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Register a new userWide field fluorescence epi-microscopy behind a scattering medium enabled by speckle correlations
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Fluorescence microscopy is widely used in biological imaging, however scattering from tissues strongly limits its applicability to a shallow depth. In this work we adapt a methodology inspired from stellar speckle interferometry, and exploit the optical memory effect to enable fluorescence microscopy through a turbid layer. We demonstrate efficient reconstruction of micrometer-size fluorescent objects behind a scattering medium in epi-microscopy, and study the specificities of this imaging modality (magnification, field of view, resolution) as compared to traditional microscopy. Using a modified phase retrieval algorithm to reconstruct fluorescent objects from speckle images, we demonstrate robust reconstructions even in relatively low signal to noise conditions. This modality is particularly appropriate for imaging in biological media, which are known to exhibit relatively large optical memory ranges compatible with tens of micrometers size field of views, and large spectral bandwidths compatible with emission fluorescence spectra of tens of nanometers widths.
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Quantitative phase microscopy (QPM) has found significant applications in the field of biomedical imaging which works on the principle of interferometry. The theory behind achieving interference in QPM with conventional light sources such as white light and lasers is very well developed. Recently, the use of dynamic speckle illumination (DSI) in QPM has attracted attention due to its advantages over conventional light sources such as high spatial phase sensitivity, single shot, scalable field of view (FOV) and resolution. However, the understanding behind obtaining interference fringes in QPM with DSI has not been convincingly covered previously. This imposes a constraint on obtaining interference fringes in QPM using DSI and limits its widespread penetration in the field of biomedical imaging. The present article provides the basic understanding of DSI through both simulation and experiments that is essential to build interference optical microscopy systems such as QPM, digital holographic microscopy and optical coherence tomography. Using the developed theory of DSI we demonstrate its capabilities of using non-identical objective lenses in both arms of the interference microscopy without degrading the interference fringe contrast and providing the flexibility to use user-defined microscope objective lens. It is also demonstrated that the interference fringes are not washed out over a large range of optical path difference (OPD) between the object and the reference arm providing competitive edge over low temporal coherence light sources. The theory and explanation developed here would enable wider penetration of DSI based QPM for applications in biology and material sciences.
Super-resolution fluorescence microscopy is an important tool in biomedical research for its ability to discern features smaller than the diffraction limit. However, due to its difficult implementation and high cost, the universal application of super-resolution microscopy is not feasible. In this paper, we propose and demonstrate a new kind of super-resolution fluorescence microscopy that can be easily implemented and requires neither additional hardware nor complex post-processing. The microscopy is based on the principle of stepwise optical saturation (SOS), where $M$ steps of raw fluorescence images are linearly combined to generate an image with a $sqrt{M}$-fold increase in resolution compared with conventional diffraction-limited images. For example, linearly combining (scaling and subtracting) two images obtained at regular powers extends resolution by a factor of $1.4$ beyond the diffraction limit. The resolution improvement in SOS microscopy is theoretically infinite but practically is limited by the signal-to-noise ratio. We perform simulations and experimentally demonstrate super-resolution microscopy with both one-photon (confocal) and multiphoton excitation fluorescence. We show that with the multiphoton modality, the SOS microscopy can provide super-resolution imaging deep in scattering samples.
The efficient delivery of light energy is a prerequisite for non-invasive imaging and stimulating of target objects embedded deep within a scattering medium. However, injected waves experience random diffusion by multiple light scattering, and only a small fraction reaches the target object. Here we present a method to counteract wave diffusion and to focus multiplescattered waves to the deeply embedded target. To realize this, we experimentally inject light to the reflection eigenchannels of a specific flight time where most of the multiple-scattered waves have interacted with the target object and maximize the intensity of the returning multiple-scattered waves at the selected time. For targets that are too deep to be visible by optical imaging, we demonstrated a more than 10-fold enhancement in light energy delivery in comparison with ordinary wave diffusion cases. This work will lay a foundation for enhancing the working depth of imaging, sensing, and light stimulation.
We develop a method based on the cross-spectrum of an intensity-modulated CW laser, which can extract a signal from an extremely noisy environment and image objects hidden in turbid media. We theoretically analyzed our scheme and performed the experiment by scanning the object placed in between two ground glass diffusers. The image of the object is retrieved by collecting the amplitudes at the modulation frequency of all the cross-spectra. Our method is non-invasive, easy-to-implement, and can work for both static and dynamic media.
Oblique plane microscopy (OPM) enables high speed, volumetric fluorescence imaging through a single-objective geometry. While these advantages have positioned OPM as a valuable tool to probe biological questions in animal models, its potential for in vivo human imaging is largely unexplored due to its typical use with exogenous fluorescent dyes. Here we introduce a scattering-contrast oblique plane microscope (sOPM) and demonstrate label-free imaging of blood cells flowing through human capillaries in vivo. The sOPM illuminates a capillary bed in the ventral tongue with an oblique light sheet, and images side- and back- scattered signal from blood cells. By synchronizing sOPM with a conventional capillaroscope, we acquire paired widefield and axial images of blood cells flowing through a capillary loop. The widefield capillaroscope image provides absorption contrast and confirms the presence of red blood cells (RBCs), while the sOPM image may aid in determining whether optical absorption gaps (OAGs) between RBCs have cellular or acellular composition. Further, we demonstrate consequential differences between fluorescence and scatteri
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