We present our latest advances in multi-wavelength label-free imaging using quantitative phase imaging. Basic QPI designs usually incorporate a single, coherent or partially coherent light source. The introduction of off-axis geometry allows for calculation of phase information from a single-exposure interferogram. For multi-wavelength imaging, it has been previously required to take sequential interferograms with different wavelengths, effectively reducing imaging throughput, or to use color cameras that have cross-talk between the color channels and artefacts caused by Bayer pattern. Alternatively, it was suggested to use spatial interferometric multiplexing in order to provide multi-wavelength data, where multiple interferograms of different off-axis angles are acquired in a single exposure. We introduce an external module for label-free QPI capable of multi-color imaging using spatial interferometric multiplexing of multiple color channels, with self-referencing that significantly reduces mechanical vibration noise. This technique has various applications such as interferometric spectroscopy of live cells and multiple wavelength phase unwrapping.
We demonstrate photothermal quantitative phase imaging of living cells with functionalized nanoparticles in a cost-efficient setup and quantify the impact of nanoparticle excitation with low modulation frequencies on cell morphology and viability.
We present highly dynamic photothermal interferometric phase microscopy for quantitative, selective contrast imaging of live cells during flow. Gold nanoparticles can be biofunctionalized to bind to specific cells, and stimulated for local temperature increase due to plasmon resonance, causing a rapid change of the optical phase. These phase changes can be recorded by interferometric phase microscopy and analyzed to form an image of the binding sites of the nanoparticles in the cells, gaining molecular specificity. Since the nanoparticle excitation frequency might overlap with the sample dynamics frequencies, photothermal phase imaging was performed on stationary or slowly dynamic samples. Furthermore, the computational analysis of the photothermal signals is time consuming. This makes photothermal imaging unsuitable for applications requiring dynamic imaging or real-time analysis, such as analyzing and sorting cells during fast flow. To overcome these drawbacks, we utilized an external interferometric module and developed new algorithms, based on discrete Fourier transform variants, enabling fast analysis of photothermal signals in highly dynamic live cells. Due to the self-interference module, the cells are imaged with and without excitation in video-rate, effectively increasing signal-to-noise ratio. Our approach holds potential for using photothermal cell imaging and depletion in flow cytometry.
Measurements of biological cells during flow are highly important for medical diagnosis based on cell sorting. In the case of cell imaging during flow, very rapid image acquisition capabilities are required to enable fast cell flow for analyzing a sufficient number of cells. We present a new flipping interferometry (FI) module for simplified off-axis close-to-common-path interferometric phase microscopy. This wide-field off-axis interferometric module provides rapid quantitative phase microscopy of biological cells during flow in a microfluidic channel, with potential of integration into cell sorting devices. Various experimental demonstrations are presented.
The vast majority of quantitative phase imaging techniques face the same major issue of phase unambiguity. The phase of light is 2π-periodic, and as such can only be extracted in a small range of typically 0.4 µm to 0.7µm in the visible range. As most of the samples in biology and metrology are much higher, phase data is wrapped within this small range. One of the more reliant solutions of phase unambiguity is phase unwrapping, where the it is unwrapped based on the surrounding data. Unwrapping, however, works best on continuous phase changes and fails when abrupt phase jumps appear, e.g steps greater than the unambiguous range. An alternative method to overcome the unambiguous phase problem is multiplexing two wavelengths to capture the same phase image. Processing together the two phase images captured by different wavelengths can produce a new image, corresponding to a phase image with a synthetic wavelength that larger than each of the original wavelengths. This effectively enlarges the unambiguous range, making costly unwrapping algorithms redundant.
We present a new dual-wavelength interferometry setup that can capture and multiplex two different phase images in an external module, portable to existing microscopy systems. The module is based on a self-interference multiplexing technique. As such, it is very flexible and can work with either transmission or reflection based microscopes. It can be used for either enlarging the unambiguous range or other dual-wavelength phase imaging applications.
We explored photothermal quantitative phase imaging (PTQPI) of living cells with functionalized nanoparticles (NPs) utilizing a cost-efficient setup based on a cell culture microscope. The excitation light was modulated by a mechanical chopper wheel with low frequencies. Quantitative phase imaging (QPI) was performed with Michelson interferometer-based off-axis digital holographic microscopy and a standard industrial camera. We present results from PTQPI observations on breast cancer cells that were incubated with functionalized gold NPs binding to the epidermal growth factor receptor. Moreover, QPI was used to quantify the impact of the NPs and the low frequency light excitation on cell morphology and viability.
We present our latest advances in highly dynamic photothermal interferometric phase microscopy for quantitative, selective contrast imaging. Gold nanoparticles can be bio-functionalized to bind specific cells. When stimulating gold nanoparticles at their plasmon-peak wavelength, local increase of temperature occurs due to plasmon resonance. This causes a rapid change of optical phase of the light beam interacting with the sample. These phase changes can be recorded by interferometric phase microscopy and analyzed to form a photothermal image of the binding sites of the nanoparticles in the cells. Furthermore, by increasing the excitation laser light, one can deplete certain cells at will. Usually, the analysis of the photothermal signals utilizes a Fourier transform, which is computational time consuming. This makes photothermal imaging not suitable for applications requiring dynamic imaging or real-time quantitative analysis, such as for analyzing and sorting cells during their fast flow. For this goal, we have developed new algorithms, based on discrete Fourier transform variants, enabling fast analysis of photothermal signals from nanoparticles in live and highly dynamic cells. For the first time, video-rate photothermal signals are obtained, which forms the basis for real-time interferometric phase microscopy with molecular specificity. This technique holds great potential for using photothermal imaging in flow cytometry.
We review our dual-modality technique for quantitative imaging and selective depletion of populations of cells based on wide-field photothermal (PT) quantitative phase imaging and simultaneous PT cell extermination. The cells are first labeled by plasmonic gold nanoparticles, which evoke local plasmonic resonance when illuminated by light in a wavelength corresponding to their specific plasmonic resonance peak. This reaction creates changes of temperature, resulting in changes of phase. This phase changes are recorded by a quantitative phase microscope (QPM), producing specific imaging contrast, and enabling bio-labeling in phase microscopy. Using this technique, we have shown discrimination of EGFR over-expressing (EGFR+) cancer cells from EGFR under-expressing (EGFR–) cancer cells. Then, we have increased the excitation power in order to evoke greater temperatures, which caused specific cell death, all under real-time phase acquisition using QPM. Close to 100% of all EGFR+ cells were immediately exterminated when illuminated with the strong excitation beam, while all EGFR– cells survived. For the second experiment, in order to simulate a condition where circulating tumor cells (CTCs) are present in blood, we have mixed the EGFR+ cancer cells with white blood cells (WBCs) from a healthy donor. Here too, we have used QPM to observe and record the phase of the cells as they were excited for selective visualization and then exterminated. The WBCs survival rate was over 95%, while the EGFR+ survival rate was under 5%. The technique may be the basis for real-time detection and controlled treatment of CTCs.
We present a method for adding molecular specificity to wide-field interferometric phase microscopy (IPM) by recording the phase signatures of gold nanoparticles (AuNPs) labeling targets of interest in biological cells. The AuNPs are excited by time-modulated light at a wavelength corresponding to their absorption spectral peak, evoking a photothermal (PT) effect due to their plasmonic resonance. This effect induces a local temperature rise, resulting in local refractive index and phase changes that can be detected optically. Using a wide-field interferometric phase microscope, we acquired an image sequence of the AuNP sample phase profile without requiring lateral scanning, and analyzed the time-dependent profile of the entire field of view using a Fourier analysis, creating a map of the locations of AuNPs in the sample. The system can image a wide-field PT phase signal from a cluster containing down to 16 isolated AuNPs. AuNPs are then conjugated to epidermal growth factor receptor (EGFR) antibodies and inserted to an EGFR-overexpressing cancer cell culture, which is imaged using IPM and verified by confocal microscopy. To the best of our knowledge, this is the first time wide-field interferometric PT imaging is performed at the subcellular level without the need for total internal reflection effects or scanning.
We present a new wide-field quantitative photothermal (PT) imaging method of gold nanoparticles (AuNPs), which is suitable for obtaining wide-field holographic molecular specificity in biological samples. To obtain this goal, we built a wide-field interferometric phase microscope and modified it for the excitation of plasmonic resonance in AuNPs, while recording their resultant phase signatures. To check the potential of the AuNPs as interferometric cellular labels, they were conjugated to a glass coverslip and excited with a laser at a wavelength corresponding to their absorption spectral peak. We then acquired an image sequence of the sample phase profile in time without the need for lateral scanning, and analyzed the entire field of view using a Fourier analysis, creating a map of the locations of the AuNPs. We obtained a strong PT signal at AuNPs central locations, exponentially dependent on the distance from their centers. This enabled identification of the central locations of the AuNPs in the chosen field of view. Moreover, these PT signals had shown a linear relation to the illumination intensity, distinguishing them from background noise and out-of-focus particles. To the best of our knowledge, we are the first to record wide-field interferometric PT signals at the subcellular level without the need of total-internal-reflection prisms or scanning.
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