Multispectral lensless digital in-line holographic microscope: LED illumination

Abstract

Holography is the process where two coherent wavefields interfere resulting in an interference pattern from which whole field information can be retrieved. Digital holography is the process where the intensity of the superposition of the two waves is recorded using a light sensitive opto-electronic detector array such as a CCD or CMOS camera. From this recorded hologram it is possible to numerically reconstruct the object wavefield.When an optical beam is focused on a pinhole whose diameter is of the order of a few times the wavelength of the illumination beam, a spherically divergent wavefield is emitted. We use the emitted optical beam to illuminate weakly scattering objects resulting in a geometrically magnified diffraction pattern at the camera face. Scattered light from the object is the called the object wavefield, while unscattered light acts as the reference wavefield. The hologram is captured digitally before numerical reconstruction is applied to yield whole field information about the object.It is possible to reconstruct the objects wavefield using this method from coherent laser or incoherent LED illumination. The emitted light from the pinhole acts a pointsource of spatially coherent light enabling holography. This, in combination with the use of multiple wavelength LED's multispectral amplitude images can be reconstructed.The multispectral lensless DIHM described here can be used to holographically image biological specimens such as cells grown for use in the biopharmaceutical industry or for research purposes. In analysing cell viability based on the trypan blue assay, the outer membrane of non-viable cells is penetrated by violet blue dye. Using such a Digital In-line Holographic Microscope as described here, automatic classification of viable and non-viable cells could be performed.