Optical Volumetric Focusing Microscopy Through Extending The Depth Of Field

High-throughput volumetric imaging technology is very important in various fields such as biology,medicine and engineering.How to acquiring 3D data quickly and sensitively is one of the main challenges in the field of modern biological microscopy.Limited by the narrow depth of field of the objective lens,most current microscopic imaging systems,such as wide-field,laser scanning confocal and light sheet fluorescence microscopes,can only image a very thin layer clearly in the z-direction and record information of only one focal plane at a time,which is the so-called plane focusing imaging.In neuroscience,the main goal is to achieve real-time high-resolution imaging of the whole-brain neurons of model organisms in freely moving behavior.Traditionally,the whole brain imaging usually requires scanning the neuron populations distributed in three dimensions layer by layer.However,the occurrence time of neural activity is generally in the order of millisecond or sub-millisecond,so the current scanning-based schemes are difficult to meet the requirements of synchronous observation of neural activity.The plane focusing imaging of traditional microscopes not only limits the neuroscience research which requires fast and high-resolution imaging,but also poses challenges to point of care testing systems.The narrow depth of focus of the imaging system necessitates precise mechanical or fluidic focus control of a sample to acquire high-quality images that can be used for downstream analysis,increasing the cost and complexity of the imaging system.This complexity represents a barrier to miniaturization and translation of point of care assays based on microscopic imaging or imaging flow cytometry.In terms of the limitation of the plane focusing imaging of traditional imaging systems,a non-paraxial approximation formula used for extending the depth of field of objective lenses with full numerical apertures is derived based on theoretical analysis.By modifying the point spread function of the system,the three-dimensional information of the sample can be obtained simultaneously without any scanning.Compared with the existing method,the original image quality can be better retained,and no subsequent image reconstruction is required.Thus,we solve the above problems well by using the developed volumetric focusing imaging method.Then,we further explore the application of this method in the research of neural activity for model organisms and point of care testing systems,providing a simple and effective solution for application scenarios requiring real-time,high-quality imaging.The main research contents and achievements of this paper are as follows:1.Based on the proposed non-paraxial approximation of defocus function in microscopy systems,a new non-paraxial approximation formula for extending the depth of focus of objective lenses with full numerical apertures is derived in this paper.Theoretical studies show that the new formula is suitable not only to objective lenses with large numerical apertures but also to objective lenses with small numerical apertures,and can completely replace the previous formula.We then constructed a volumetric focusing microscopic imaging system based on a spatial light modulator,and tested the imaging performance of the system using the proposed new formula on different samples such as multi-layered beads sample,thick brain tissue slices of transgenic mouse,and pan-neuronal nucleus-labelled Caenorhabditis elegans.Those results show that the non-paraxial approximation formula can extend the depth of field of the conventional microscopy by about tenfold while maintaining the quality of the original image which can be used directly.Compared with the paraxial design,the new design leads to brighter PSFs and thus images with higher contrast.2.In this paper,a highly transparent aspheric phase plate is designed and fabricated to replace the spatial light modulator since our design is circularly symmetric and easy to be fabricated,which further reduces the cost of system and improves the efficiency of photon utilization.Based on this phase plate,a newly home-built imaging system was constructed and verified on different samples such as fluorescent microspheres and Caenorhabditis elegans.The imaging results show that the phase plate can effectively improve the light efficiency,reduce the exposure time and greatly improve the imaging speed while extending the depth of field of the imaging system like the spatial light modulator,which lays a solid foundation for the subsequent research.3.By applying the processed phase mask to neuroscience research and combining with the self-developed dark-field tracking module,the multimodal volumetric focusing imaging system was established which can simultaneously record information of the neural activity and behavior of freely-behaving Caenorhabditis elegans.Through experiments of calcium imaging on the interneurons of the Caenorhabditis elegans under free behavior,the obtained data of behavior and calcium fluorescent signal were coupled to link neural activity with the behavior of Caenorhabditis elegans in real time,which confirmed the practical performance of this system in solving relevant problems of neuroscience,and provided a powerful observation method for studying the functional connection of neurons in the whole brain of Caenorhabditis elegans.4.To solve the problem of low throughput and high complexity in conventional imaging system due to complex mechanical or fluidic focus control required by traditional microscopic imaging and imaging flow cytometry,this paper developed a portable and low-cost volumetric focusing imaging system suitable for point of care assays by using a simple drop-in phase mask,extending the depth of field of the system by more than fivefold while largely preserving the image quality compared to other depth extending methods.First,the focus-extending system overcomes manufacturing tolerances in low-cost sample chambers,thus making the system easier to be built and operated.Secondly,the system can effectively overcome the field curvature and astigmatic aberration commonly occurs in low-cost objective lens,and enlarge the useable field of view of low-cost objectives.Then,the concept of extending the depth of field is introduced into the traditional bright field imaging system,and experiments show that the portable volumetric focusing imaging system can extend the depth of field of the traditional bright field imaging system by about 5-fold.In addition,experimental results proved that the system can simultaneously image the freely flowing microspheres and cells randomly distributed in the microfluidic tube with a height of 200 microns without flow focusing,which greatly improves the throughput,precision and practicability of the imaging flow systems,and reduces the cost and complexity of the system at the same time.Finally,blood cell samples mixed with cancer cells(HeLa cells)were studied using this focus-extending system,which proved that the system can simultaneously image all cells in the whole three-dimensional chamber with high quality,thus enabling simple and rapid classification of different cells based on morphology.As the image quality is preserved without the need of post processing,our solution is also highly appropriate for on-line applications such as cell sorting.