence imaging that replaces lenses with an optimized amplitude mask placed a few hundred micrometers above the sensor and an efficient algorithm that can convert a single frame of captured sensor data into high-resolution 3D images. The result is FlatScope: perhaps the world's tiniest and lightest microscope. FlatScope is a lensless microscope that is scarcely larger than an image sensor (roughly 0.2 g in weight and less than 1 mm thick) and yet able to produce micrometer-resolution, high-frame rate, 3D fluorescence movies covering a total volume of several cubic millimeters. The ability of FlatScope to reconstruct full 3D images from a single frame of captured sensor data allows us to image 3D volumes roughly 40,000 times faster than a laser scanning confocal microscope while providing comparable resolution. We envision that this new flat fluorescence microscopy paradigm will lead to implantable endoscopes that minimize tissue damage, arrays of imagers that cover large areas, and bendable, flexible microscopes that conform to complex topographies.

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Figures

Fig. 1

Fig. 1. Traditional microscope versus FlatScope.

(A) Traditional microscopes capture the scene through an objective and tube lens (~20 to 460 mm), resulting in a quality image directly on the imaging sensor. (B) FlatScope captures the scene through an amplitude mask and spacer (~0.2 mm) and computationally reconstructs the image. Scale bars, 100 μm (inset, 50 μm). (C) Comparison of form factor and resolution for traditional lensed research microscopes, GRIN lens microscope, and FlatScope. FlatScope achieves high-resolution imaging while maintaining a large ratio of FOV relative to the cross-sectional area of the device (see Materials and Methods for elaboration). Microscope objectives are Olympus MPlanFL N (1.25×/2.5×/5×, NA = 0.04/0.08/0.15), Nikon Apochromat (1×/2×/4×, NA = 0.04/0.1/0.2), and Zeiss Fluar (2.5×/5×, NA = 0.12/0.25). (D) FlatScope prototype (shown without absorptive filter). Scale bars, 100 μm.

Fig. 2

Fig. 2. T2S model.

(A) Illustration of the FlatScope model using a single fluorescent bead as the scene. (B) Fluorescent point source at depth d, represented as input image X d. (C) FlatScope measurement Y. FlatScope measurement can be decomposed as a superposition of two patterns: (D) pattern when there is no mask in place (open) and (E) pattern due to the coding of the mask. Each of the patterns is separable along x and y directions and can be written as (F and G) two separable transfer functions. The FlatScope model, which we call as the T2S, is the superposition of the two separable transfer functions.

Fig. 3

Fig. 3. Resolution tests with the FlatScope prototype.

(A) Double slit with a 1.6-μm gap imaged with a 10× objective. (B) Captured FlatScope image. (C) FlatScope reconstruction of the double slit with a 1.6-μm gap. (D to F) FlatScope reconstructions of USAF resolution target at distances from the mask surface of 200 μm (D), 525 μm (E), and 1025 μm (F). Scale bar, 100 μm.

Fig. 4

Fig. 4. 3D volume reconstruction of 10-μm fluorescent beads suspended in agarose.

(A) FlatScope reconstruction as a maximum intensity projection along the z axis as well as a ZY slice (blue box) and an XZ slice (red box). (B) Estimated 3D positions of beads from the FlatScope reconstruction. (C) Ground truth data captured by confocal microscope (10× objective). (D) Depth profile of reconstructed beads compared to ground truth confocal images. Empirically, we can see that the axial spread of 10-μm beads is around 15 μm in FlatScope reconstruction. That is, FlatScope’s depth resolution is less than 15 μm. The three beads shown are at depths of 255, 270, and 310 μm from the top surface (filter) of the FlatScope.

Fig. 5

Fig. 5. 3D volumetric video reconstruction of moving 10-μm fluorescent beads.

(A) Subsection of FlatScope time-lapse reconstruction of 3D volume with 10-μm beads flowing in microfluidic channels (approximate location of channels drawn to highlight bead path and depth). Scale bar, 50 μm (FlatScope prototype graphic at the top not to scale). (B) Captured images of frames 1, 3, and 5 (false-colored to match time progression). (C and D) FlatScope reconstructions of frames 1, 3, and 5 at estimated depths of 265 and 355 μm, respectively (dashed lines indicate approximate location of microfluidic channels). Reconstructed beads false-colored to match time progression. Scale bar, 50 μm.

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