Single-cell Micro-spectrum And Mass Spectrometry Automatic Sampling And Detection System Based On Microscopic Fluorescence Guidance

Mass spectrometry is widely adopted to measure single-cell proteome,lipidome and metabolome.However,full-scan mass spectrometry imaging is inefficient for detecting sparse single-cell samples.Some studies have suggested that the introduction of optical microscopy into mass spectrometry detection can increase the detection efficiency of single cells,by performing a mass spectrometry detection method oriented to the location of cells in the optical image.However,this method is still limited to brightfield imaging.In order to combined with fluorescence imaging with wider application requirements,and supplemented by micro-spectrum detection of electromagnetic radiation of cells,the goal of this paper rests upon automatic,modular and multimodal detection,designs and implements an automatic sampling and detection system of single cell micro-spectrum and mass spectrometry guided by fluorescence microscope.Specific contents are as follows:1)The hardware of the detection system has been designed and developed:the wide-field fluorescence imaging device realized fluorescence imaging in the range of 737×491μm~2through 10x mirror and 395 nm LED illumination;the 3D displacement module enabled scanning in the range of the 2×2 cm~2and displacement with 200 nm accuracy by combining XY displacement,Z displacement and stage.The combination of above two achieved large-scale fluorescence image acquisition and high-precision cell-guided displacement.On that basis,the micro-spectrum device succeeded in spectral detection with a sampling diameter of about 9μm and a wavelength range of 450 nm to 1100 nm through a 395 nm semiconductor laser and4f spatial filtering.The mass spectrometry detection module effectively performed mass spectrometry with a sampling diameter of about 23μm and a long-distance transmission of 20cm by coupling the nanospray desorption electrospray ionization source,the nitrogen-protected accelerated ion device,the air flow assisted ionization device as well as the mass analyzer and detector.The combination of above two continuously detected single-cell micro-spectrum and mass spectrometry.2)The software of the detection system has been designed and developed:software of highly integrated human-computer interaction was written,which realized the underlying hardware automatic control,single-cell automatic detection and multi-modal data storage.In the single-cell automatic detection algorithm,firstly,large-scale fluorescence image scanning and stitching were carried out through template matching;secondly,cell segmentation and localization under stitched images were conducted successfully through transfer learning and key point-based detection network;finally,the shortest path planning and the accurate micro-spectrum and mass spectrometry detection of cells were effectively performed through the self-organizing mapping network and the secondary calibration.3)The comprehensive performance of the detection system was tested:through performing scanning at 5μm intervals on subtype bacterial specimens in the range of 1.25×1.25mm~2,a 250×250×2048 high-dimensional spectral image was acquired.Through single-point mass spectrometry detection of Rhodamine B,the prominent 443 Da peak of Rhodamine B was obtained.The above results unveil that the detection system can detect micro-spectrum and mass spectrometry.In addition,the specific segmentation model was generated by transfer learning,and a total of 230 cells were segmented by performing wide-field fluorescence scanning on a fluorescent single-cell sample in the range of 1.4×1.4 mm~2,and labeling the cell with 100 images.After path planning and automatic detection,the fluorescence morphology,micro-spectrum and mass spectrometry of cells were reserved.The result demonstrates that the detection system can automatically detect single cells.Finally,rhodamine B signal with a sampling diameter of 12μm was acquired through replacing nanospray desorption electrospray ionization source with laser desorption ionization source;the fluorescence imaging of brain organoid slice was realized by replacing ultraviolet illumination with blue light illumination,.The above results suggest that the detection system can be flexibly configured.In conclusion,the micro-spectrum and mass spectrometry automatic sampling and detection system based on microscopic fluorescence guidance has been successfully established,which preliminarily realizes the specific detection of single cell fluorescence morphology,micro-spectrum and mass spectrometry,and fulfils application needs in different scenarios.The follow-up research will be further optimized and modified under this framework.