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), Gradient Light Interference Microscopy (GLIM) and Whitelight Diffraction Phase Microscopy (wDPM). SLIM, GLIM and wDPM 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, GLIM and wDPM 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, GLIM and wDPM 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, GLIM and wDPM were invented at University of Illinois at Urbana Champaign by the Professor Gabriel Popescu QLI group. Phi Optics is commercializing SLIM, GLIM and wDPM as add-on modules for commercial phase contrast, DIC and brightfield 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 attached to a Nikon inverted phase contrast 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 Pro module attached to a Leica inverted DIC microscope. (Microscope sold separately.)

The GLIM module is connected to the imaging port of a Diffraction Interference Contrast (DIC) microscope, with a camera connected at its output. The microscope separates the illumination into a sample and a reference beam with a polarization 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) such that they always interfere with high contrast.

WHITELIGHT DIFFRACTION PHASE MICROSCOPY (wDPM)

The wDPM module is connected to the imaging port of a regular brightfield microscope, with a camera connected at its output. A diffraction grating (G)  placed in the output image plane of the microscope separates the imaging field into many copies of itself, which diffract from the grating at various angles. A pinhole filter blocks all diffraction orders except for the 0th and 1st orders. The zero order (sample beam) remains unfiltered and travels directly down the optical axis of the wDPM module, minimizing the aberrations present in the final image. The first order (reference beam) is filtered by the pinhole such that the field approaches a plane wave at the camera plane. The two beams interfere at camera plane and the resulting interferogram is Fourier-transformed to output the phase image of the object observed under the microscope.

This quasi-common-path configuration makes the approach single shot, meaning that the wDPM acquisition speed is limited only by the speed
of the camera employed, while still benefiting from the noise cancellation properties of common-path interferometric systems.

References:

The power of imaging with phase (2017): http://light.ece.illinois.edu/index.html/archives/3182

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

Gradient light interference microscopy (2017) http://light.ece.illinois.edu/index.html/archives/3247

Whitelight diffraction phase microscopy (2014): http://light.ece.illinois.edu/index.html/archives/2414

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), GLIM (US Patent 10,132,609), wDPM (US 8,837,045 B2 and 9,404,857)

Selected Publications

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

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