ABSTRACT
Cellular metabolism is a key regulator of energetics, cell growth, regeneration, and homeostasis. Spatially mapping the heterogeneity of cellular metabolic activity is of great importance for unraveling the overall cell and tissue health. In this regard, imaging the endogenous metabolic cofactors, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD), with subcellular resolution and in a noninvasive manner would be useful to determine tissue and cell viability in a clinical environment, but practical use is limited by current imaging techniques. In this paper, we demonstrate the use of phasor-based hyperspectral light-sheet (HS-LS) microscopy using a single UVA excitation wavelength as a route to mapping metabolism in three dimensions. We show that excitation solely at a UVA wavelength of 375 nm can simultaneously excite NAD(P)H and FAD autofluorescence, while their relative contributions can be readily quantified using a hardware-based spectral phasor analysis. We demonstrate the potential of our HS-LS system by capturing dynamic changes in metabolic activity during preimplantation embryo development. To validate our approach, we delineate metabolic changes during preimplantation embryo development from volumetric maps of metabolic activity. Importantly, our approach overcomes the need for multiple excitation wavelengths, two-photon imaging, or significant postprocessing of data, paving the way toward clinical translation, such as in situ, noninvasive assessment of embryo viability.
ABSTRACT
Embryo quality is a crucial factor affecting live birth outcomes. However, an accurate diagnostic for embryo quality remains elusive in the in vitro fertilization clinic. Determining physical parameters of the embryo may offer key information for this purpose. Here, we demonstrate that digital holographic microscopy (DHM) can rapidly and non-invasively assess the refractive index of mouse embryos. Murine embryos were cultured in either low- or high-lipid containing media and digital holograms recorded at various stages of development. The phase of the recorded hologram was numerically retrieved, from which the refractive index of the embryo was calculated. We showed that DHM can detect spatio-temporal changes in refractive index during embryo development that are reflective of its lipid content. As accumulation of intracellular lipid is known to compromise embryo health, DHM may prove beneficial in developing an accurate, non-invasive, multimodal diagnostic.
ABSTRACT
The direct detection and imaging of exoplanets requires the use of high-contrast adaptive optics (AO). In these systems quasi-static aberrations need to be highly corrected and calibrated. To achieve this, the pupil-modulated point-diffraction interferometer (m-PDI) was presented in an earlier paper. This present paper focuses on m-PDI concept validation through three experiments. First, the instrument's accuracy and dynamic range are characterized by measuring the spatial transfer function at all spatial frequencies and at different amplitudes. Then, using visible monochromatic light, an AO control loop is closed on the system's systematic bias to test for precision and completeness. In a central section of the pupil with 72% of the total radius, the residual error is 7.7 nm rms. Finally, the control loop is run using polychromatic light with a spectral FWHM of 77 nm around the R-band. The control loop shows no drop in performance with respect to the monochromatic case, reaching a final Strehl ratio larger than 0.7.
ABSTRACT
The direct detection and imaging of exoplanets requires the use of high-contrast adaptive optics (AO). In these systems quasi-static aberrations need to be highly corrected and calibrated. In order to achieve this, a high-sensitivity wavefront sensor, the pupil-modulated point-diffraction interferometer (m-PDI), is presented. This sensor modulates and retrieves both the phase and the amplitude of an incoming electric field. The theory behind the wavefront reconstruction, the visibility of fringes, chromatic effects and noise propagation are developed. Results show this interferometer has a wide chromatic bandwidth. For a bandwidth of Δλ = 50% in units of central wavelength, the visibility of fringes and the response of the WFS to low and high-order aberrations are almost unaffected with respect to the monochromatic case. The WFS is, in contrast, very sensitive to variations in the size of its pinhole. The size of the pinhole is shown to affect the sensor's linearity, the dynamic range and the amount of noise. Larger pinholes make the sensor less sensitive to low-order aberrations, but in turn also decrease the effects of misalignments.
