Eight-wavelength Digital Inline Holography and Brightfield Imaging in a single microscope

Microscopic examination is a key step of the early diagnostic of microbiological infections. In the case of biofluid examination, specific staining facilitates the discrimination of the microbes in presence. While (incoherent, RGB) Brightfield imaging is currently the reference method for an eye-based interpretation, stained object discrimination on images could benefit from an imaging technology bringing both higher standardization of image collection and higher informative contrast [1]. We propose to assess Digital Inline Holographic Microscopy (DIHM) [2], a quantitative phase imaging modality based on coherent light, and to implement it for the first time with more than three spectral bands. DIHM improves the standardization and automation of the image collection as it is based on the acquisition of out-of-focus holograms. Moreover, both absorbance and phase of the objects are retrieved after a posteriori numerical reconstruction of a single hologram, for each wavelength. The reconstructions are based on physical models of diffraction of light and require a precise setting of the optical setup, as well as precise calibration of its parameters [3]. The inline configuration enabled us to implement, on the same microscope, an 8-wavelength DIHM as well as the Brightfield modality, with the goal to compare the informative contrast allowed by both modalities in the context of stained microbe discrimination in a biofluid, based on the same field of view. Another advantage of inline holography is that it does not require a very high coherence (compared to off-axis, for instance, which requires lasers) [4]; the possibility to use LEDs highly facilitates the implementation of multiple wavelengths. In the proposed prototype (Fig. 1), the challenge relied mainly in making both the coherent multi-wavelength light (needed for DIHM) and the incoherent white illumination (required for Brightfield) to co-exist in the simplest possible design. A Köhler illumination with adjustable irises for field and aperture diaphragms has been used to control and optimize the spatial coherence of the DIHM illumination, while a custom array of white LEDs has been placed after the aperture diaphragm for the Brightfield modality (A-B). An objective lens (100X, oil,
$\text{NA}=1.4$
), a beamsplitter (70T/30R), two lens tubes and two CMOS cameras (in-focus and color for Brightfield, out-of-focus and monochrome for DIHM) form the detection part (B). An example of the (in-focus) brightfield image and the (out-of-focus)
$8-\lambda$
hologram are shown for a stained mixture of red blood cells and bacteria (C). Here the reconstructed 8-channel absorbance and phase are shown after recombining the channels in a RGB (i.e. 3 channel) format, more conventional for the eye (D).