Technology

What is Quantitative Phase Imaging (QPI)?

Optical path length (OPL) changes are subtle differences in thickness and refractive index in a transparent object. Quantitative Phase Imaging (QPI) refers to a group of techniques that include Spatial Light Interference Microscopy (SLIM) and Gradient Light Interference Microscopy (GLIM). SLIM and GLIM measure the OPL changes introduced by the sample and record it as pixel values in the generated image. Pixel intensity is proportional to the optical path length (OPL) change and enables direct measurement of the morphology, refractive index and dry mass of the object.

SLIM and GLIM combine phase imaging with low-coherence interferometry and holography in a common-path geometry. This provides high signal to noise ratio (nanometer phase sensitivity) and low noise floor (temporal stability) when compared to Phase Contrast (PC), Differential Interference Contrast (DIC) or other QPI methods. SLIM and GLIM use the white light illumination of the microscope which avoids the speckles that plagues laser illumination-based QPI techniques and improves the optical sectioning due to the low-coherence length of the light source. Measurement throughput is high (up to 100 microliters measurement volume per minute) while simultaneously providing submicron resolution at high sample density (up to 10^10 particles/mL).

SLIM and GLIM were invented at University of Illinois at Urbana Champaign by the Professor Gabriel Popescu QLI group. Phi Optics is commercializing SLIM and GLIM as add-on modules for commercial PC and respectively DIC microscope frames in a 4f-optical relay design that provides high-fidelity optical field reconstruction between the object and image planes with near diffraction-limited operation.                              

                                                            

Rat-Hippocampal-Neurons-SLIM CHO-K1 cells - SLIM image
Features      Benefits
Real-time quantitative phase imaging and display Watch the data acquisition scan as its happening
Label-free, low-illumination power capable Non-destructive, excellent for long-term live cell, tissue and assay investigation
Programmed 2D and 3D scanning in a large field of view See large surface areas and capture mass, volume and surface area, tracking the evolution of cells and tissues over time
Seamless overlay with other microscopy channels All images captured on the same camera – great compliment for fluorescence and other channels
Auto segmentation tool Allows for quick and easy sorting

                   

SPATIAL LIGHT INTERFERENCE MICROSCOPY

 

SLIM-Nikon-AndorSLIM Pro module, as shown attached to a Nikon inverted DIC microscope. (Microscope sold separately.)

The SLIM module is connected to the imaging port of a Phase Contrast microscope, with a camera connected at its output. The microscope separates the illumination into a sample and a reference beam that pass through the sample and are collected at the imaging port with a phase shift between them. The beams pass through the SLIM module where an electro-optical system introduces four accurately controlled phase delays between them. The SLIM camera acquires an intensity image for each phase delay. The intensity images are combined by interference and a recombination algorithm outputs the quantitative OPL map of the entire field of view of the microscope objective. The OPL map is converted to specimen height/volume, dry mass, and refractive index.

GRADIENT LIGHT INTERFERENCE MICROSCOPY (GLIM)

 

GLIM-Leica-HamGLIM Basic module, as shown attached to a Leica inverted DIC microscope. (Microscope sold separately.)

GLIM

The GLIM module is connected to the imaging port of a Phase Contrast microscope, with a camera connected at its output. The microscope separates the illumination into a sample and a reference beam with a phase shear between them that pass through the sample and are collected at the imaging port. The beams pass through the GLIM module where an electro-optical system introduces four accurately controlled phase delays between them. The GLIM camera acquires an intensity image for each phase delay. The intensity images are combined by interference and a recombination algorithm outputs the quantitative OPL map of the entire field of view of the microscope objective. The OPL map is converted to specimen height/volume, dry mass, and refractive index.

GLIM rejects much of the multiple scattering contributions present in an optically thick specimen (e.g. embryos and 3D organoids): the two imaging beams are always equal in power, and suffer equal degradation (that is, the same background noise) because of multiple scattering in the sample such that they interfere with high contrast.

CellVista Imaging Acquisition Software

Phi Optics QPI instruments come standard with proprietary CellVista™ imaging software. CellVista is Windows-based and easy to use, but it can handle complex experiments and help optimize your time in the lab. CellVista interfaces directly with the microscope’s software and acquires data from multiple channels simultaneously, with multiple iterations in time. All channels are captured with a single camera, and every pixel is encoded with optical density information. Images are exported as TIFF files for post processing and are suitable machine learning or big data projects. There is also an available SDK for integration to other software platforms.

References:

Spatial light interference microscopy (2011) http://light.ece.illinois.edu/wp-content/uploads/2011/08/2011_OE_SLIM.pdf

Quantitative Phase Imaging (2012) http://light.ece.illinois.edu/wp-content/uploads/2012/08/Progress-in-Optics-2012.pdf

GLIM Paper – Nature Communications (2017) https://www.nature.com/articles/s41467-017-00190-7

Quantitative Microscopy/Drug Discovery: BioOptics World (2018) https://www.bioopticsworld.com/articles/print/volume-11/issue-5/quantitative-microscopy-drug-discovery-adding-on-deep-tissue-subcellular-quantitative-phase-imaging.html

Quantitative phase imaging in biomedicine, Nature Photonics 12,(2018) http://light.ece.illinois.edu/index.html/archives/3563

Patents:

SLIM (US Patents 8,184,298, 8,520,213, 9,052,180, and 9,404,857) and GLIM (US Patent 10,132,609) were invented at University of Illinois at Urbana Champaign.

Selected Publications

See the latest publications from QLI Labs at Beckman Institute at UIUC here: http://light.ece.illinois.edu/index.html/publications/articles