Developing a Reliable Holographic Flow Cyto-Tomography Apparatus by Optimizing the Experimental Layout and Computational Processing

Abstract

Digital Holographic Tomography (DHT) has recently been established as a means of retrieving the 3D refractive index mapping of single cells. To make DHT a viable system, it is necessary to develop a reliable and robust holographic apparatus in order that such technology can be utilized outside of specialized optics laboratories and operated in the in-flow modality. In this paper, we propose a quasi-common-path lateral-shearing holographic optical set-up to be used, for the first time, for DHT in a flow-cytometer modality. The proposed solution is able to withstand environmental vibrations that can severely affect the interference process. Furthermore, we have scaled down the system while ensuring that a full 360° rotation of the cells occurs in the field-of-view, in order to retrieve 3D phase-contrast tomograms of single cells flowing along a microfluidic channel. This was achieved by setting the camera sensor at 45° with respect to the microfluidic direction. Additional optimizations were made to the computational elements to ensure the reliable retrieval of 3D refractive index distributions by demonstrating an effective method of tomographic reconstruction, based on high-order total variation. The results were first demonstrated using realistic 3D numerical phantom cells to assess the performance of the proposed high-order total variation method in comparison with the gold-standard algorithm for tomographic reconstructions: namely, filtered back projection. Then, the proposed DHT system and the processing pipeline were experimentally validated for monocytes and mouse embryonic fibroblast NIH-3T3 cells lines. Moreover, the repeatability of these tomographic measurements was also investigated by recording the same cell multiple times and quantifying the ability to provide reliable and comparable tomographic reconstructions, as confirmed by a correlation coefficient greater than 95%. The reported results represent various steps forward in several key aspects of in-flow DHT, thus paving the way for its use in real-world applications.

Computational methodologies for reliable tomographic phase imaging flow cytometry

“…It is possible to achieve over 40X in processing acceleration. c) displays the isolevel representation of a simulated tomographic cell phantom and the corresponding reconstruction obtained with the conventional FBP and the MHOTV 15 ). The overall tomograms quality is preserved while the FBP artifacts are removed thanks to the regularization ability of MHOTV.…”

“…The most used one is the Filtered Back Projection (FBP) but it can suffers from artifacts mainly due to the limited number and non-uniform orientations. To overcome this issue, we have implemented the MHOTV algorithm 15 that is able to remove the FBP artifacts preserving the resolution of the tomographic data. As mentioned above, the TPI-FC system is able to collect potentially thousands of cells' tomograms per minute, opening a problem about their storage and managing.…”

“…Here we report the selected computational solutions for a reliable tomographic reconstruction in TPI-FC, considering the four main steps of processing, i.e. (i) fast quantitative phase maps (QPMs) recovery from holographic sequences 14 , (ii) 3D pose estimation of flowing cells through holographic tracking, (iii) tomograms reconstruction using the Multiscale High-order Total Variation (MHOTV) algorithm 15 and (iv) tomograms representation via 3D Zernike descriptors 16 .…”

Optimizing the experimental layout and the computational processing for lab-on-chip-based tomographic flow cytometry system

“…The digital holograms are acquired through the implemented setup (Figure 1a) and numerically processed to obtain the QPMs that are used to perform the tomographic reconstruction of the cell. The angular sequence is retrieved resorting to the approach proposed by [15] adapted to our 45° tilted field of view [13] (see Figure 1b). For the considered setup the average number of frames available for a full rotation is 35, with non-uniform angular steps between two consecutive frames.…”

“…In these solutions the two interfering waves share similar optical paths, thus compensating for environmental vibrations, making them more adapt to be incorporated into robust holographic setups employable in principle in common life. Recently, we demonstrated the possibility to implement a quasi-common-path lateral-shearing architecture for DHT in flow cytometry condition [13]. In addition, to increase the available field of view for guaranteeing a full-360 rotation of imaged cells, the camera performs the imaging on a 45° tilted microfluidic channel.…”