Microscopy

The phase contrast microscope is a vital instrument in biological and medical research; it facilitates the observation of living cells by using spatial light modulation to enhance the intrinsic contrast of transparent and colorless components in a cell. It can reveal cell structure without the need of contrast agents. However, the information obtained is only qualitative.

Jones Phase Microscopy (JPM) and White Light Quantitative Phase Microscopy (PC++) of transparent and anisotropic samples are novel interferometric techniques that are quantitative and polarization sensitive. They are capable of quantifying the optical phase delays associated with live cell samples while providing information about the morphology and dynamics.

Quantifying these optical path length shifts allows for nanometer scale measurements with broad applications in nanotechnology and life sciences, e.g. from inspecting semiconductor wafers to studying processes in live cells.

Description/Details 

Jones Phase Microscopy (JPM) technique is capable of extracting the spatially-resolved Jones matrix associated with transparent and anisotropic samples. It can provide the quantitative phase image of the cells as well as the intrinsic structure details and dynamics. JPM setup consists of a modified Hilbert Phase microscope; the quantitative phase image is correlated to the sample using a 2D spatial Hilbert transform. The full Jones matrix in each point within the field associated with the transparent sample is extracted and the embedded quantitative information of phase delay is used to reconstruct its image.

The White Light Quantitative Phase Microscope (PC++) interfaces with a commercial phase contrast microscope and transforms it into a quantitative instrument by performing two additional measurements (hence the name PC++). In a conventional phase microscope the additional phase delay is introduced by the phase objective, the PC image is delivered at the image plane and recorded by a CCD. However, in PC++, the optical field at the image plane is further modulated before detection which is equivalent to spatial filtering with three different phase masks and reconstruction of image by addition of fields containing information about the unscattered light (forming the uniform background) and the fine structural details (the contrast). 

Applications

• Optical microscopy and imaging of: live cells and thin tissue slices; microorganisms; lithographic patterns and nanoparticles
• Study of cell dynamics and morphology

Benefits

• These techniques provide improved contrast in unstained biological specimens and can be utilized to examine dynamic events in living cells.
• Quantitative Phase delay information
• Polarization sensitive imaging: while Jones Phase Microscope is a modified Hilbert Phase microscope, the PC++ is an add-on to a commercial phase contrast microscope. 

The Frequency Domain Dynamic Light Scattering (FD-DLS) is a new methodology that facilitates study of dynamics in arbitrarily scattering samples. Dynamic light scattering (DLS) measures the intensity of light scattered by particles in the sample over a broad range of temporal scales. DLS instruments obtain a correlation factor from the intensity versus time profile for situations where light scatters no more than once in the medium. The principle of DLS is extended to Diffusing Wave Spectroscopy (DWS), for highly scattering media, by taking into account the path-length distribution P(s) of the scattered light. However, this requires a priori knowledge of P(s), significantly restricting the applicability of dynamic measurements in highly scattering media. FD-DLS, thus overcomes the limitations of both DLS and DWS.

The Fourier Transform Angular Light Scattering (FTALS) is the spatial equivalent of Fourier Transform Infrared Spectroscopy (FTIR). Light scattering is combined with a typical inverted microscope and all the spatial frequencies are simultaneously detected. It provides ultrasensitive detection of angular scattering as well as fast measurement from live cells and tissues. It combines the high spatial resolution associated with optical microscopy and intrinsic averaging of light scattering techniques. The enhanced sensitivity is granted by the image-plane measurement of the optical phase and amplitude and the spatial resolution of the scatterer positions is well preserved.

Description/Details

The Frequency Domain Dynamic Light Scattering (FD-DLS) technique employs a typical Fourier domain optical coherence tomography system to measure the temporal correlation (between the scattering wave vector and the diffusion coefficient D) as well as the path length distribution of the scattered light, P(s). It addresses the limitations of both the DLS and the DWS since it does not require the investigators to make assumptions about the sample under observation and it can be used in complex media. The underlying principle of Fourier Transform Angular Light Scattering (FT-ALS) is to retrieve the phase and amplitude associated with a coherent microscope image and numerically propagate this field to the scattering plane. Since all the spatial frequencies are simultaneously detected it provides high throughput along with much higher sensitivity to angular scattering than the conventional FTIR. The experimental setup combines light scattering with an inverted microscope. A fast Fourier transform is applied to obtain the angular scattering in the spatial domain. Accurate phase retrieval for elastic light scattering (ELS) measurements and fast acquisition speed for dynamic light scattering (DLS) studies are achieved by diffraction phase microscopy (DPM), which provides the phase shift associated with transparent structures from a single interferogram measurement. The phase measurement is performed in the image plane of a microscope rather than the Fourier plane which offers important advantages in the case of the thin samples.

Applications 

FD-DLS In-vivo blood viscometry, which is critical for patients with heart disease. Quantifying the dynamics of blood perfusion. Potential diagnostic capability in certain types of cancer where cancer tissue experiences increased blood perfusion. FT-ALS It provides ultrasensitive measurements of light scattering from live cells and tissues Cell sorting Stain-free histology

Benefits

FD-DLS technique has broad application to characterizing scattering and dynamic media. It is an absolute measurement, where knowledge of the composition of the suspended particles is not needed. It is especially helpful if the optical properties of the suspended particles are not known or if the suspension is made up of particles with different optical properties. The Frequency Domain Dynamic Light Scattering (FD-DLS) is not limited by the complexity of the scattering medium. No a priori information about the path length distribution function is needed. Based on commercially available Optical Coherence Tomography (OCT) system.

FT-ALS provides significantly higher sensitivity to angular scattering than the traditional goniometer based measurement. Capable of measuring single particles. Measurements on live cells over a broad range of angles High throughput technique with exhaustive information in a single CCD exposure. In the image plane of a thin and transparent sample, such as live cells, the intensity is evenly distributed, which efficiently utilizes the limited dynamic range of the CCD. Ability to detect weak scattering signals over broad temporal (milliseconds to hours) and spatial (fraction of microns to centimeters) scales. 

Professor Dana Dlott and Natalia Garcia Rey have designed a spectra-electrochemical cell that is user and environmental friendly. The material used is chemically resistant for cleaning and electrochemical experiments. Previous designs presented a complicated assembly with many different parts, large volume, and a liquid gap between the window and the electrode that could not be controlled. 

Their findings show how important it is to have control of the gap for the ultrafast spectroscopic analysis and for understanding of the electrochemical reactions occurring on the electrolyte-electrode. This design also allows one to move the electrode sample to refresh the solution and repeat the experiment, setting the sample back to the same position with micrometer precision, which is very important in laser experiments and for comparing between results. The design uses commercial electrodes that can be polished, electrochemically cleaned and assembled in the cell. Previous designs use evaporated metallic thin fill on glass, increasing the cost of each experiment, and limitations of the electrodes to study (ie. single crystals). In addition, this design does not require a special sample holder; it can fit a commercially available 2" diameter mirror mount.

Dr. Popescu from the University of IL has developed a Gradient Light Interference Microscopy (GLIM) as an add-on module for commercial microscopes that enables 3D imaging of unlabeled specimens. This method is not limited to photobleaching and phototoxicity commonly associated with fluorescence microscopy. Both thin and thick samples have been imaged successfully including beads, Hela cells, bovine embryos over broad temporal and spatial scales.