PhaseRMiC: phase real-time microscope camera for live cell imaging

: We design a novel phase real-time microscope camera (PhaseRMiC) for live cell phase imaging. PhaseRMiC has a simple and cost-effective configuration only consisting of a beam splitter and a board-level camera with two CMOS imaging chips. Moreover, integrated with 3-D printed structures, PhaseRMiC has a compact size of 136 × 91 × 60 mm 3 , comparable to many commercial microscope cameras, and can be directly connected to the microscope side port. Additionally, PhaseRMiC can be well adopted in real-time phase imaging proved with satisfied accuracy, good stability and large field of view. Considering its compact and cost-effective device design as well as real-time phase imaging capability, PhaseRMiC is a preferred solution for live cell imaging.


Introduction
Quantitative phase microscopy (QPM) provides a way to observe label-free biological specimens with high imaging contrast but totally different from conventionally used bright-/dark-field or fluorescence microscopy [1]. Many QPM techniques have been successfully used in various applications, unfortunately, most of them rely on bulky optical systems and require expensive cost, limiting their practical applications in wide fields. In order to solve the problem, compact or even handheld microscopes, especially those for phase imaging, have been proposed. Based on ptychography, Raspberry Pi based ptychographic microscope [2] proposed by Harvey group, EmSight [3] developed by Yang group, FPscope [4] and lensless on-chip ptychographic microscope [5] both designed by Zheng group have been reported. These designs can provide phase imaging with extremely large field of view, but they are not preferred solution for real-time phase imaging since they rely on heavy scanning and iterative phase reconstruction. Waller group updated the Cellscope [6] using a domed LED array for phase imaging [7] relying on differential phase contrast, though it significantly accelerates the phase imaging speed, it is still not real-time phase imaging since it requires multiple shots for phase reconstruction. Compact holographic microscopes such as lensfree ones [8,9] designed by Ozcan group, off-axis one reported by Ferraro group [10], shearing ones [11,12] proposed by Javidi group as well as τ [13] and flipping [14,15] interferometers reported by Shaked group can retrieve sample phase in single shot, therefore, these designs can be used in real-time phase imaging applications. Unfortunately, phase unwrapping is often time-consuming, and coherent light is required not convenient for practical applications. Park group proposed white-light quantitative phase imaging unit [16] directly connected to the microscope for phase imaging aiming at promoting compact holographic camera to practical applications, while it only can deal with samples sparsely located as compact shearing holographic microscopes. Quadriwave lateral shearing interferometer [17][18][19][20] can be used for phase imaging directly connected to the microscope, and it does not require coherent illumination, but it is too expensive. Therefore, these above designs are not suitable solutions for real-time phase imaging with fast speed, simple configuration and cost-effective price.
Different from ptychography, differential phase contrast, holography and quadriwave lateral shearing interferometry, transport of intensity phase microscopy (TIPM) [21] does not rely on coherent sources, scanning or iteration, therefore TIPM can support quantitative phase imaging with simple and cost-effective setups as well as fast phase retrieval speed such as our previously proposed smartphone based TIPM [22]. Unfortunately, TIPM requires multi-focus images for phase reconstruction, and it often relies on mechanical or electrical focus adjusting, not only making the imaging system complicated and unstable, but also restricting its applications mostly in static imaging. In order to extend TIPM to dynamic imaging applications, single camera based tactics such as achromatic method [23], point spreading function engineering methods [24][25][26] and beam splitting methods [27][28][29] as well as multiple camera based tactics such as dual/multi-view methods [30][31][32][33] have been designed for real-time phase imaging. Unfortunately, they all suffer from complicated systems, for example, achromatic method requires specific design and precise calibration on the imaging system; point spreading function engineering methods need distorted diffraction gratings, volume holographic gratings or even spatial light modulator; beam splitting methods using single camera relies on spatial light modulator, and those methods using multiple cameras complicate the TIPM system as well as increase the system size and cost. Therefore, these methods are often implemented relying on temporally built systems, and there is still not a compact or simple device for real-time TIPM.
In order to provide compact phase sensing microscope modules such as τ interferometer [13], flipping interferometer [14,15] and white-light quantitative phase imaging unit [16] or even camera-like devices as quadriwave lateral shearing interferometer [17][18][19][20] and Shack-Hartmann wavefront sensor, but not relying on highly coherent light, expensive devices or special sample requirement, we propose a new design named PhaseRMiC, which is a Phase Real-time Microscope Camera based on transport of intensity phase imaging. PhaseRMiC has a simple and cost-effective configuration integrated with 3-D printed structures in a compact size of 136×91×60 mm 3 comparable to many commercial microscope cameras, and can be directly connected to the microscope side port for real-time phase imaging proved with satisfied accuracy, good stability and large field of view. Considering its compact and cost-effective device design as well as real-time phase imaging capability, PhaseRMiC is a preferred solution for live cell phase imaging.

PhaseRMiC design
PhaseRMiC is shown in Fig. 1(a), and it can be directly connected to a commercial microscope (Nikon TiE2, Japan) for simultaneous multi-focus image recording and a personal computer for phase reconstruction as revealed in Fig. 1(D). Figure 1(b) describes both the optical and mechanical configurations of the PhaseRMiC: the wavefront from the microscope is first divided using a prism beam splitter and then collected with a board-level camera integrated with two CMOS imaging chips (1280×1024, 4.8 µm, Daheng Imaging, China) running under a laptop. The distances between two CMOS imaging chips and the beam splitter are designed as 11.5 mm and 9.5 mm as shown in the scheme in Fig. 1(c), respectively. Therefore, two under-and over-focus images with defocus distance of 1 mm can be simultaneously recorded for phase reconstruction via solving the transport of intensity equation, in which the in-focus image can be approximated as the average of the under-and over-focus images. All the elements are integrated using 3-D printed structures to construct a camera-like device as shown in Fig. 1(a), which has a compact size of 136×91×60 mm 3 and is also cost-effective comparable to many commercial microscope cameras. Additionally, PhaseRMiC has a large field of view (FoV) since it does not need FoV segmentation. After constructing the PhaseRMiC, the FoV mismatch including translation, rotation and scaling between two CMOS imaging chips should be corrected. Instead of using difficult hardware correction during assembly and adjusting, here the FoV mismatch is determined and numerically compensated using our previously adopted phase correlation method [34]. Moreover, the defocus interval between the under-and over-focus images was precisely calibrated using a single-lens imaging system. The PhaseRMiC and a standard target fixed on a precision translation stage were respectively placed on the object and image spaces of the lens with known parameters, and the target was shifted along the optical axis to obtain in-focus images corresponding to two CMOS imaging chips. Since their object distances could be precisely measured, their corresponding image distances were decided according to the Gaussian function in geometrical optics, and the difference between these computed image distances was equal to the focus interval, which was used in the following phase retrieval. Before using PhaseRMiC for specimen phase imaging, the in-focus specimen position should be first determined by adjusting the specimen stage of the upright microscope or the micro-objective of the inverted microscope according to the average of the simultaneously captured under-and over-focus images using in-focus criterion: when the specimen imaging plane is just located in the center of two CMOS imaging chips, the computed average is actually the in-focus image. Then, a series of under-and over-focus images can be recorded using PhaseRMiC running under a laptop. Finally, their phases can be reconstructed via solving the transport of intensity equation [21] as shown in Eq. (1), in which φ is the specimen phase, k is the wave number as 2π/λ, λ is the wavelength, I is the image intensity, ∂I/∂z represents the intensity derivative, ∇ ⊥ is the lateral gradient operator, x and y indicate the lateral spatial coordinate, and z indicates the axial axis.
Equation (1) illustrates that specimen phase can be reconstructed through solving the partial differential equation. It is worth noting that fast Fourier transform (FFT) is a preferred solution in dealing with partial differential equation since the lateral gradient operator can be easily computed in spectral domain as illustrated in Eq. (2), in which F represents FFT, f(x,y) is a function in spatial domain, x and y describe the spatial coordinate, while u and v describe the spectral coordinate.
Afterwards, the specimen phase can be finally reconstructed via Eq. (6).
In order to pursue real-time in-focus phase imaging as well as simple and cost-effective device, only two defocus images as under-and over-focus ones as I(x,y;-∆) and I(x,y;∆) are captured for phase retrieval, and the intensity derivative can be computed according to Eq. (7). Moreover, the in-focus image I(x,y;0) is obtained as the average of under-and over-focus ones explained by Eq. (8) as our previous works [31,32,34].
Compared to those methods relying on more multi-focus image recording [35][36][37][38], the phase retrieval accuracy of PhaseRMiC is inevitably limited. Moreover, both CMOS sensors are fixed thus losing the adaption to different imaging signal to noise ratios (SNRs) [39]. Therefore, in order to test the phase imaging performance, experiments using standard samples, fixed cells and live cells were implemented in the following section.

Results
To test the phase imaging performance of PhaseRMiC, a standard random phase mask (fabricated by Shanghai Institute of Optics and Fine Mechanics, Chinese Academy of Sciences) with the binary phase values of 0 and π at 632.8 nm was measured. An interference filter (Daheng Optics, China) with the central wavelength of 632.8 nm and the full width at half maximum (FWHM) of 10 nm was inserted in the microscope and the Kohler illumination with the condenser aperture as ∼40% of the objective aperture was set to respectively improve the temporal and spatial coherence of the illumination to ensure phase imaging. Moreover, a 10× micro-objective was adopted for specimen magnification. Figures 2(a) and 2(b) reveal the captured under-and over-focus intensities after FoV correction, and Fig. 2(c) is the numerically computed in-focus intensity as the average of those in Figs. 2(a) and 2(b). Figure 2(d) shows the reconstructed phase in the whole FoV of 0.21 mm 2 , which exhibited the binary distribution with the phase difference of ∼π. Figure 2(e) reveals the zoomed-in phase distributions in two different regions of interests (RoIs), and Fig. 2(f) plots their cross sectional phase distributions, both clearly proving that the specimen phase can be measured using PhaseRMiC. Moreover, to test the phase imaging stability of PhaseRMiC, 1800 pairs of under-and over-focal images were captured during 120 s in 15 fps, and the phase fluctuations at the positions marked in Fig. 2(e) during 120 s are plotted in Fig. 2(g) with the statistical phases as 0 and π, respectively. The rather low standard deviations in phase fluctuations proved good phase imaging stability of PhaseRMiC. Additionally, the extremely large FoV can support massive cell imaging without any FoV scanning or splicing. It is worth noting that PhaseRMiC only captures two defocus image for phase retrieval, therefore, it can hardly reach the phase retrieval accuracy of those TIPM relying on multi-focus image recording. Moreover, both imaging sensors are fixed losing the adaption to different imaging SNRs. However, according to the experiments in Fig. 2, PhaseRMiC could still obtain the phase distribution very close to the actual one, thus PhaseRMiC can still be treated as a quasi-quantitative one the same as those TIPM methods relying on few (3 or less) multi-focus image recording [23][24][25][26][27][28][29][30][31][32][33][34]. Moreover, the verifications on phase imaging stability and FoV demonstrated that PhaseRMiC can be well used in phase imaging applications. After phase imaging performance testing, PhaseRMiC was first adopted for phase imaging on fixed biological specimens as human red blood cells and Vero cells. During these measurements, an interference filter with the central wavelength of 532 nm and the FWHM of 10 nm was used; and a 10× micro-objective was adopted. Figures 3(a) and 3(b) reveal the captured under-and over-focus images of a blood smear after FoV correction, and Fig. 3(c) is the computed in-focus image. Figure 3(d) shows the reconstructed phase with massive red blood cells supported by the extremely large FoV of 0.21 mm 2 . In order to analyze the cellular details, Fig. 3 several individual cells, illustrating that most of these red blood cells had the specific biconcave configurations. In addition, fixed Vero cells using 4% paraformaldehyde were also observed: Figs. 3(f) and 3(g) reveal the captured under-and over-focus images after FoV correction and Fig. 3(h) is the computed in-focus image. Figure 3(i) shows the retrieved cellular phase and Fig. 3(j) lists zoom-in phases in two different RoIs. According to these results, PhaseRMiC could provide phase images which often had higher imaging contrast than in-focus images.

(e) lists
Finally, PhaseRMiC was adopted in live cell phase imaging. Vero cells were cultured with 10 mL dulbecco's modified eagle medium containing 0.05% fetal bovine serum for 12-14 h until the cells were completely adherent and extended. After removing the culture medium, 2 mL phosphate buffer saline was added for cell cleaning for three times and finally sucked out. In order to promote rapid cell change, trypsin solution was introduced, and the cell shapes changed from rhombic to round in 3 min, which was recorded using PhaseRMiC with the frame rate of 20 fps. Figure 4(a) shows partial time-series under-and over-focus images, and Fig. 4(b) reveals their reconstructed phases. Figure 4(c) as well as Visualization 1 clearly exhibits the cell changes from rhombic to round due to the introduced trypsin solution. The application proves that PhaseRMiC can be well used in live cell phase imaging.

Discussions
Since phase imaging can significantly improve the imaging contrast especially for label-free biological samples, many designs, especially those compact ones, have been proposed as listed in Table 1. Portable Fourier ptychographic microscopes can simultaneously obtain high spatial resolution and large FoV, but they suffer from slow speed in both image recording due to the required multi-angular scanning as well as phase reconstruction because of the iterative retrieval algorithm. Also relying on programmable illuminations, portable microscopes based on differential phase contrast significantly simplify both the recording and reconstruction procedures, while the multi-shot requirement still limits their real-time phase imaging applications. Many digital holographic microscopes can provide sample phase distributions only from single-shot interferogram/hologram, thus they successfully solve the real-time phase imaging problem. However, many of them require laser for illumination, and the often required phase unwrapping remarkably slows down the phase retrieval speed. Additionally, both Shack Hartmann wavefront sensor and quadriwave lateral shearing interferometry can be integrated to camera-like devices, and used for real-time phase imaging; while their spatial resolution is limited due to their respectively used micro-lens array and chessboard grating, and moreover, they are extremely expensive. Compared to the above techniques, compact TIPM based devices are preferred solutions for real-time phase imaging. With special designs such as achromatic, beam splitting and dual/multi-view tactics, they can reconstruct sample phase in both high spatial and temporal resolutions, and moreover, phase reconstruction is also fast avoiding time consumed phase unwrapping. But these designs still suffer from several disadvantages, achromatic tactic requires specially designed achromatic lens, beam splitting tactic often limits the imaging FoV and dual/multi-view tactic requires more image recorders thus increasing the costs and complicating the system. While our proposed PhaseRMiC integrates a beam splitter and a board-level camera to a compact device, and it employs two CMOS sensors for under-and over-focus imaging, respectively. Therefore, our proposed PhaseRMiC has several advantages: (1) it can reach high phase imaging speed as 20 fps used in live cell imaging (or even higher as 30 fps limited by the camera); (2) it has a simple and cost-effective configuration only with few optical elements; (3) it has a large FoV since it does not rely on FoV division for multi-focus imaging; and (4) it is an integrated camera-like device and can be directly used combining with commercial microscopes. Therefore, it can be potentially used as a phase camera especially in biological applications.
But unfortunately, PhaseRMiC still has some shortcomings. (1) PhaseRMiC only captures two defocus (under-and over-focus) images for phase retrieval, therefore, its accuracy is limited. Many works have been reported that capturing more multi-focus images can definitely improve the phase imaging performance [35][36][37][38], but multi-focus image stack recording inevitably complicates the imaging system, and more importantly decreases the imaging speed. While in this work, the proposed PhaseRMiC aims at pursuing fast phase imaging speed as well as simple and cost-effective device, which shares the similar purpose of those single-shot/real-time TIPM works also recording few (3 or even less) multi-focus images [23][24][25][26][27][28][29][30][31][32][33][34]. But proved by experiments on imaging the standard random phase mask, PhaseRMiC provided the phase distribution very close to the actual one, therefore PhaseRMiC can still be treated as a quasi-quantitative phase imaging technique. (2) Both image sensors are fixed in PhaseRMiC, thus the defocus interval cannot be changed. A manual displacement module can be useful since different displacement is adaptive to various conditions in different imaging SNRs. While the addition of displacement module inevitably complicates the PhaseRMiC device, and the complicated focal calibration should be implemented again if the CMOS sensor positions are adjusted. Moreover, since PhaseRMiC is often used combining with commercial microscopes, and the imaging SNR can be easily guaranteed by adjusting the illumination light intensity. Therefore, even defocus spacing is fixed, satisfied phase can still be reconstructed. But compact precision motorized translation devices can be considered to be integrated in the updated PhaseRMiC to solve such problem. (3) The present version of PhaseRMiC still requires extra computer for phase reconstruction. Therefore, in future works, PhaseRMiC should still be updated to equip with FPGA computation for fast phase imaging and display.

Conclusions
To conclude, we design the PhaseRMiC aiming at promoting TIPM to practical applications. PhaseRMiC could retrieve sample phase with satisfied accuracy, good stability and large FoV proved using standard specimens. More importantly, PhaseRMiC has been successfully used in live cell phase imaging with high frame rate. Considering its compact and cost-effective design as well as real-time phase imaging capability, PhaseRMiC is a preferred practical solution for many phase imaging applications, such as live cell imaging, cytometry, and so on.