1. 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