Quantitative Detection Technology Based On Phase Real-time Microscopic Imaging

Since the development of microscopy imaging technology,people have explored a fascinating microcosm of the microscopic world.However,with the advancement of technology and the progress of society,there is a growing demand for a quantitative,non-contact method to obtain three-dimensional information from samples.By comparing contemporary microscopy imaging techniques,it has been found that methods based on biological fluorescence can achieve quantitative measurements but can damage or kill sample cells,making non-contact measurements impossible.On the other hand,although digital holography technology can meet current demands,the requirement for coherent light and complex equipment significantly increases manufacturing costs.Therefore,the method based on the optical intensity transmission equation has been chosen to recover the phase and achieve quantitative,non-contact sample measurements.However,there are currently few phase microscopes available on the market.These devices are expensive,costing tens of thousands of yuan,and have a large footprint(approximately 50 cm * 50 cm * 60cm).They still require manual focusing and are mostly used in cell imaging applications.This article addresses the existing issues with phase microscopy imaging technology by proposing the design of a real-time quantitative phase microscopy imaging system named Phase Microimaging Box(referred to as P.M.Box).From the overall system design,optical path construction,component selection,to the autonomous design of the mechanical structure,the development cost is ensured to be within 5,000 yuan,significantly reducing the size of the imaging system.The final overall dimensions are controlled to be within 25cm10cm20 cm.To achieve better human-machine interaction,a complementary interactive software is developed based on the Py Charm platform using pyqt5 technology,enabling enhanced system control.Based on the advantage of phase recovery based on solving the optical intensity transmission equation,this article aims to further accelerate the imaging speed of the system by using a stereo camera to simultaneously capture two defocused images required for phase recovery.An autofocus scheme suitable for P.M.Box is designed,and an image sharpness evaluation function is employed to quickly locate the focus position.Through testing,the autofocus stability error is within 0.05 mm.Subsequently,the same defocus distance(0.5mm in this article)is set for phase recovery.During the practical implementation,it was observed that external factors such as camera positioning and system errors could lead to inconsistent field of view in the captured images,significantly reducing the accuracy of the final phase recovery.Therefore,this study investigates and implements two field-of-view correction algorithms: feature-based field-of-view correction and transform-domain field-of-view correction.However,experimental results revealed that the feature-based method exhibited mismatches when dealing with samples that have few distinctive features,such as transparent or semi-transparent samples.Consequently,the transform-domain field-of-view correction method,which achieves sub-pixel level accuracy,was chosen.It should be noted that this algorithm involves iterations and requires significant computational resources.However,it only needs to be performed once after the system is installed.Ultimately,P.M.Box was used to perform phase recovery on cells and determine the height distribution of the samples,thus validating the feasibility of phase recovery using P.M.Box.To ensure the performance of the system,tests were conducted on the system’s resolution and accuracy of phase recovery.Using the USAF-1951 resolution target at different objective magnifications,it was observed that the system achieved a resolution of 2.19μm at 10 x magnification,meeting the requirements of the system.Testing with a random phase plate revealed that the accuracy of phase recovery also met the standard.To test the application scenarios of P.M.Box,observations were made on the phase distribution of A549 lung cancer cells during enzymatic processes and quantitative measurements of glass scratches.The results demonstrated that the system is capable of real-time observation of live cells and is also significant in detecting transparent or semi-transparent surface defects.Overall,the system proved to be suitable for real-time observation of live cells and plays an important role in detecting defects on transparent or semi-transparent surfaces.