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Self-interference digital holography (SIDH) can image incoherently emitting objects over large axial ranges from three two-dimensional images. By combining SIDH with single-molecule localization microscopy (SMLM), incoherently emitting objects can be localized with nanometer precision over a wide axial range without mechanical refocusing. However, background light substantially degrades the performance of SIDH due to the relatively large size of the hologram. To optimize the performance of SIDH, we performed simulations to study the optimal hologram radius (Rh) for different levels of background photons. The results show that by reducing the size of the hologram, we can achieve a localization precision of better than 60 nm laterally and 80 nm axially over a 10 µm axial range under the conditions of low signal level (6000 photons) with 10 photons/pixel of background noise. We then performed experiments to demonstrate our optimized SIDH system. The results show that point sources emitting as few as 2120 photons can be successfully detected. We further demonstrated that we can successfully reconstruct point-like sources emitting 4200 photons over a 10 µm axial range by light-sheet SIDH.
RESUMO
Introduction: Mitochondria are extremely important organelles in the regulation of bone marrow and brain activity. However, live imaging of these subcellular features with high resolution in scattering tissues like brain or bone has proven challenging. Methods: In this study, we developed a two-photon fluorescence microscope with adaptive optics (TPFM-AO) for high-resolution imaging, which uses a home-built Shack-Hartmann wavefront sensor (SHWFS) to correct system aberrations and a sensorless approach for correcting low order tissue aberrations. Results: Using AO increases the fluorescence intensity of the point spread function (PSF) and achieves fast imaging of subcellular organelles with 400 nm resolution through 85 µm of highly scattering tissue. We achieved ~1.55×, ~3.58×, and ~1.77× intensity increases using AO, and a reduction of the PSF width by ~0.83×, ~0.74×, and ~0.9× at the depths of 0, 50 µm and 85 µm in living mouse bone marrow respectively, allowing us to characterize mitochondrial health and the survival of functioning cells with a field of view of 67.5× 67.5 µm. We also investigate the role of initial signal and background levels in sample correction quality by varying the laser power and camera exposure time and develop an intensity-based criteria for sample correction. Discussion: This study demonstrates a promising tool for imaging of mitochondria and other organelles in optically distorting biological environments, which could facilitate the study of a variety of diseases connected to mitochondrial morphology and activity in a range of biological tissues.
RESUMO
This work reports the realization of Gd3+ persistent luminescence in the narrowband ultraviolet-B (NB-UVB; 310-313 nm) through persistent energy transfer from a sensitizer of Pr3+, Pb2+ or Bi3+. We propose a general design concept to develop Gd3+-activated NB-UVB persistent phosphors from Pr3+-, Pb2+- or Bi3+-activated ultraviolet-C (200-280 nm) or ultraviolet-B (280-315 nm) persistent phosphors, leading to the discovery of ten Gd3+ NB-UVB persistent phosphors such as Sr3Gd2Si6O18:Pr3+, Sr3Gd2Si6O18:Pb2+ and Y2GdAl2Ga3O12:Bi3+ as well as five ultraviolet-B persistent phosphors such as Y3Al2Ga3O12:Pr3+, Sr3Y2Si6O18:Pb2+ and Y3Al2Ga3O12:Bi3+. The persistent energy transfer from the sensitizers to Gd3+ is very efficient and the Gd3+ NB-UVB afterglow can last for more than 10 hours. This study expands the persistent luminescence research to the NB-UVB as well as the broader ultraviolet-B spectral regions. The NB-UVB persistent phosphors may act as self-sustained glowing NB-UVB radiation sources for dermatological therapy.