Dual-Illumination Fourier Modulation Microscopy: New Techniques for Multimodal Light Imaging

The term Internal Darkfield can be applied to microscopy methods that employ Fourier stops to block or attenuate undiffracted zero-order light, with optional superposition of adjustably attenuated brightfield to achieve Variable Graded-Field images. Existing Luminance Contrast and Single-Sideband Edge Enhancement or Schlieren methods may be improved by using a secondary illumination beam to generate Internal Darkfield, preferably in color, along with attenuated primary bright illumination that ideally uses a wide-aperture asymmetric beam. The main embodiment of the Dual-Illumination Fourier Modulation Microscopy methods uses a thin strip-stop to block a narrow secondary pencil beam at variable angles of incidence. Axial darkfield manifests luminous visualization of internal detail with reduced edge-blooming artifacts, whereas peripheral angles give enhanced directional resolution. Methods are described using semicircular Fourier stops including a pure Variable Rejection Internal Darkfield method that allows adjustable positioning of a peripheral pencil deep inside the stop zone to reject most of the diffuse background along with low spatial frequencies. At smaller angles, more low spatial frequencies are admitted, allowing increased brightness. Fourier modulation can also be applied to various methods of Multiple Oblique Beam Illumination, condenser-free LED microscopy including multi-color modes, Spatial Light Modulator innovations including split-aperture phase retrieval, and high-pass filter innovations. Suggestions are also offered for fluorescence and autofluorescence applications, along with hyperspectral imaging using variable wavelength secondary illumination. The underlying principles of Schlieren microscopy and split-aperture methods are explored, along with the mechanisms that generate relief contrast due to sideband suppression or asymmetry.