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Live Cell Imaging


Figure 5: Example of NIH-3T3 cell research showing (left) a quantitative phase image, (center) a 3D fluorescence (GFP-Mito and mCherry-Golgi) image, and (right) an overlay image. Images were obtained using a Tomocube HT-2 HT plus fluorescence microscope.


Discussion Without damaging cells or interfering with normal molec-


ular activities, HT has begun to be successfully applied to sin- gle-cell analysis, cytology, haematology, cytopathology, and drug and toxicity reactions. Tis method is particularly useful when combined with fluorescence microscopy to provide the specificity of the organelles and molecules under study. CAR-T cells. Te analysis of CAR-T cells and the study


of the immunological synapse is an example of the power of this method. Although fluorescence-based techniques have proven useful in imaging the junction between immune cells


and their targets, the problems of photo-bleaching, photo- toxicity, and slow imaging limit their ability to address sin- gle-cell dynamic imaging successfully. So the Tomocube and KAIST teams are now working with the HT-2 microscope’s correlative fluorescence imaging to improve the precision and accuracy of the technique and provide the chemical speci- ficities necessary to elucidate the remaining questions about IS formation mechanisms. Te method could also be modi- fied to study other areas in immunological research, ranging from T-cell receptor (TCR) signaling to cytotoxicity of innate immune cells.


Future outlook. HT and other QPI tech-


niques have much to offer researchers seek- ing to understand the complex interactions within living cells. Together with the molec- ular specificity of fluorescence as seen in the Tomocube’s HT-2 microscope, the speed and quantitative approach of QPI are beginning to have an impact in correlative imaging. We now are witnessing the harnessing of raw computing power in the creation of


large-


format stitched images and the introduction of artificial intelligence and machine learning. Together, these approaches can play an impor- tant role in enhancing our understanding of the physiology and pathology of cells and tis- sues. Perhaps they will also make a critical contribution to the diagnosis and treatment of disease.


Conclusion QPI and HT microscopy, enabled by


Figure 6: Label-free immunological synapse (IS) reconstruction and quantification of the IS forma- tion kinetics of CAR-T cells: (top) A reconstructed RI map is used as the input for the deep learning model, which segments CART19 and K562-CD19 cells and defines the immunological synapse. The color maps are based on the 2D ranges of RI and the RI gradient. (bottom) 2D projections of the 3D protein density distributions of the two cells. The immunological synapse was extracted at the cell-cell interface. The color scale provides the maximum projection of 3D protein density distributions (pg/fL) and the maximum projection of surface protein of the IS (pg/μm2


). 22


today’s sophisticated laser-illuminated imaging systems and computing power, are breaking new ground in LCI using the same fundamental property of light discovered in 1934. In the same way that Zernike’s phase- contrast microscope detected variations in light passing through transparent specimens,


www.microscopy-today.com • 2020 January


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