On-Chip Holographic Microscopy and its Application for Automated Semen Analysis - Biomedical Optical Phase Microscopy and Nanoscopy - page 150

[19 26] , laser Doppler velocimetry [27 32] , and photon correlation spectroscopy [33 35] ,

have also been proposed to automatically analyze semen and avoid the labor intensive
nature of this manual method. However, these approaches can only indirectly estimate the
overall sperm concentration or motility and therefore are not widely adopted.

Computer-assisted semen analysis (CASA) systems are currently considered as one of the
most promising technologies to replace the traditional manual semen analysis methods with
pattern analysis algorithms that automatically process the images recorded with a
conventional optical microscope [36 42] . CASA systems are important because of their

ability to provide quantitative information about sperm motility (e.g., the speed distribution
of individual sperm), which can be used to predict fertilization rate [43,44] and to evaluate
the effect of various drugs on sperm quality [45,46] . Although the state-of-the-art CASA
systems are very efficient and versatile, their widespread use in fertility clinics is partially
hindered by their relatively large dimensions, high costs, and maintenance needs. For the
same reason, CASA platforms have limited applications to field use in veterinary medicine
such as stud farming and animal breeding [43,47 78] .

Through a color change due to chemical staining or labeling of sperm-specific proteins,
commercially available male fertility test kits for personal home use, such as FertilMARQ
[50] or SpermCheck [51] , also aim to indirectly quantify sperm concentration. However,
sperm motility or the concentration of motile sperm cannot be quantified by these tests.
Recently, an alternative semen analysis platform has also been reported by utilizing a
compact microfluidic device that can measure electrical impedance changes due to sperm
movement [52] . Unfortunately, only the total number of the sperm in the sample can be
provided by this lab-on-a-chip platform, that is, motile and immotile sperm cannot be
differentiated from each other.

To conduct automated semen analysis using our lensless holographic microscope
( Figure 8.1A ), digital summation of sequentially captured lensless frames enables us to
rapidly count only the immobile sperm based on the reconstructed phase images. On the
other hand, digital subtraction of these consecutive holographic frames permits
quantification of the speed and the trajectories of individual motile sperm within an FOV of
B 24 mm 2 .

In this chapter, we first present a detailed analysis on the fundamental principles of on-chip
incoherent in-line holography which is at the heart of the presented semen analysis
platform. Next, implementation of such a lensless holographic imaging device toward
automated semen analysis is explained together with the digital algorithms that we
employed for automatic quantification of sperm density and motility. Lastly, we present
results of this high-throughput on-chip semen analysis platform and its potential
applications. Such a compact and cost-effective automated semen analysis platform running
on a wide-field lensless on-chip microscope would be a very valuable tool for andrology