Quantitative orientation-independent differential interference contrast (OI-DIC) microscope

The orientation-independent differential interference contrast (OI-DIC) module is an add-on, which can be used for extension the functionality of research grade Olympus microscopes without their modification. The module provides significant improvements to widely used regular DIC technique allows to obtain the quantitative label-free images of dry mass (protein). The module attunes bias and switch shear directions rapidly without mechanically rotating the specimen or any optical components.

Main components: The module consists of two DIC beam-shearing assemblies, 546/20 nm bandpath optical filter, controller of liquid crystal (LC) cells, digital camera, and custom software. The DIC assemblies were custom-built in the MBL. Each assembly employs a pair of crossed regular Olympus DIC prisms with TN (twisted-nematic) liquid crystal cell in between. One of the assemblies also contains ECB (electrically controlled birefringence) liquid crystal cell. The bandpath filter was purchased from Semrock (www.semrock.com). The controller was purchased from Arcoptix (http://www.arcoptix.com). The camera is Lumenera Infinity 3-1M (www.lumenera.com). The software is based on MATLAB (www.MathWorks.com). It was developed in collaboration with KB Imaging (http://www.kbimaging.com).

Optional components: additional OI-DIC assemblies for objective lenses with other magnifications and immersions, bandpath optical filters for other wavelengths.

We implemented OI-DIC set-up on upright microscope Olympus BX61 (Olympus, Tokyo, Japan). The microscope was equipped with 100W halogen lamp, objective lens UPlan Fl 100x/1.30 Oil, condenser lens U-TLO, and video camera adapter U-TV0.5xC-3.

A schematic of the principal components of the OI-DIC module installed into a microscope is shown in Figure 2. The microscope is transformed to the OI-DIC modality by adding two beam-shearing assemblies. The first assembly consists of pair of identical DIC prisms DIC1 and DIC2, and polarization rotator PolRot1 (TN LC cell) sandwiched between the prisms. The shear directions of the DIC prisms are orthogonal. The first prism has the shear direction at 0º and the second prism has the shear direction at 90º. The polarization rotator works as a bi-stable element that rotates the beam polarization by 0º or 90º, called state OFF and state ON, accordingly. In operation the assembly shear direction can be oriented either at -45º or at 45º. Similarly, the second beam-shearing assembly consists of pair of identical DIC prisms DIC3 and DIC4, and polarization rotator PolRot2 (TN LC cell). Orientation of the prisms DIC3and DIC4 is 270º and 180º. The second assembly also includes a phase shifter (ECB LC cell), which allows to vary the bias between the two interfering beams. Its principal axis is oriented at 0º. Optical properties of the LC cells are determined by the amplitude of square wave voltage signals V₁ and V₂, which are generated by LC-cell controller (signal generator). In particular, signal V₁ simultaneously switches the shear direction of assemblies, and signal V₂varies the bias. A desktop computer with custom written software sequentially establishes several settings of output signals of the LC-cell controller and takes the corresponding set of 4 or 6 raw DIC images from CCD camera. The images with orthogonal shear directions and different biases are processed according to a computation algorithm. In the result we receive a quantitative image (map) of the optical path difference (OPD) gradient vector, which then integrated in order to get an OPD map. The obtained OPD images can be enhanced by using deconvolution or the inverse Riesz transform.

Figure 3 shows standard high-resolution Olympus DIC slider U-DICTHR (objective-side) and prism U-DIC40HR (condenser-side), and new sliders with the beam-shearing assemblies, OI-DIC-A (objective-side) and OI-DIC-B (condenser-side), in the transmitted white light. The prism U-DIC40HR is mounted in DIC slider for Olympus DICD condenser. There are two crossed polarizers at the entrance and exit of the beam. The DIC prisms and the beam-shearing assemblies split the input light into two output orthogonally polarized beams with a small shearing angle. The output beams create interference fringes, which are perpendicular to the shear plane. The top row shows that the shear plane of the Olympus DIC prisms is oriented at 0º. The shear plane of the OI-DIC beam-shearing assemblies is oriented at -45º in the OFF state (central row) and at +45º in the ON state (bottom). The distance between the interference fringes is inversely proportional to the shear angle. Therefore interference fringes in the assembly OI-DIC-A are located about 1.5 times closer to each other in comparison to the Olympus DIC slider U-DICTHR. Because of small shear angles Olympus DIC prism U-DIC40HR and condenser-side assembly OI-DIC-B display a single interference fringe that appears alone because the fringe period is greater than the width of the aperture.