Multimodal discrimination of immune cells using a combination of Raman spectroscopy and digital holographic microscopy

This work was supported by the UK Engineering and Physical Sciences Research Council under grant EP/J01771X/1, A European Union FAMOS project (FP7 ICT, 317744), and the ’BRAINS’ 600th anniversary appeal, and Dr. E. Killick. We would also like to thank The RS Macdonald Charitable Trust for funding support. KD acknowledges support of a Royal Society Leverhulme Trust Senior Fellowship. This work was also supported by the PreDiCT-TB consortium [IMI Joint undertaking grant agreement number 115337, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution (www.imi.europa.eu)]The ability to identify and characterise individual cells of the immune system under label-free conditions would be a significant advantage in biomedical and clinical studies where untouched and unmodified cells are required. We present a multi-modal system capable of simultaneously acquiring both single point Raman spectra and digital holographic images of single cells. We use this combined approach to identify and discriminate between immune cell populations CD4+ T cells, B cells and monocytes. We investigate several approaches to interpret the phase images including signal intensity histograms and texture analysis. Both modalities are independently able to discriminate between cell subsets and dual-modality may therefore be used a means for validation. We demonstrate here sensitivities achieved in the range of 86.8% to 100%, and specificities in the range of 85.4% to 100%. Additionally each modality provides information not available from the other providing both a molecular and a morphological signature of each cell.Publisher PDFPeer reviewe

Portable advanced off-axis camera for quantitative phase microscopy

We propose and experimentally demonstrate a device in which common-path interferometry combined with off-axis holographic geometry is used to realize a digital holographic camera which can be attached to the camera port of a conventional transmission microscope for complex wavefront analysis. A thick transmission volume grating recorded holographically into thick photosensitive glass splits the beam containing the sample information in two beams. The untouched transmitted beam creates the sample arm of the interferometer. The Bragg diffracted order of the grating is spectrally and spatially filtered by diffraction to generate a clean reference beam. Double passing the diffracted order through the grating using a retroreflector device provides filtering in two dimensions. The spatial filtering done by the grating which works based on high angular selectivity of thick volume gratings, reduces the alignment spatial sensitivity which is an advantage over the conventional spatial filtering done by pinholes. Besides, using a second thick grating, we introduce a desired coherence plane tilt in the reference beam which is sufficient to create high-visibility interference over the entire field of view. The full-field off-axis interferograms are created from which the amplitude and phase can be reconstructed. The advantage of the proposed camera is the insensitivity to the alignment, thus can be the basis for a standalone camera mountable on a standard optical microscope

Towards an incoherent off-axis digital holographic microscope

We propose and experimentally demonstrate a system in which off-axis digital holographic microscopy is realized using a broadband illumination source. Single-shot holographic measurements are enabled, while the coherence noise is removed thanks to the broad bandwidth of the illuminating source. The proposed digital holographic camera is portable and can be attached to the camera port of a conventional optical microscope. This camera is capable of obtaining the complex wavefront i.e the intensity and phase information of the light transmitted or reflected from a sample. A combination of a thick transmission volume grating recorded holographically into thick photosensitive glass and thin transmission phase gratings recorded holographically into thin photopolymers, spatially filters the beam of light containing the sample information in two dimensions through diffraction. This filtered beam creates the reference arm of the interferometer. The untouched transmitted beam creates the sample arm of the interferometer. The spatial filtering performed by the combination of gratings above reduces the alignment spatial sensitivity which is an advantage over conventional spatial filtering done by pinholes. Besides, using a second thin grating, we introduce a desired coherence plane tilt in the reference beam which is sufficient to create high-visibility interference over the entire field of view in off-axis configuration. Full-field off-axis interferograms are thus created from which the phase information can be extracted