Quantitative Stain-free Imaging of Sperm Cells for In-vitro Fertilization using Digital Holographic Microscopy

We present a new stain-free imaging platform for simultaneous measurement of human spermatozoa thickness and integral-refractive index (RI) profiles during flow cytometry.

Estimation of these physical parameters in light microscopy is not feasible, as biological cells are semi-transparent. Digital holographic microscopy (DHM) records the phase difference of light through the sample, which is linearly proportional to the optical path delay (OPD). The OPD quantitatively encodes for both of the above parameters for each pixel in the recorded image.

Motivated by our recent findings that suggest a high correlation between stain-free recordings of OPD maps by DHM and DNA fragmentation in sperm cells, we aim to decouple this optical relation for live specimens.

Previously, we constructed an automated sperm cell selection system by utilizing cell-specific features for classification by means of machine learning. The current conventional decoupling methods are based on multimodal imaging, such as combining fluorescence, atomic-force (AFM) microscopy with DHM or applying geometrical constraint often combined with confocal microscopy. These methods include use of toxic stains, invasive measurements, inherent image registration problems, and inefficient fitted models supported with slow and bulky imaging modality, respectively.

A more efficient decoupling technique, but an impractical one for dynamic samples such as sperm cells, is measuring the same cell is two culture mediums. To solve this problem, we have designed a single exposure dual-wavelength microscopy taking into advantage the RI spectral shift of the surrounding dispersive medium. This yields the possibility for dynamic RI imaging of live human sperm cells during flow cytometry.