Abstract
We present our study regarding a compact system design for cell counting and simultaneous 3D imaging, based on digital in-line holographic microscopy configuration. The system is built around the known experimental configuration which includes a pinhole but we also investigate the configuration with a monomode fiber as a light source. Considered samples consist of a very low concentration of cells in flow in a microchannel. The main challenge in our design is to obtain the digital hologram of one cell on a regular video camera sensor in proper resolution conditions, as opposed to the usual configurations where the aim is to visualize a large area. This fact is possible with shorter distances between pinhole and sample and with pinholes with diameters slightly larger than 1micron. These can now be realized by considering the microtechnological processes for microchannel and pinhole fabrication on the same substrate with high refractive index - to increase the numerical aperture of the system The geometrical parameters are established after the numerical analysis of the diffracted field from a single cell and of the entire system numerical aperture values.
Highlights
Starting from Denis Gabor’s principle introduced for holography, many branches have since been developed with different applications: digital in-line holographic microscopy [1][3], digital off-axis holographic microscopy [4, 5], holographic interferometry [6], holographic memories [7], to name but a few
We propose a compact system where a low concentration of red blood cells (RBCs) flows in a microchannel which is illuminated by a quasipoint source
Some other conditions must be taken into account in order to design our compact system for cell visualization and count: Eq (1) in order to avoid multiple cell superposition in flow in the microchannel, its thickness is considered in the range 30-40 μm Eq (2) in order to record on the CCD camera sensor the hologram of a single cell, we must choose a proper value for the cone angle with its top on the pinhole Eq (3) the area occupied by the cell must be less than approximately half from the illuminating area in the sample plane [20]
Summary
Starting from Denis Gabor’s principle introduced for holography, many branches have since been developed with different applications: digital in-line holographic microscopy [1][3], digital off-axis holographic microscopy [4, 5], holographic interferometry [6], holographic memories [7], to name but a few. DIHM configuration offers in the reconstructed function quantitative information along the propagation axis with nanometer accuracy in certain conditions [15]-[18], which completes the transversal dimensions for 3D object images This technique allows high speed image acquisition suitable for studying cells involved in fast processes like flow in microchannels. To simulate the case when we visualize each cell separately, in our system we will decrease the distance between the pinhole and the sample plane and we will consider pinholes greater than 1 micron For these diameters, the numerical aperture (an important parameter for setups in DIHM configuration) decreases. An optical fiber was used in other setups for DIHM configuration, but with the aim to visualize a large portion of the sample volume [20]
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