OBJECTIVE:To demonstrate nanoscale motion tracing of spermatozoa and present analysis of the motion traces to characterize the consistency of motion of spermatozoa as a complement to progressive motility analysis. DESIGN:Anonymized sperm samples were videographed under a quantitative phase microscope, followed by generating and analyzing superresolution motion traces of individual spermatozoa. SETTING:Not applicable. PATIENT(S):Centrifuged human sperm samples. INTERVENTION(S):Not applicable. MAIN OUTCOME MEASURE(S):Precision of motion trace of individual sperms, presence of a helical pattern in the motion trace, mean and standard deviations of helical periods and radii of sperm motion traces, speed of progression. RESULT(S):Spatially sensitive quantitative phase imaging with a superresolution computational technique MUltiple SIgnal Classification ALgorithm allowed achieving motion precision of 340 nm using ×10, 0.25 numerical aperture lens whereas the diffraction-limited resolution at this setting was 1,320 nm. The motion traces thus derived facilitated new kinematic features of sperm, namely the statistics of helix period and radii per sperm. Through the analysis, 47 sperms with a speed >25 μm/s were randomly selected from the same healthy donor semen sample, it is seen that the kinematic features did not correlate with the speed of the sperms. In addition, it is noted that spermatozoa may experience changes in the periodicity and radius of the helical path over time. Further, some very fast sperms (e.g., >70 μm/s) may demonstrate irregular motion and need further investigation. Presented computational analysis can be used directly for sperm samples from both fertility patients with normal and abnormal sperm cell conditions. We note that MUltiple SIgnal Classification ALgorithm is an image analysis technique that may vaguely fall under the machine learning category, but the conventional metrics for reporting found in Enhancing the QUAlity and Transparency Of health Research network do not apply. Alternative suitable metrics are reported, and bias is avoided through random selection of regions for analysis. Detailed methods are included for reproducibility. CONCLUSION(S):Kinematic features derived from nanoscale motion traces of spermatozoa contain information complementary to the speed of the sperms, allowing further distinction among the progressively motile sperms. Some highly progressive spermatozoa may have irregular motion patterns, and whether irregularity of motion indicates poor quality regarding artificial insemination needs further investigation. The presented technique can be generalized for sperm analysis for a variety of fertility conditions.

Motility of cells plays an important role to determine the cell growth, health and to monitor the gene transformation. The aim of the current study is to propose a combination of a high-contrast label-free imaging method and a computational approach (conventionally used for super-resolution) which can be used as a tool in tracing the motion of the cells and organelles. Here, we integrate quantitative phase microscopy (QPM) with waveguide-based trapping (WT) and applied multiple signal classification algorithm (MUSICAL) to analyze the motion of the trapped particle. We successfully trapped and displaced a 1 µm polystyrene bead particle on a strip waveguide using a 1064 nm laser beam. While propelling the polystyrene bead particle, we recorded time-lapsed interferometric images using a partially spatially coherent (PSC) light-based off-axis QPM system. The reconstruction of time-lapsed phase images of the trapped particle is accomplished using the Fourier transform and transport of intensity algorithm, which further used in MUSICAL for the motion trace analysis. Here, we traced the motion of a trapped bead particle with scale finer than the size of the object i.e., diffraction limit of the system. We show super-resolved motion trace even though the particle’s image is itself diffraction limited in each frame. The proposed study could be useful in different biological applications such as cell monitoring, cell tracking, manipulation, and classification between healthy and unhealthy cells.

Structured illumination microscopy (SIM) is a most popular super-resolution technique used in cell biology and bio-imaging. Here, we present a novel approach to realize multiscale super-resolution SIM by swapping the non-linearity between instrumentation and reconstruction algorithm to achieve super-resolution. Our goal is to overcome two conventional limitations of SIM i.e., fixed resolution and the need of precise knowledge of illumination pattern. The optical system encodes higher order frequencies of the sample by projecting PSF-modulated binary patterns for illuminating the sample plane, which do not have clean Fourier peaks conventionally used in SIM. These patterns fold high frequency content of sample into the measurements in an obfuscated manner, which are de-obfuscated using multiple signal classification algorithm. Our approach eliminates the need of clean peaks in the illumination pattern, which have multiple advantages i.e., simple instrumentation and the flexibility of using different collection lenses. The reconstruction algorithm used in the proposed work does not require known illumination. Finally, we reduce the sensitivity of reconstruction algorithm to the signal to background ratio. Here, we acquired patterned illumination images of the same sample using different collection objective lenses, and obtained diffraction limited as well as super-resolved images, supporting 4 different resolution in the same system through SIM. Our experimental results with multiple collection objective lens show wider applicability of the proposed system at signal to background ration as small as <3.

Multifocus microscopy enables recording of entire volumes in a single camera exposure. In dense samples, multifocus microscopy is severely hampered by background haze. Here, we introduce a scalable multifocus method that incorporates optical sectioning and offers axial superresolution capabilities. In our method, a dithered oblique light-sheet scans the sample volume during a single exposure, while generated fluorescence is linearised onto the camera with a multifocus optical element. A synchronised rolling shutter readout realised optical sectioning. We describe the technique theoretically and verify its optical sectioning and superresolution capabilities. We demonstrate a prototype system with a multifocus beam splitter cascade and record monolayers of endothelial cells at 35 volumes per second. We furthermore image uncleared engineered human heart tissue and visualise the distribution of mitochondria at axial superresolution. Our method manages to capture sub-diffraction sized mitochondria-derived vesicles up to 30 um deep into the tissue.

Structured illumination microscopy suffers from the need of sophisticated instrumentation and precise calibration. This makes structured illumination microscopes costly and skill-dependent. We present a novel approach to realize super-resolution structured illumination microscopy using an alignment non-critical illumination system and a reconstruction algorithm that does not need illumination information. The optical system is designed to encode higher order frequency components of the specimen by projecting PSF-modulated binary patterns for illuminating the sample plane, which do not have clean Fourier peaks conventionally used in structured illumination microscopy. These patterns fold high frequency content of sample into the measurements in an obfuscated manner, which are de-obfuscated using multiple signal classification algorithm. This algorithm eliminates the need of clean peaks in illumination and the knowledge of illumination patterns, which makes instrumentation simple and flexible for use with a variety of microscope objective lenses. We present a variety of experimental results on beads and cell samples to demonstrate resolution enhancement by a factor of 2.6 to 3.4 times, which is better than the enhancement supported by the conventional linear structure illumination microscopy where the same objective lens is used for structured illumination as well as collection of light. We show that the same system can be used in SIM configuration with different collection objective lenses without any careful re-calibration or realignment, thereby supporting a range of resolutions with the same system.