A schematic diagram of our lensfree multi-height imaging set-up can be seen in . The set-up is composed of a partially-coherent light source (~5 nm bandwidth centered at 550 nm), glass cover slips with different thicknesses and a CMOS detector-array. The set-up is rather simple to operate without any complicated alignment. For reconstructing dense samples where object-support based phase-recovery approaches face challenges, different lensfree intensity measurements of the sample are acquired at different Z2 distances.
distance is controlled by placing glass cover slips with different thicknesses between the sample and the sensor-chip. The thicknesses of our glass cover slips varied between 50 µm and 250 µm, hence the corresponding Z2
distances varied between ~0.7 mm and ~1 mm. Each lensfree intensity measurement is sampled by our CMOS sensor with 2.2 µm pixel size. This relatively large pixel size can cause undersampling issues; therefore, a PSR method is applied in order to effectively decrease the detector pixel size [31
]. For each Z2
-distance a lower-resolution (LR) image stack is captured, where each image in this stack is sub-pixel shifted with respect to the other images in the stack. These sub-pixel shifts are achieved by a slight translation of the fiber-tip position between two sequential images (see . upper left inset).
depicts the image processing steps after image acquisition. For each Z2-distance, one SR hologram is synthesized from the LR image-stack (typically 16 images in each stack). Then, these M SR images are registered to each other (see Section 4) to account for rotation, translation and shear that may occur during placing/replacing the glass cover slips between each lensfree measurement. For the reconstruction procedure (iterative phase recovery, see Section 5), these M SR intensity measurements are utilized as amplitude constraints (typically 1-70 iterations are required for satisfactory results). Once the phase is iteratively recovered, any one of these M SR images is low-pass filtered (Butterworth 2nd order) to eliminate high frequency noise and the resulting complex image is back propagated to the object plane, retrieving both phase and amplitude images of the specimens on the chip (see ).
illustrates the benefits of using the above outlined multi-height lensfree imagingapproach for a blood smear sample. shows a full FOV (~24 mm2
) LR lensfree hologram as captured by the CMOS sensor. The green dashed rectangle focuses on an area that is rather dense; however the blood cells are still organized as a mono-layer, suitable for imaging. The reconstruction results of this dense blood smear using five different Z2
-distances (711μm, 767μm, 821μm, 876μm and 946μm) are shown in . These five Z2
-distances/heights are automatically evaluated by using an auto-focus algorithm [68
]. The reconstruction results of provide a decent agreement to a 10 × microscope objective comparison image shown in . , and provide images of zoomed areas (taken from the dashed blue rectangle in ) of single height back propagation image, multi-height reconstruction image and a 20 × microscope objective comparison image, respectively. The back propagated single height image () has lower contrast, and it is hard to evaluate the locations of the RBCs for spatial masking purposes. Therefore support-based phase recovery would not be effective in this case. On the other hand, the multi-height amplitude image () has significantly improved contrast, and individual RBCs can be identified and resolved even in dense clusters. It is important to emphasize that these multi-height reconstruction images shown in
are obtained without the use of any spatial masking or any other prior information regarding the sample.
Fig. 3 (a) Full FOV, LR hologram. (b) Multi-height based PSR lensfree amplitude image of a dense RBC smear is shown. This lensfree image was reconstructed using five different heights (λ = 550nm). The FOV corresponds to the green dashed rectangular in (more ...)
After these blood smear experiments, next we imaged Pap smears (based on SurePath automated slide preparation [70
]) using the same multi-height imaging set-up.
summarizes our imaging results for this Pap smear. Because of the density of the specimen, the reconstruction of this image is a challenging task for any phase recovery method. shows the multi-height phase image, which is recovered using lensfree measurements from five different heights (754μm, 769μm, 857μm, 906μm and 996μm - these Z2
-distances were automatically determined using an auto-focus algorithm [68
]). and show zoomed images of the same Pap smear sample, for amplitude and phase channels, respectively. In these reconstructed multi-height images the cell morphology is clear and their boundaries can clearly be seen and separated from the background. Moreover, minor overlaps among the cells do not constitute a limitation in this method. As a comparison, depicts a single height back propagated phase image corresponding to one of the Z2
measurements (the FOV is the same as in ). It is evident that distinguishing the cells from the background is a difficult task in this dense reconstructed image. To better provide a comparison, and (g,l) also show zoomed images of the same Pap smear sample, for phase and amplitude channels, respectively, calculated using back propagation of a single height image. Compared to and , these single height back projection images show significant spatial distortion due to the density of the cells. and also provide 40 × objective lens (0.65NA) microscope comparison images for the same zoomed regions, clearly providing a decent match to our multi-height reconstruction results shown in and (c,h). Especially note the enhanced contrast of the cell boundaries in our phase images (), which is complementary to the spatial information coming from our amplitude images ()
. This complementary set of information that is conveyed by the amplitude and phase images might facilitate detection of abnormal cells within a Pap test that are characterized for instance by a high nuclear-cytoplasmic ratio. It is also important to note that all the phase images reported in our manuscript are wrapped; hence, for the multi-height reconstructed phase images, phase jumps should be expected in absorbing areas of the cells (e.g. nuclei), where phase would not be properly defined. Contrary to the phase images, these absorbing areas will be of high contrast in their corresponding lensfree amplitude images, which once again emphasizes the complementary nature of phase and amplitude lensfree image channels.
Fig. 4 (a) Multi-height based PSR lensfree phase image of a Pap test is shown. This image was reconstructed using five heights. 36 iterations were used during phase recovery (λ = 550nm). (b) Single height back propagated PSR phase image is shown. (c) (more ...)
Next we investigated how the number of intensity measurements used in our iterative reconstruction process affects the image quality (see
). To provide a fair comparison (i.e., to better isolate the source of improvement in image quality), a total of 144 Fourier transform pairs were used in each case, regardless of the number of intensity measurements employed in the multi-height based phase recovery. Moreover, all the phase images are wrapped and the same global phase was assigned to all of the images to avoid different phase jumps in different images. shows a single height back propagated phase image. When a second intensity measurement is added, multi-height based iterative phase recovery approach can be utilized. Consequently, the recovered phase image after 72 iterations (see ) looks significantly better than the phase image of . A further improvement in image quality is achieved by adding a third intensity measurement to the multi-height phase recovery process (). After 36 iterations (i.e., corresponding to a total of 144 Fourier transform pairs as before), the cells that were hidden in the noisy background are now visible (see white arrows in ). A moderate improvement is noticed in the image contrast when adding more intensity measurements, as can be seen in the reconstructed multi-height phase images from four and five heights ( and , respectively). Note that in these two cases, 24 and 16 iterations were used, respectively, so that the total number of Fourier transform operations remains the same in all reconstructions shown in , which helps us to isolate the source of the phase reconstruction improvement and relate it to multiple height measurements rather than the number of back-and-forth digital propagation operations. shows a microscope comparison image (10 × , 0.25NA) for the same region of interest. Note also that the cell’s boundaries are more visible in our phase images, while the absorbing nuclei of the cells are better visualized in our amplitude images as illustrated in .
Fig. 5 Pap smear reconstruction results acquired for different number of lensfree diffraction intensities. (a) Back propagated image from one PSR lensfree hologram. (b), (c), (d) and (e) Multi-height based PSR lensfree phase images from two, three, four and (more ...)
After validating the usefulness of pixel super-resolved multi-height based phase recovery approach with dense blood smears and Pap tests, we experimentally tested its impact on the reconstructed image quality. An important question that we aimed to address with this additional experiment was whether or not the digital cross registration process among different Z2 lensfree holograms results in spatial smearing of our reconstructed images. Therefore we compared the imaging performance of our multi-height reconstruction results against a single back-propagated super resolved hologram. For this end, we imaged an isolated ‘UCLA’ pattern that was etched on a glass slide using focused ion beam (FIB) milling, where the letters ‘U’ and ‘C’ are ~1 µm apart. We emphasize here that for such an isolated object multi-height based image reconstruction is not necessary. Since we aim to understand the impact of multi-height cross registration related issues, in this final experiment we chose an isolated object (‘UCLA’) so that the back-propagation result of a single height SR hologram could work for comparison purposes. This is quite different from the dense objects/specimens reported in -, where back-propagation of a single height lensfree PSR hologram fails, requiring the use of multiple height measurements.
For this final experiment, the single height back propagated SR holographic image is shown in
, where the letters ‘U’ and ‘C’ are clearly separated. The ‘UCLA’ pattern is spatially isolated from nearby objects, and therefore for this small isolated FOV phase recovery is not necessary as emphasized earlier. , , and show multi-height based reconstructed amplitude images, for two, three, four and five different heights, respectively (λ = 490nm). For fair comparison among these recoveries, once again the number of Fourier transform pairs was kept constant in each case, as a result of which each reconstruction used a different number of iterations (60, 30, 20 and 15 iterations, respectively). It is evident that the letters ‘U’ and ‘C’ are clearly separated in all of these images, which is an indication of our success in cross registration of different height super-resolved holograms to each other so that spatial smearing affects due to possible inconsistencies among different Z2 lensless holograms are minimized. A microscope comparison image of the same “UCLA” pattern can also be seen in , acquired using a 40x objective lens (0.65 NA).
Fig. 6 Adding intensity measurements from different Z2 distances does not degrade the image resolution. An important question that we aimed to address with this additional experiment was whether or not the digital cross registration process among different height (more ...)