Compact, lensless digital holographic microscope for remote microbiology

In situ investigation of microbial life in extreme environments can be carried out with microscopes capable of imaging 3-dimensional volumes and tracking particle motion. Here we present a lensless digital holographic microscope approach that provides roughly 1.5 micron resolution in a compact, robust package suitable for remote deployment. High resolution is achieved by generating high numerical-aperture input beams with radial gradient-index rod lenses. The ability to detect and track prokaryotes was explored using bacterial strains of two different sizes. In the larger strain, a variety of motions were seen, while the smaller strain was used to demonstrate a detection capability down to micron scales. © 2016 Optical Society of America OCIS codes: (090.1995) Digital holography; (100.3175) Interferometric imaging; (120.4570) Optical design of


Introduction
Conventional high-resolution microscopes capable of imaging bacteria have limited depth-offield and typically require complex objective lenses with tight alignment tolerances.As a result, direct observation of prokaryotes (bacteria and archaea) in their native environments has yet to be performed in most parts of the Arctic and Antarctic, around hydrothermal vents, and in the majority of the open ocean [1].Quantifying prokaryotic behavior in situ is important for understanding large-scale marine processes such as carbon cycling [2,3].It is also of great interest for the investigation of the possibility of life in extraterrestrial Ocean Worlds, such as Enceladus and Europa [4], but instruments for unambiguous detection of prokaryotes in planetary environments don't yet exist.Some microscopes have flown in space, but as yet no microscopic observations have been made on Mars with the resolution required to detect bacteria [5,6].For all such remote deployment scenarios, a compact, robust microscope capable of operating in an ambient environment is required.
Digital holographic microscopy [7] has a number of advantages over conventional microscopy for remote autonomous deployment, including robustness (no moving parts, as no focus mechanism is needed), high throughput, and compressed sensing (i.e., the entire 3-d sample volume is encoded in each 2-d frame capture).As a result, this technique is beginning to see application beyond the laboratory [8,9].Our previous "common path" digital holographic microscope [DHM] design [10] reported microbial imaging in Greenland sea ice [11], but its reliance on classical lens-based optics left this prototype larger than desirable for robotic deployment.On the other hand, lensless holography, with no imaging optics between the light source and detector [12][13][14][15][16], can enable compact, lightweight systems.
Several lensless holographic and tomographic microscope approaches exist [8,9,[13][14][15][16][17][18][19][20][21][22][23][24][25][26], with differing advantages and disadvantages.For example, use of incoherent light provides speckle noise reduction [15], but also reduced fringe visibility and depth of field.Tomography [17][18][19][20] can provide high resolution, but multiple reads are needed to acquire a full information set.Conversely, motion tracking does not necessarily require high-resolution imaging [8,21].On-chip systems can provide a large field of view (FOV) by situating the sample very close to the detector array [21][22][23], but thermal issues can arise from differing ambient-sample and powered-detector temperatures.A more classical lensless DHM configuration with a somewhat larger sample-to-detector distance may thus be more suited to microscopic imaging in extreme environments.Of these, laser-based systems [14,[24][25][26] have tended to include pinholes and additional fore-optics to increase the laser-beam numerical aperture (NA), thereby increasing system volume and alignment complexity.For robustness and compactness, ideally any difficult-to-align elements such as small pinholes should be avoided, and any high-NA laser beam (or beams) should be provided without greatly impacting system volume.This paper provides a solution to both of these issues by making use of small radial gradient-index (GRIN) rod lenses [27] to inject high NA laser beams into a lensless DHM.

Lensless digital holographic microscopy with GRIN lenses
In lensless holography, reference and source beams directly illuminate the detector array, without any intervening optics.However, as in standard microscopy, high-resolution holographic microscopy also requires high NA, as the linear resolution is still given by λ/(2NA).High-NA laser beams must then be launched from just in front of the detector array, i.e., from a distance, f, of ≈1 -3 cm for a typical cm-scale array.Moreover, in the off-axis holography case, the fringes on the detector array due to the two-beam interference pattern must have a spatial period, L, of at least twice the pixel pitch, p, where b is the separation baseline between the two source points.For a pitch of a few µm, a roughly mm-sized source baseline is required.While a beamsplitter could be used to separate the two launch points into separate beam paths [28], in either case a number of components must be crowded together: both point sources of light (or their reimaged foci), the sample, and the camera (as well as a beamsplitter if one is employed).Launching the beams from single-mode optical fibers is an attractive possibility, as fiber tips can be located relatively freely, and unconnectorized fibers can be used to save space.However, typically available single-mode fibers have lower NAs than is necessary for µm-scale resolution, especially at shorter (blue/violet) wavelengths.The goal for off-axis holography is thus to provide, with minimal optics, a pair of high NA laser beams in close proximity to each other.We achieve this by using small GRIN rod lenses to increase the NA of a single-mode (SM) fiber output beam.GRIN lenses with mm-scale diameters and lengths are readily available at low cost, and can be used to produce a high NA output focus from a collimated input beam [27].A schematic of our conceptual design is shown in Fig. 1(a).A single SM fiber output beam is first collimated by a small fiber-collimating lens, and the collimated beam then over-illuminates a pair of GRIN lenses, yielding a pair of high-NA focal spots in their common removes the n thereby elimi susceptible.F the sample a between the s Although enable a singl the use of GR beam to the G enables the s straightforwar of great value off-axis holo because of the permits recon configuration introduces a s overlap shoul small single-c distances, and extended.On       the extended Air Force target in the complementary image.This background structure would be much reduced if these larger-scale opaque structures did not surround the region of interest, as will be the case for biological targets.For this reason, Fig. 2(a) is far from a best case.Figures 2(b) and 2(c) shows two horizontal cross-cuts through the image of Fig. 2(a), where it can be seen that the vertical bars in element 3 of group 8 show fairly deep modulation, while element 4 of group 8 has a reduced fractional modulation, and is closer to the background fluctuation level near these elements.With line widths in the two cases of 1.55 and 1.38 μm, respectively, and allowing for the fact that some residual astigmatism and background contamination may be present in the image, in rough terms this demonstrates an ability to resolve features (line widths) down to ~1.5 μm.This is roughly as expected, as a source NA of approximately 0.17 for s ≈20 mm yields an expected resolution of ≈1.2 μm.Moreover, as all instrumental parameters and correction techniques available have not yet been fully explored or exploited (in particular, the integral C-mount on the front of the camera constrained the source-detector distance to be larger than the C-mount length), and as the mottled background due to the second beam is significant for the case of Air Force targets, the ultimate performance of this approach has likely not yet been reached.
With no optical elements between the GRIN foci and the camera, the depth of field extends essentially from the GRIN focal plane to the detector plane, with the resolution degrading from ≈1.5 μm at our sample location to 3.45 μm (the camera pixel size) at the detector plane.This extended depth of field was verified by translating the sample along the optical axis as much as allowed by the GRIN disk assembly and the camera C-mount, with sample motions over a range of ≈6 mm yielding similar performances.As to the field of view, similar triangles defined by the angle subtended by the array diameter, d, from the GRIN lens focus give With a 7 mm array, s ≈20 mm and f ≈25 mm yields FOV ≈1.4 mm.It should be possible to increase the FOV by removing the camera's C-mount to access smaller values of s.Finally, with the aberration-correction step described above, the resolution did not degrade noticeably across the image, e.g., Fig. 3(b).

Demonstration prokaryote observations
The ability to image and track prokaryotes was demonstrated on two test strains of different sizes, morphologies, and motility patterns.A sample chamber depth of 0.8 mm was made using optical quality cell culture wells (Electron Microscopy Sciences, part no.70326-30) topped with a microscope slide or coverslip.The reference and object beams both passed through the sample.Hologram time series of 30-60 s at 7-15 frames per second were recorded and reconstructed as described.The resultant intensity images were median subtracted using Fiji [32], and the phase images cropped to exclude the edges, but otherwise unprocessed.Figures 3 (a) & (b) show single-plane amplitude and phase images of the larger bacterial strain (Bacillus subtilis ATCC 6051) that was fully resolved by our DHM.The cells were grown to mid-log phase in Lysogeny Broth (LB), and then immediately before imaging, diluted to a concentration of ~10 7 /mL in motility medium (10 mM potassium phosphate, 10mM NaCl, 0.1mM EDTA, 0.1 mM glucose, pH 7.0).As is readily apparent in Visualization 1 and Visualization 2, the cells were highly motile, with a swimming speed of ~5-10 µm/s.For this larger bacterium, translational, rotational, bending and oscillatory motions are all very evident in Visualization 1 and Visualization 2. Some stationary cells, presumably adhering to the slide surfaces, are also seen.Next, the ability to detect unresolved cells in dilute solutions was demonstrated using a smaller bacterial test species.For this we used the marine psychrophile Colwellia psychrerythraea strain 34H, which measures approximately 1 x 0.5 µm and demonstrates rapid motility (up to 100 µm/s) [33].The cells were maintained in ½ strength Marine Broth (Difco) at 6 °C, and diluted into the same culture medium for imaging.Their existence and motility was readily observed -when in focus, the cells appeared as bright spots in both intensity and phase.Figure 3(c) shows a single-plane amplitude reconstruction of organisms at a density of ~10 7 cells/mL, and Fig. 3(d) shows a time track constructed from amplitude data.Visualization 3 and Visualization 4 show amplitude and phase videos of a single-plane reconstruction sequence, in which a number of cells are seen to move across the field, thus demonstrating that such a microscope can be used for remote life searches down to somewhat below the instrumental resolution.
The choice of 800 µm deep sample chambers represented a compromise between SNR and freedom of axial movement.While the instrument's performance did not degrade substantially through a thicker air path, the signal-to-noise ratio of a particle suspension at high particle densities is inversely proportional to the product of the particle number density and the sample chamber depth [34].Figures 3(e Fig. 1 (c) Ph at the A solid m assembled in mounted side the "GRIN di diameter).Th diameter) were of ≈25 mm, pr single collimat mode until the fiber system w high NA to pas other than the f is indeed lensl this approach RIN lens.In eit ne pinholes, as[7], off-axis ho l images, allo ase images.Th n amplitude.In han in-line ho r GRIN system ur particular d ample plane, w bjects more diff r a sparse popu opposite recon d hence very search region.l of our DHM, and fiber laser input is e) is to the right.

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Fig. 3 .
Fig. 3. Bacterial strains imaged with our lensless DHM.(a) B. subtilis in intensity (video shown in Visualization 1).(b) B. subtilis in phase (video in Visualization 2).(c) Colwellia in intensity (video in Visualization 3; a Colwellia phase video is shown in Visualization 4).The arrows indicate cells in or near focus.(d) A Colwellia time track.(e) Zoomed-in image of B. subtilis in a 100 µm deep chamber.(f) Zoomed in image of B. subtilis in an 800 µm deep chamber.
) and 3(f) shows a comparison of B. subtilis cells in the 800 µm deep chamber vs. cells in a chamber 100 µm deep, illustrating the increased contrast seen with thinner samples.If only 2-dimensional images are required, thinner chambers can be employed.

Fig. 4 .
Fig. 4. 3D visualizations.4a) xz image showing a cross section through the reconstructed PSF of a B. subtilis cell.(b) Image of B. subtilis culture at a single time.Individual cells can be identified by their extended PSFs.(c) 3D tracks of 30 s of cell motion of a Colwellia culture.Cells were identified using a maximum intensity projection.Non-motile cells (green) clustered near z = 0, while trajectories of motile cells spanned the volume.Time is color-coded.