Gradient Light Interference Microscopy (GLIM) METHODS FOR MEASURING DRY MASS CHANGE IN TIME-LAPSE GRADIENT LIGHT INTERFERENCE MICROSCOPY Brittani L. Carroll1,2; Mikhail E. Kandel2,3; Ghazal Naseri Kouzehgarani3,4; Martha U. Gillette3,4,5 Gabriel Popescu2,3 1University of Evansville, 2Dept. of Electrical and Computer Engineering, 3Beckman Institute for Advanced Science and Technology, 4Neuroscience Program, and 5Cell and Developmental Biology, University of Illinois at Urbana-Champaign, 61801 Motivation Gradient Light Interference Microscopy (GLIM) Phase Object DIC Phase Shifting Gradient Light Interference Microscopy (GLIM) is a new quantitative phase imaging (QPI) method that can be utilized for thick sample up to hundreds of microns deep. Although QPI instruments are unlike traditional microscope due to the fact that they give you a phase value, GLIM varies even more by joining differential interference contrast (DIC) with a low-coherence interferometry and holography. DIC microscopy, which GLIM is an extension of, is a quantitative label-free technique that has been utilized enormously throughout the years to study cells without the need for any contrast substance. The way that GLIM is set up causes the gradient of the phase to be measured, meaning the image needs to be reconstructed and the data needs to be integrated for quantitative analysis. The overall goal of this project is to test different systems, techniques, and algorithms, as well as write another algorithm to integrate the image using line integration, and apply the techniques to acute brain slices to see if the dry mass of the cells change over time to prove that GLIM is a successful microscopy method. Lamp 0π 0.5π P1 φ NP1 Sample π 1.5π NP2 Phase (Radians) High Visibility Optical setup of GLIM Intensity (eV) Variable Retarder Low Visibility P2 Camera GLIM is an add on module to DIC microscopy. The polarizer is removed which means it is no longer able to interfere and a spatial light modulator is used to introduce four modulations with different deflection patterns, or shifts, which are combined to obtain a quantitative mass of phase gradient across a sample with high visibility. Summary & Future Directions Summary GLIM is a new microscopy method useful when imaging thick samples. Images produced by GLIM are a gradient of the phase and need to be reconstructed. There are many useful techniques, including line integration and the Hilbert transform, that can be used to reconstruct or integrate the image. Future Direction Utilize GLIM to observe and image optically thick samples such as nano pillars, cells, and embryos Analyze and create more methods of integration on the brain slices to discover the most efficient way to reproduce the sample image Compare two different phase shifting DIC systems using GLIM, transmitting light verse backscattering techniques Image Reconstruction Time-lapse Imaging on Brain Slices Line Integration FOV3 Time-Lapse DIC FT FILTER FINAL t0 t5 t10 t15 t20 X 44µm LINE INTEGRATION 𝐼𝑀𝐺 𝑥,𝑦 = 𝑖=1 𝑥 𝐼𝑀𝐺(𝑖,𝑦) t25 t30 t35 t40 t44 Before beginning line integration, the image must have the background cleared and be rotated so the Fourier transform is upright. BANDPASS *Scale bar applies to all images The time between each image is 43 minutes with a total of 44 images per sample. Hilbert Transform GLIM FT IM(FINAL) X Ø Acknowledgements I would like to acknowledge and thank NSF for funding the Discoveries in Bioimaging REU, as well as Professor Marina Marjanovic and Joanne Li for running the Discoveries in Bioimaging REU program and providing constant support and advice. I would also like to thank UIUC Graduate College Summer Research Opportunities Program (SROP). Lastly, I would like to Professor Gabriel Popescu and Mikhail Kandel for allowing me to work in their lab with them this summer and for helping me with my project. The most important part to the Hilbert transform is finding the angle which intensifies the cells the most. Mouse brain slice