Speckle fluctuation spectroscopy of intracellular motion in living tissue using coherence-domain digital holography

Abstract

Abstract. Dynamic speckle from 3-D coherence-gated optical sections provides a sensitive label-free measure of cellular activity up to 1 mm deep in living tissue. However, specificity to cellular functionality has not previously been demonstrated. In this work, we perform fluctuation spectroscopy on dynamic light scattering captured using coherence-domain digital holography to obtain the spectral response of tissue that is perturbed by temperature, osmolarity, and antimitotic cytoskeletal drugs. Different perturbations induce specific spectrogram response signatures that can show simultaneous enhancement and suppression in different spectral ranges. Biological speckle has a dual character. On one hand, it is a parasitic effect that degrades the contrast of biomedical imaging, and many approaches seek speckle reduction.1 On the other hand, speckle provides strong statistical information about scattering media and can be used for interferometric imaging of cells.2 Dynamic speckle, in particular, has considerable information content. For instance, dynamic speckle can be used to monitor blood flow 3 and can assess the health of living tissue using intracellular motion as a fully endogenous imaging contrast agent.4 3-D imaging approaches are particularly important for assessing tissue viability, pharmacological toxicity, and cancer progression, 5 and can be provided by coherence-domain techniques 6 and digital holography. 7 Holography captures high-contrast depth-gated speckle statistics because of broad-field illumination. 8 We previously used coherence-gated digital holography to capture intracellular motion in 3-D tissue as an imaging contrast agent 9 based on the statistical fluctuations of dynamic speckle. However, there are many functional causes of subcellular motion, and an overall motility metric does not capture specific functions. In this work, we perform fluctuation spectroscopy on depth-gated dynamic speckle to generate frequency versus time spectrograms of tissue responding to various environmental and pharmacological perturbations. The spectral responses depend on the specific type of perturbation, and these spectral responses can serve as functional fingerprints for tissue-based screening.Holograms in short coherence digital holography were recorded and reconstructed from the experimental setup shown in Fig. 1. Lenses L1 and L2 expanded a reference beam, and lens L3 performed the Fourier transform of the object beam. Details of the experimental configuration are discussed in Ref.9. We used rat osteogenic sarcoma tumor spheroids as the target tissue samples.10 Multicellular tumor spheroids have approximately spherical geometry that facilitates comparison of structure to function. A typical pseudo-B-scan image is shown in Fig. 1, which is produced from 3-D holographic images of an 800-m-diam rat tumor spheroid.To generate frequency versus time spectrograms, we acquired 300 successive digital holograms at two acquisition rates ͑200 holograms for 20 s and 100 holograms for 200 s͒ every 4 min for three hou...

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“…Biodynamic imaging [5][6][7] is an optical imaging technology closely related to en face OCT [8] , but with enhanced partially-coherent speckle generated by broad-area illumination with coherence detection through digital holography [9][10][11] .…”

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