What is Quantitative Phase Imaging (QPI)?
Live cells are notoriously difficult to observe under a microscope due to their high water content and lack of natural pigmentation.
Fluorescence imaging provides improved contrast and can be used for quantitative measurements. Contrast agents are inserted into the cell (dyes, nanoparticles) or induced via genetic mutation. The agents attach to specific cell structures and processes (i.e. labeling) and are excited with specialized light to highlight them. The excitation/emission are time limited (photobleaching), can reduce cell viability and stress the cell physiology (photoxicity).
Cell imaging with brightfield (left) and fluorescence (right)
Quantitative Phase Imaging (QPI) refers to a number of techniques that provide contrast by quantifying the changes in the wavefront (phase shift) when light propagates through a transparent specimen. A high-performance group of these techniques have been invented at University of Illinois at Urbana Champaign by Professor Gabriel Popescu’s Quantitative Light Imaging (QLI) laboratory: Spatial Light Interference Microscopy (SLIM), Gradient Light Interference Microscopy (GLIM) and Whitelight Diffraction Phase Microscopy (wDPM).
Live U-2 OS cell culture imaged with Phi Optics CellVista SLIM Pro. Colormap indicates local phase shift (higher = more red) (courtesy Lippincott-Schwartz NIH Lab)
When compared to classical phase imaging methods (phase contrast and differential interference contrast) SLIM, GLIM and wDPM measure quantitatively the phase shifts. They provide excellent contrast, long term, and label free mapping (refractive index, thickness and dry mass) of 2D/3D live cell and tissue cultures.
Microtubules imaging using SLIM and Phase Contrast (with permission from Kandel et al, ACS Nano 2016)
Accute mouse brain tissue imaged with GLIM and DIC (courtesy UIUC Beckman QLI group)
How does Quantitative Phase Imaging work?
The basic principle of QPI relies on two beams of light: a portion of the light travels through the specimen (sample beam) and carries the information, while the second does not (reference beam).
Contrast is generated from local optical pathlength changes (OPL) or phase shift between the beams caused by subtle variations in thickness and refractive index of the sample. QPI methods generate a quantitative map of local phase shifts in the sample.
Left: QPI measures the OPL difference (i.e. phase shift) Φ through a sample of height h and refractive index n, floating in a medium of refractive index n0. Right: Live HeLa cells imaged with SLIM (courtesy Northwestern Biological Imaging Facility). Colormap indicates local phase shift changes (in radians).
SLIM, GLIM and wDPM combine phase imaging with low-coherence interferometry and holography in a common-path geometry. Phase imaging microscopes (phase contrast, DIC, diffraction phase) employ regular white light (low-coherence) and provide the two illumination beams. The beams pass through the same optical elements from sample plane to the camera plane. The beams superposition is measured in every pixel (common-path interferometry) and provides high signal to noise ratio (nanometer phase sensitivity).
The direct quantitative phase map of the specimen (holography) is then recovered: pixel intensity in a QPI image is a local measurement of the phase shift (in radians). This phase map can be converted to local thickness, refractive index and dry mass maps of the specimen.
Specimen topography: SLIM height profile of carbon film (with permission from W. Zhang et al, Optics Express 2011)
Specimen refractometry: 3D refractive index profile of HT-29 live cell (with permission from T. Kim, R. Zhou et al, PNAS 2014)
Specimen dry mass: E-coli growth measured with SLIM (with permission from M. Mir et al, PNAS 2011)
The use of white light illumination provides a few advantages to SLIM, GLIM and wDPM techniques that makes them a perfect companion for fluorescence imaging.
It avoids the speckles that plague laser-based illumination QPI techniques and provides a low noise floor therefore a flat measurement background. Combined with common-path interferometry SLIM, GLIM and wDPM impart nanometer phase sensitivity for minute features: lipid nanoparticles, microtubules, viral capsids aggregates, mycoplasma contamination.
Phase measurement noise level in a background image (i.e. no sample) for SLIM vs. laser-based QPI. Color bars in nanometers (modified with permission from Z. Wang et al, Opt. Exp., (2011))
Coherence gating due to the white light illumination source improves the optical sectioning afforded by high NA objectives for accurate 3D tomography.
Through focus SLIM Z-stack of HT-29 live cell. Color bar is phase in radians (with permission from T. Kim, R. Zhou et al, PNAS 2014)
The low-power density (compared to laser sources) of the white light illumination allows continuous, long term imaging (from seconds to days) without photobleaching and phototxicity. SLIM, GLIM and wDPM can measure various assays (proliferation, cytotoxicity, immune cell killing, and so on) while co-localized fluorescence is used sparsely for dynamic segmentation of regions of interest (ROI).
Colocalized quantitative phase and fluorescence channels
Phi Optics QPI modules: SLIM, GLIM and wDPM
Phi Optics developed SLIM, GLIM and wDPM to seamlessly upgrade new and existing commercial (Zeiss, Nikon, Leica, Olympus) microscopes with any magnification available (immersion or dry, 5X to 100X). Samples can be loaded in standard holders (glass slides, single and multi-well plates) and fields of view are limited only by the microscope stage movement.
Phi Optics modules can measure samples in various holders
Phi Optics modules use the same camera for all channels of imaging from the microscope therefore insuring seamless overlays. High end sCMOS and EMCCD cameras can be integrated for high sensitivity fluorescence co-localization. Any fluorescence channels present on the microscope can be used for imaging.
Phi Optics CellVista Pro for 4D, multi-ROI and multichannel imaging
Phi Optics CellVista acquisition software platform can control the microscope frame motorization for programmed 4D (3D and time lapse) multichannel (QPI and fluorescence) monitoring of live and fixed cultures.
Spatial Light Interference Microscopy
CellVista SLIM Pro module attached to a Leica DMi8 microscope (Microscope sold separately)
The SLIM module is connected to the imaging port of any commercial phase contrast microscope. The microscope separates geometrically the two beams by collecting the reference beam (unscattered light) through the annular phase plate of the objective, while the sample beam (scattered light) is collected through the rest of the objective aperture.
The beams continue 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.
Phi Optics SLIM is an optimal tool for for 2D cell- and tissue-based assays that can rapidly assess the inhomogeneous characteristics on a cell-by-cell basis across large populations.
Gradient Light Interference Microscopy (GLIM)
CellVista GLIM Pro module attached to a Nikon Ti2 microscope (Microscope sold separately)
The GLIM module is connected to the imaging port of any Diffraction Interference Contrast (DIC) microscope. The microscope separates the illumination into a sample and a reference beam with a polarization shear between them.
The beams continue 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.
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, i.e. the same background noise, such that they always interfere with high contrast.
Whitelight Diffraction Phase Microscopy (wDPM)
CellVista wDPM Pro module attached to a Zeiss Axio Observer microscope (Microscope sold separately)
The wDPM module is connected to the imaging port of any regular brightfield microscope, with a camera connected at its output.
A diffraction grating is placed in the output image plane of the microscope and separates the imaging field into many copies of itself that leave the grating at various angles.
A pinhole filter blocks all diffraction orders except for the 0th and 1st orders. The 1st order (reference beam) is filtered by the pinhole such that the field approaches a plane wave at the camera plane. The 0th order (sample beam) remains unfiltered and travels along the optical axis of the wDPM module, minimizing the aberrations present in the final image.
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. The OPL map is converted to specimen height/volume, dry mass, and refractive index.
This quasi-common-path configuration makes the approach single shot. This means 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)
Spatial light interference microscopy (2011)
Gradient light interference microscopy (2017)
Whitelight diffraction phase microscopy (2014)
Quantitative Microscopy/Drug Discovery: BioOptics World (2018)
Quantitative phase imaging in biomedicine, Nature Photonics 12,(2018)
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.
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