illustrates the reconstruction procedures of our lensfree off-axis reflection holographic microscope shown in . To quantify the performance of its resolution and FOV, we first imaged a US Air Force resolution test target (US-AFT), i.e., USAF 1951 Test Chart from Edmund Optics. The raw lensfree hologram of the US-AFT is recorded using the reflection set-up of as shown in . Note that the interference fringes between the reference and the reflected object waves are clearly visible within the expanded frame of . This lensfree reflection hologram is then digitally interpolated and padded with zeros (see ) and its corresponding 2D Fourier spectrum is also shown in . Note that as a result of the off-axis recording geometry, the spatial frequencies of the real and twin images are clearly separated from the 0th order [2
]. Furthermore, some of the parasitic reflections [6
] occurring at various interfaces in our experimental setup also appear in , marked with yellow dashed arrows. After spatial filtering of these unwanted frequency components, our lensfree reflection hologram can be rapidly reconstructed using the Fresnel approximation [6
] to yield the image of the AFT over a rather large FOV of ~9 mm2
, as shown in . also shows a digitally enlarged version of the central section (taken from indicated by the yellow dashed rectangular box), which demonstrates ≤2 µm lateral resolution.
Fig. 3 Reconstruction procedures for off-axis geometry using the lensfree reflection DHM shown in are described. (a) The raw off-axis lensfree reflection hologram of US-AFT. The interference fringes of off-axis geometry are shown in the expanded version (more ...)
Next, using the same off-axis lensfree reflection microscopy mode (i.e., ), we imaged a histopathology slide (prepared using standard sample preparation protocols [46
]) corresponding to a human skin tissue, the results of which are summarized in
. To minimize multiple reflection interference artifacts in our reflection hologram, we used a right angle prism behind the glass sample holding the tissue slide with refractive index matching oil between the two. Since the intensity of the reflected wave from the skin tissue is quite weak, a regular thin cover glass (thickness ~100 µm) is used as a reference mirror in to balance the intensity between the object and the reference waves. shows the lensfree off-axis reflection hologram of this skin tissue, where a digitally expanded version of it is also shown in . The reconstruction result of this off-axis reflection hologram is shown in . For comparison purposes, also illustrates the results of a conventional reflection mode bench-top microscope imaging the same specimen taken with a 4X objective lens (0.1 NA), which agrees well with our field-portable lensfree reflection microscope results. Note that due to their limited FOV, higher magnification objective lenses (e.g., 10X or 20X) would not be able to capture the same comparison image.
Fig. 4 Reflection imaging of a histopathology slide corresponding to skin tissue using lensfree off-axis holography. (a) The raw off-axis reflection hologram of skin tissue. (b) The digitally zoomed hologram region specified with the blue rectangle in (more ...)
Following this, we tested the imaging performance of our transmission mode field-portable microscope shown in . To demonstrate its resolving power, lensfree in-line hologram of an AFT is recorded using the set-up shown in , where the raw holograms of groups 2 & 3, groups 4 & 5, and groups 6 & 7 are shown in
, , and , respectively. The corresponding reconstructed amplitude images of these lensfree transmission holograms are shown in , , and , respectively. These images are digitally obtained by an iterative reconstruction process (~15 iterations) that is based on object-support constrained phase recovery, which effectively removes the twin image artifact of in-line holography [3
]. The reconstructed images shown in demonstrate a resolution of ≤2μm over a wide imaging FOV of ~24 mm2
which is due to our unit-fringe-magnification hologram-recording geometry. It is important to note that our off-axis lensfree reflection microscope provides a similar spatial resolution, however, over a smaller FOV of ~9 mm2
. This relatively reduced FOV of the reflection mode is due to its increased fringe magnification (F
~1.67 compared to F
~1 in transmission mode) as well as due to its off-axis geometry with θ ~5°.
Fig. 5 Demonstration of the imaging performance of our in-line lensfree transmission microscope shown in . (a-c) Raw lensfree in-line transmission holograms for (a) groups 2-3, (b) groups 4-5, and (c) groups 6-7 of the US-AFT. The corresponding reconstruction (more ...)
And finally, we imaged 4-μm-sized micro-particles using the same lensless holographic transmission microscope.
illustrates our lensfree holographic imaging results and also provides comparison images of the same objects obtained by a 40X objective lens (NA = 0.6) of a conventional bench-top bright-field microscope, which provide a decent agreement to our reconstruction results.
Fig. 6 (a) Raw lensfree transmission hologram for 4 µm beads and (b) its corresponding reconstructed amplitude image are illustrated. (c) Conventional bright-field microscope image of the same objects (40X objective lens – NA: 0.6) is also provided (more ...)
We should note that this presented field-portable microscope could be used to monitor e.g., water samples as well as various bodily fluids such as semen or blood. In its transmission geometry, a major advantage of this platform is its imaging volume. The large FOV (~24 mm2) combined with a long depth-of-field (e.g., ~1-2 mm) can enable rapid screening of large sample volumes. For water quality monitoring applications, for instance, the reflection mode would also be quite relevant, especially for sample concentration steps that involve e.g., porous silicon membranes, where the reflection mode geometry could be of great interest with its large imaging FOV (e.g., ~9 mm2)