Laplace field microscopy for label-free imaging of dynamic biological structures

We present Laplace field microscopy as a method for generating intrinsic contrast of transparent specimens. This technique uses a spatial light modulator to perform the Laplacian of the field in the Fourier plane of a microscope image. The resulting image incorporates phase information and thus renders high contrast images from phase objects. We demonstrate the potential of the method by imaging index-matched beads, unlabeled tissue slices, and dynamic live cells.     

Phase Differential Enhancement of FLIM to Distinguish FRET Components of a Biosensor for Monitoring Molecular Activity of Membrane Type 1 Matrix Metalloproteinase in Live Cells

Fluorescence lifetime-resolved imaging microscopy (FLIM) has been used to monitor the enzymatic activity of a proteolytic enzyme, Membrane Type 1 Matrix Metalloproteinase (MT1-MMP), with a recently developed FRET-based biosensor in vitro and in live HeLa and HT1080 cells. MT1-MMP is a collagenaise that is involved in the destruction of extra-cellular matrix (ECM) proteins, as well as in various cellular functions including migration. The increased expression of MT1-MMP has been positively correlated with the invasive potential of tumor cells. However, the precise spatiotemporal activation patterns of MT1-MMP in live cells are still not well-established. The activity of MT1-MMP was examined with our biosensor in live cells. Imaging of live cells was performed with full-field frequency-domain FLIM. Image analysis was carried out both with polar plots and phase differential enhancement. Phase differential enhancement, which is similar to phase suppression, is shown to facilitate the differentiation between different conformations of the MT1-MMP biosensor in live cells when the lifetime differences are small. FLIM carried out in differential enhancement or phase suppression modes, requires only two acquired phase images, and permits rapid imaging of the activity of MT1-MMP in live cells.     

Optical scatter imaging: subcellular morphometry in situ with Fourier filtering

We demonstrate a quantitative optical scatter imaging (OSI) technique, based on Fourier filtering, for detecting alterations in the size of particles with wavelength-scale dimensions. We generate our scatter image by taking the ratio of images collected at high and low numerical aperture in central dark-field microscopy. Such an image spatially encodes the ratio of wide to narrow angle scatter and hence provides a measure of local particle size. We validated OSI on sphere suspensions and live cells. In live cells, OSI revealed biochemically induced morphological changes that were not apparent in unprocessed differential interference contrast images. Unlike high-resolution imaging methods, OSI can provide size information for particles smaller than the camera's spatial resolution.     

Confocal diffraction phase microscopy of live cells

We present a new quantitative phase microscopy technique, confocal diffraction phase microscopy, which provides quantitative phase measurements from localized sites on a sample with high sensitivity. The technique combines common-path interferometry with confocal microscopy in a transmission geometry. The capability of the technique for static imaging is demonstrated by imaging polystyrene microspheres and live HT29 cells, while dynamic imaging is demonstrated by quantifying the nanometer scale fluctuations of red blood cell membranes.     

Scanning ion conductance microscopy for imaging biological samples in liquid: a comparative study with atomic force microscopy and scanning electron microscopy

The present study was designed to show the applicability of scanning ion conductance microscopy (SICM) for imaging different types of biological samples. For this purpose, we first applied SICM to image collagen fibrils and showed the usefulness of the approach-retract scanning (ARS)/hopping mode for such samples with steep slopes. Comparison of SICM images with those obtained by AFM revealed that the ARS/hopping SICM mode can probe the surface topography of collagen fibrils and chromosomes at nanoscale resolution under liquid conditions. In addition, we successfully imaged cultured HeLa cells, with 15 μm in height by ARS/hopping SICM mode. Because SICM can obtain non-contact (or force-free) images, delicate cellular projections were visualized on the surface of the fixed cell. SICM imaging of live HeLa cells further demonstrated its applicability to study the morphological dynamics associated with biological processes on the time scale of minutes under liquid conditions. We further applied SICM for imaging the luminal surface of the trachea and succeeded in visualizing the surface of both ciliated and non-ciliated cells. These SICM images were comparable with those obtained by scanning electron microscopy. Although the dynamic mode of AFM provides better resolution than the ARS/hopping mode of SICM in some samples, only the latter can obtain contact-free images of samples with steep slopes, rendering it an important tool for observing live cells as well as unfixed or fixed soft samples with complicated shapes. Taken together, we demonstrate that SICM imaging, especially using an ARS/hopping mode, is a useful technique with unique capabilities for imaging the three-dimensional topography of a range of biological samples under physiologically relevant aqueous conditions.     

Deep-learning-based prediction of living cells mitosis via quantitative phase microscopy

We present a deep learning approach for living cells mitosis classification based on label-free quantitative phase imaging with transport of intensity equation methods. In the approach, we applied a pretrained deep convolutional neural network using transfer learning for binary classification of mitosis and non-mitosis. As a validation, we demonstrated the performances of the network trained by phase images and intensity images, respectively. The convolutional neural network trained by phase images achieved an average accuracy of 98.9% on the validation data, which outperforms the average accuracy 89.6% obtained by the network trained by intensity images. We believe that the quantitative phase microscopy in combination with deep learning enables researchers to predict the mitotic status of living cells noninvasively and efficiently.     

Multifocal multiphoton microscopy

We present a real-time, direct-view multiphoton excitation fluorescence microscope that provides three-dimensional imaging at high resolution. Using a rotating microlens disk, we split the near-infrared light of a mode-locked titanium-sapphire laser into an array of beams that are transformed into an array of high-aperture foci at the object. We typically scan at 225 frames per second and image the fluorescence with a camera that reads out the images at video rate. For 1.4 aperture oil and 1.2 water immersion lenses at 780-nm excitation we obtained axial resolutions of 0.84 and 1.4mum , respectively, which are similar to that of a single-beam two-photon microscope. Compared with the latter setup, our system represents a 40-100-fold increase in efficiency, or imaging speed. Moreover, it permits the observation with the eye of high-resolution two-photon images of (live) samples.     

Dual-wavelength linear regression phase unwrapping in three-dimensional microscopic images of cancer cells

We present a study of the three-dimensional structure of cancer cells using dual-wavelength phase-imaging digital holographic microscopy. Phase imaging of objects with optical height variation greater than the wavelength of light is ambiguous and causes phase wrapping. By comparing two phase images recorded at different wavelengths, the images can be accurately unwrapped. The unwrapping method is computationally fast and straightforward, and it can process complex topologies. Additionally, the limitations on the total optical height are significantly relaxed. This new methodology is widely applicable to other phase-imaging techniques as well as in applications beyond optical microscopy.     

MR phase imaging: sensitive and contrast-enhancing visualization in cellular imaging

The successful translation of stem-cell therapies requires a detailed understanding of the fate of transplanted cells. Magnetic resonance imaging (MRI) has provided a noninvasive means of imaging cell dynamics in vivo by prelabeling cell with T(2) shortening iron oxide particles. However, this approach suffers from a gradual loss of sensitivity since active cell mitosis could decrease the cellular contrast agent (CA) concentration below detection level. In addition, the interpretation of images may be confounded by hypointensities induced by factors other than this CA susceptibility effect (CASE). We therefore examined the feasibility of exploiting the phase information in MRI to increase the sensitivity of cellular imaging and to differentiate the CASE from endogenous image hypointensity. Phase aliasing and the B(0) field inhomogeneity effect were removed by applying a reliable unwrapping algorithm and a high-pass filter, respectively, thus delineating phase variations originating from high spatial frequencies due to the CASE. We found that the filtered phase map detects labeled cells with high sensitivity and can readily differentiate the cell migration track from the white matter, both of which are hypointense in T(2)-weighted magnitude images. Furthermore, an approximate fivefold contrast-to-noise ratio enhancement can be achieved with an MRI phase map over conventional T(2)-weighted magnitude images.     

En face imaging of single cell layers by differential phase-contrast optical coherence microscopy

Optical coherence microscopy (OCM) is capable of imaging the backscattering potential of a sample with high transversal and axial resolution. We report on a combination of OCM with a differential phase-contrast technique that permits imaging of the subwavelength optical path differences that occur between a narrow beam probing a sample and its surrounding. This technique allows small transversal refractive-index variations close to a selected interface to be seen. We report on the method and present first images of a test sample and a single cell layer. The cells act as phase objects; imaging the phase properties improves the contrast compared with that of intensity images.     

Field-based angle-resolved light-scattering study of single live cells

We perform field-based angle-resolved light-scattering measurements from single live cells. We use a laser interferometer to acquire phase and amplitude images of cells at the image plane. The angular scattering spectrum is calculated from the Fourier transform of the field transmitted through the cells. A concurrent 3D refractive index distribution of the same cells is measured using tomographic phase microscopy. By measuring transient increases in light scattering by single cells during exposure to acetic acid, we correlate the scattering properties of single cells with their refractive index distributions and show that results are in good agreement with a model based on the Born approximation.     

Diffraction phase microscopy for quantifying cell structure and dynamics

We have developed diffraction phase microscopy as a new technique for quantitative phase imaging of biological structures. The method combines the principles of common path interferometry and single-shot phase imaging and is characterized by subnanometer path-length stability and millisecond-scale acquisition time. The potential of the technique for quantifying nanoscale motions in live cells is demonstrated by experiments on red blood cells.     

Spectral-domain optical coherence phase microscopy for quantitative phase-contrast imaging

We describe a novel microscopy technique for quantitative phase-contrast imaging of a transparent specimen. The technique is based on depth-resolved phase information provided by common path spectral-domain optical coherence tomography and can measure minute phase variations caused by changes in refractive index and thickness inside the specimen. We demonstrate subnanometer level path-length sensitivity and present images obtained on reflection from a known phase object and human epithelial cheek cells.     

Phase-resolved optical coherence tomography and optical Doppler tomography for imaging blood flow in human skin with fast scanning speed and high velocity sensitivity

We have developed a novel phase-resolved optical coherence tomography (OCT) and optical Doppler tomography (ODT) system that uses phase information derived from a Hilbert transformation to image blood flow in human skin with fast scanning speed and high velocity sensitivity. Using the phase change between sequential scans to construct flow-velocity imaging, this technique decouples spatial resolution and velocity sensitivity in flow images and increases imaging speed by more than 2 orders of magnitude without compromising spatial resolution or velocity sensitivity. The minimum flow velocity that can be detected with an axial-line scanning speed of 400 Hz and an average phase change over eight sequential scans is as low as 10 microm/s, while a spatial resolution of 10 microm is maintained. Using this technique, we present what are to our knowledge the first phase-resolved OCT/ODT images of blood flow in human skin.     

Live cell refractometry using microfluidic devices

Using Hilbert phase microscopy for extracting quantitative phase images, we measured the average refractive index associated with live cells in culture. To decouple the contributions to the phase signal from the cell refractive index and thickness, we confined the cells in microchannels. The results are confirmed by comparison with measurements of spherical cells in suspension.     

Continuous wave terahertz phase imaging with three-step phase-shifting

We demonstrated a continuous wave terahertz (THz) phase imaging system which can present the depth information of an object's surface interior accurately and non-invasively. With the three-step phase-shifting method, the phase images of samples which can provide the relative optical depth profile of the sample's surface and interior have been achieved. Two samples with different thickness and materials were measured to demonstrate the phase imaging ability of the system. The results show that this method is effective and the measured values agree well with the actual ones. The longitudinal resolution of the system was also measured. To exhibit the advantages of the three-step phase shifting method, the multi-wavelength imaging method was also employed and the results are compared.     

Two-photon near- and far-field fluorescence microscopy with continuous-wave excitation

We report on scanning far- and near-field two-photon microscopy of cell nuclei stained with DAPI and bisbenzimidazole Hoechst 33342 (BBI-342) with the 647-nm laser line of a cw ArKr mixed-gas laser. Two-photon-excited fluorescence images are obtained for 50-200 mW of average power at the sample. A nearly quadratic dependence of fluorescence intensity on laser power confirmed the two-photon effect. The nonlinearity was further supported by evidence of three-dimensional sectioning in a scanning far-field microscope. We find that the cw two-photon irradiation sufficient for imaging within typically 5 s does not significantly impair cell cycling of BBI-342-labeled live cells. Finally, high-resolution imaging in scanning near-field microscopy with good contrast is demonstrated.     

Three-dimensional cylindrical X-band ESR imaging by a combined pulsed gradient Fourier and static gradient projection reconstruction method

Static gradient electron spin echo projection reconstruction imaging is favourable for X-band material science applications requiring temperature variation with a metal cryostat. To prevent imaging artefacts due to the high conduction electron diffusion coefficient in the preferred conduction direction of quasi-one-dimensional conductors, only pulsed gradient phase encoding for that direction can be tolerated. We present results of an appropriate cylindrical imaging scheme combining both methods. Conduction electron spin density images with 13 x 13 x 17 microm(3) volume element size or spin-lattice relaxation time images with inversion recovery sequence and 13 x 13 x 68 microm(3) volume element size are presented for fluoranthene radical cation salt single crystals of typical sizes of 0.4 x 0.4 x 1 mm(3).     

High-resolution imaging using Hadamard encoding

High-resolution imaging techniques using noninvasive modalities such as magnetic resonance (MR) imaging are being pursued as in vivo cancer screening techniques in an attempt to eliminate the invasive nature of surgical biopsy. When acquiring high-resolution MR images for tissue screening, image fields of view have in the past been limited by the matrix sizes available in conventional MR scanners. We present here a technique that uses aliasing to produce high resolution images with larger matrix sizes than are currently available. The image is allowed to alias in both the frequency encoding and phase encoding dimensions, and the individual, aliased fields of view are recovered by Hadamard encoding methods. These fields may then be tiled to obtain a composite image with high spatial resolution and a large field of view. The technique is demonstrated using two-dimensional and three-dimensional in vivo imaging of the human brain and breast.     

Development of neutron spin phase contrast imaging

We present an imaging technique utilizing a neutron spin interferometer. Neutron spin phase contrast is achieved in spatial resolved measurements of the phase difference between two superposed neutron spin states introduced by passing through a magnetic sample. Since the phase difference of spin states parallel and anti-parallel to the magnetic field is proportional to the magnetic field integral, it is possible to record images of the internal magnetic field distribution of the sample. Taking advantage of high transmission probabilities, neutron spin phase contrast provides non-destructive images of internal magnetic structures.