The technique is able to recover phase even in the presence of amplitude modulation, making it significantly more powerful than existing methods of phase microscopy. The most critical components of the fluorescence microscope for quantitative imaging. In quantitative phase microscopy images cells appear as peaks. The hologram results from the interference of the object beam with a reference wave that can be kept separate from. Quantitative reflection phase mesoscopy by remote coherence. Combining a technique called quantitative phase microscopy with a trained neural network avoids the need to use chemical stains to visualize constituents of biological tissue samples. Holography derives from the greek words holos meaning whole or entire, and graphy meaning a process of writing or recording. Quantitative phase microscopy qpm, a technique combining phase imaging and microscopy, enables visualization of the 3d topography in reflective samples, as well as the inner structure or refractive index distribution of transparent and translucent samples. The method, colormultiplexed differential phase contrast cdpc, is a singleshot variant of differential phase contrast dpc, which. Our system consists of a commercial brightfield microscope with one hardware modificationan inexpensive 3d printed condenser insert. Once the image series has been captured, all other processing is performed in the software environment. Quantitative phase microscopy qpm, a technique combining phase imaging and microscopy, enables visualization of the 3d topography in reflective samples, as well as the inner structure or.
Refractive index measurement in viable cells using quantitative phase. Review of quantitative phasedigital holographic microscopy. Quantitative phase imaging of live cells using fast fourier phase microscopy niyom lue, wonshik choi, gabriel popescu, takahiro ikeda, ramachandra r. Especially in cases where conventional preparation techniques, such as fixation, staining, or fluorescent tagging, may affect cellular functions and limit biological insight. Pdf quantitative phasecontrast confocal microscope. Moses 1,2 1 vascular biology program, boston childrens hospital, 2 department of surgery, harvard medical school and boston childrens hospital.
Osa quantitative phase microscopy with asynchronous digital. Quantitative phase microscopy solution for cell imaging, accurate. Quantitative fluorescence the phenomenon of fluorescence has revolutionized research in biology and medicine. Pdf quantitative phase imaging in microscopy colin. This combination can merge the merits of these two different imaging modalities. Contrary to conventional phase contrast images, phase shift images of living cells are suitable to be processed by image analysis software. Quantitative phase contrast microscopy or quantitative phase imaging are the collective names.
The phasics technology introduces a new modality in light microscopy. Quantitative phase imaging with jaminlebedeff microscope. Multimodal quantitative phase imaging with digital. Since the patented invention of dhm, lyncee team has worked in close cooperation with the biologist of the swiss federal institute of technology of lausanne epfl to discover with them the various interpretation of qpm in cell biology. Schematic representation of the phase retardation of light as it passes through a sample. Thus we developed software that can emulate phase contrast and differential interference contrast microscopy images by numerically processing fpm images. The technique very easily combines to fluorescence microscopy to observed labelled molecules in their environment. The phase map generation, based on the set of three brightfield images captured, involved application of a software. Quantitative phase profiles of cells allow determination of cellular structure and biophysical parameters with minimal sample manipulation. Qpccm can obtain the quantitative phase profiles at an even better noise level than dh. Benchmarks new quantitative phase target qpt tm is the first pure phase artifact available for evaluation and calibration of phase microscopy systems. In cytology this method can be used as an easy way to determine the phase difference and therefore the dry mass per area of cells, microorganisms, etc. We compare the proposed single and alternating illumination dpc with the trimode dpc experimentally using a phase resolution target quantitative phase microscopy target qpt tm, benchmark technologies corporation, u. Which is the reason why the holomonitor app suite software enables fully.
The quantitative phase images of specimens are captured in high precision by the processing of filtering and phase compensation. This has led to the development of noninvasive live cell imaging and automated cell culture analysis systems based on quantitative phase contrast microscopy. Quantitative microstructure imaging of cement, using scanning electron microscopy in combination with xray microanalysis and image processing, is a more recent development in compositional analysis of cement, although its roots may be traced back to optical microscopy techniques first used by le chatelier. Singleshot isotropic quantitative phase microscopy based on. Highresolution transportofintensity quantitative phase.
More than 100 years ago, paul ehrlich began to develop specific chemical staining methods and the latest development of lightcontrolled proteins optogenetics will surely not mark the end of this progression. We present a quantitative phase contrast confocal microscope qpccm by combining a linescanning confocal system with digital holography dh. Quantitative phase imaging with jaminlebedeff microscope and. The technique produces quantitative images of the phase profile of the sample without phase unwrapping. Accuracy has been improved more than twofold and this improvement is sufficient to establish the dynamics of cell growth and adherence to simple growth laws. Traditional phase contrast or bright field images of living unstained biological cells, figure 3 right, have proved themselves to be very difficult to analyze with.
Refractive index measurement in viable cells using. Introduction to phase contrast microscopy microscopyu enus. Quantitative phase contrast microscopy or quantitative phase imaging are the collective names for a group of microscopy methods that quantify the phase shift that occurs when light waves pass through a more optically dense object. Study on quantitative phase imaging by dualwavelength. A new approach to phase microscopy, quantitative phase. We present a singleshot isotropic quantitative phase imaging method based on colormultiplexed differential phase contrast.
Quantitative phase microscopy anew tool for investigating. Below each image, the image is also displayed as a 3dimensional image, where the height is determined by the brightness of each pixel. Quantitative phase microscopy is socalled holography microscope which can. By recording the phase retardation of a light wave when transmitted through the observed transparent specimen, quantitative phase qp microscopy has recently emerged as a powerful labelfree technique in the field of cell imaging. We show this concept at work for enhanced resolution imaging of a calibrated test target amplitude object, a blazed phase grating with known. Singleshot isotropic quantitative phase microscopy based. We have introduced the technique of siqpm which combines structured illumination microscopy with quantitative phase microscopy to allow extended resolution imaging of phase and amplitude samples. Quantitative phase contrast confocal microscope changgeng liu, 1 stefano marchesini, 2 and myung k. This combination can merge the merits of these two. Jul 28, 2014 we have experimentally demonstrated a quantitative phase contrast confocal microscope that is fundamentally faster and simpler compared to the pointscanning digital confocal system 16, 17. Quantitative phase microscopy how is quantitative phase. High resolution quantitative phase microscopy phasics. Quantitative phase microscopy and spectroscopy techniques.
In summary, a spectral domain phase microscopy capable of highdynamicrange quantitative phase contrast imaging was developed. It offers the advantage of acquiring quantitative phase information in. Quantitative phase microscopy phase shift and is measured in radians. Recent efforts have focused on tying phase data and associated metrics to cell morphology. Dhm is a phase microscopy technique that, additionally to providing an intensity contrast, provides quantitative phase measurements qpm. Quantitative phase microscopy for labelfree cell imaging. Light waves arein phase beforepassing through the spec. Singleshot quantitative phase microscopy with color. Highresolution quantitative phasecontrast microscopy by. Quantitative phase imaging of biological cells and tissues using singleshot white light interference microscopy and phase subtraction method for extended range of measurement paper 971851 authors. Using a deep neural network, we demonstrate a digital staining technique, which we term phasestain, to transform the quantitative phase images qpi. Department of physiology, university of melbourne, parkville, victoria, australia. Osa quantitative phasecontrast confocal microscope.
We present a quantitative phasecontrast confocal microscope qpccm by combining a linescanning confocal system with digital holography dh. Quantitative phase imaging phi phase holographic imaging. A dedicated software package computes the camera image in real time to deliver the. Pdf quantitative phase imaging of live cells using fast fourier. It provides biologists with a tool to study static and dynamic physical properties of live cells quantitatively, without exogenous contrast agents. The solution consists in a cameralike instrument, compatible with all standard microscopes and with existing modalities such as fluorescence, and a comprehensive software suite tailored to many applications. Computationally enhanced quantitative phase microscopy. Translucent objects, like a living human cell, absorb and scatter small amounts of light. Quantitative phase microscopy qpm with digital holographic microscope by.
Once the image series has been captured, all other processing is performed in. Motivation quantitative phase imaging is a new optical approach that promises to revolutionize microscopy studies in cell biology. Quantitative phase imaging 7 component of k, say k. Quantitative phase imaging of live cells using fast. Our setup was built based on an inverted microscope ix83, olympus, in which a high contrast amorphous silicon asi thin. Osa resolution enhancement in quantitative phase microscopy. Kim 1 1 digital holography and microscopy laboratory, department of physics, university of. The quantitative phase microscopy qpm based on differential interference contrast dic offers significant advantages in comparison to other currently available qpm techniques. Apr 10, 2012 classical phase contrast microscopy techniques allow only non quantitative partial visualization of transparent samples. Colorcoded led microscopy for quantitative phase imaging. We demonstrate a new method of measuring quantitative phase in imaging of biological materials. In recent years, a variety of techniques have appeared for complete phase determination with different levels of complexity. Phase analysis of portland cement by combined quantitative x. Since the introduction of the digital camera in the mid1990s, several new digital phase imaging methods have been developed, collectively known as quantitative phase contrast microscopy.
The large number of measurements needed is a disadvantage of these methods. The technique uses 1 camera acquisition for 1 phase image. Quantitative phase imaging of cells and tissues qli. A timelapse, labelfree, quantitative phase imaging study of dormant and active human cancer cells jing huang 1,2, peng guo 1,2, marsha a. Quantitative phase microscopy provides a labelfree mechanism to. Quantitative phase imaging in microscopy springerlink. Moreover, our software uses the intensity and phase. Implementation and application to sperm motility analysis wonchan lee, junho choi, suho ryu, daeseong jung. Noninterferometric singleshot quantitative phase microscopy. Jaminlebedeff polarizing interference microscopy is a classical method for determining the refractive index and thickness of transparent tissues. Highresolution quantitative phase contrast microscopy by digital holography christopher j. A dualwavelength digital holographic microscopy with premagnification is proposed to obtain the object surface measurements over the large gradient. Lyncee tec is particularly proud to be the pioneer company developing the use of quantitative phase microscopy qpm in the field of cellular biology. Nov 23, 2009 mehta, s, sheppard, cjr 2008 asymmetric illumination based differential phase contrast aidpc for fullfield transmission imaging of phase information in biological specimens, focus on microscopy fom08, awaji island, japan, 16 april, p.
The method can be used to obtain quantitative phase images of dynamic biological samples on. Quantitative phase microscopy 2012 wiley analytical. Digital holographic microscopy thus makes it possible to visualize and quantify transparent objects and is therefore also referred to as quantitative phase contrast microscopy. Mar 30, 2005 brightfield images were subsequently processed to generate phase maps using qpm 2.
Single cell labelfree imaging with quantitative phase. Quantitative phase imaging for microscopy by phasics life. The hologram is recorded by an image sensor and computer processed to produce the phase image. A new approach to phase microscopy, quantitative phase amplitude microscopy qpam has recently been proposed that relies on observing the manner in which intensity images change with small. Quantitative phase microscopy target benchmark technologies. The phase images are acquired without phase unwrapping, which is necessary in traditional digital holographic. Determine the area s c that is covered by the cells in the quantitative dhm phase images by image segmentation, which can be performed using the free software cell profiler. Quantitative phase imaging qpi or quantitative phase contrast microscopy provides both quantitative and beautiful images of living cells, transforming phase microscopy into a quantitative tool for detailed cell analysis. The technique is labelfree so it enables noninvasive timelapse microscopy. Resolution enhancement in quantitative phase microscopy.
Structured illumination quantitative phase microscopy for. Using a deep neural network, we demonstrate a digital staining technique, which we term phasestain, to transform the quantitative phase images qpi of labelfree tissue sections into images that. Many different type cells and protocols have been measured and experimented. The software offers a simple and guided user interface, with selfexplanatory work flow, from setup of the experiment to result analysis. Quantitative phase imaging quantitative light imaging. Jul 28, 2015 conventional lowmagnification phase contrast microscopy is an invaluable, yet a qualitative, imaging tool for the interrogation of transparent objects over a mesoscopic millimeterscale fieldof. Phase contrast microscopy, first described in 1934 by dutch physicist frits zernike, is a contrastenhancing optical technique that can be utilized to produce highcontrast images of transparent specimens such as living cells, microorganisms, thin tissue slices, lithographic patterns, and subcellular particles such as nuclei and other organelles. The sid4bio software is designed to assist image acquisition and analysis. The software captures conventional bright field images and produces independent intensity and phase information. We present a new technique for quantitative phase and amplitude microscopy from a single color image with coded illumination. The laser biomedical research center lbrc has been one of the leaders in interferometric microscopy, including widefield quantitative phase microscopy qpm, tomographic phase microscopy tpm, and depthresolved reflection phase microscopy with applications in labelfree quantification of cellular morphology including height, footprint, surface area, and volume, cell biomechanics, cell.
Quantitative phase imaging is an emerging technique used in live cell microscopy whereby the phase of transparent materials is captured and then computationally rendered into useable images. The phase stability of the system was measured to be 0. The image covering a sample area of 150m m was acquired in 0. Hemisphere loop, suite 146, tucson, arizona 85706, usa. A product for quantitative phase imaging iatia has developed a selfcontained product 9, to implement quantitative phase imaging for optical microscopy. A timelapse, labelfree, quantitative phase imaging study of. We address these limitations by demonstrating a new computationally enhanced methodology for quantitative phase microscopy ceqpm for adherent cells, using improved image processing algorithms and automated cell tracking software. In our method, the illumination source is modulated by an lcd into an annular colormultiplexed pattern matching the numerical aperture of the objective precisely to maximize the frequency response for both low and high frequencies from 0 to 2 n a o b j. Used in conjunction with dedicated software, it enables easy and efficient. To further illustrate the benefit of quantitative phase imaging, the composite image above shows cell images taken with conventional phase contrast microscopy left and quantitative phase microscopy right. Classical phase contrast microscopy techniques allow only non quantitative partial visualization of transparent samples.
Normalize each series of quantitative phase images to the image with maximum phase contrast. Some applications concern large scientific communities. Methods methods in quantitative phase imaging in life. The holomonitor app suite software is a powerful tool designed to extract quantitative data about your cells from the cell images captured by the holomonitor system. Quantitative phasecontrast microscopy wikimili, the best. It delivers a complete set of parameters based on morphometric and phase information contained in the sid4bio camera image. Traditional phase contrast methods enhance contrast optically, blending brightness and phase information in single image. Using a deep neural network, we demonstrate a digital staining technique, which we term phasestain, to transform the quantitative phase images qpi of labelfree tissue sections into images that are equivalent to the brightfield microscopy images of. Quantitative phase microscopy 2012 wiley analytical science.
A timelapse, labelfree, quantitative phase imaging study. Quantitative phase imaging of live cells using fast fourier. Quantitative phasecontrast microscopy wikimili, the. It works with any microscope and enables measuring valuable numerical parameters on live cells. This method, asynchronous digital holography, employs knowledge of a moving fringe created by acoustooptic modulators to execute phase shifting interferometry using two nearsimultaneous interferograms. Mar 12, 2014 the continued development of hardware and associated image processing techniques for quantitative phase microscopy has allowed superior phase data to be acquired that readily shows dynamic optical volume changes and enables particle tracking.
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