Compact and light-weight automated semen analysis platform using lensfree on-chip microscopy

Abstract

We demonstrate a compact and lightweight platform to conduct automated semen analysis using a lensfree on-chip microscope. This holographic on-chip imaging platform weighs ∼46 g, measures ∼4.2 × 4.2 × 5.8 cm, and does not require any lenses, lasers or other bulky optical components to achieve phase and amplitude imaging of sperms over ∼24 mm(2) field-of-view with an effective numerical aperture of ∼0.2. Using this wide-field lensfree on-chip microscope, semen samples are imaged for ∼10 s, capturing a total of ∼20 holographic frames. Digital subtraction of these consecutive lensfree frames, followed by appropriate processing of the reconstructed images, enables automated quantification of the count, the speed and the dynamic trajectories of motile sperms, while summation of the same frames permits counting of immotile sperms. Such a compact and lightweight automated semen analysis platform running on a wide-field lensfree on-chip microscope could be especially important for fertility clinics, personal male fertility tests, as well as for field use in veterinary medicine such as in stud farming and animal breeding applications.

Figure 1

(a) A lensfree holographic on-chip microscope that measures 4.2 × 4.2 × 5.8 cm and weighs 46 g is shown. The embedded light source (an LED filtered by a pinhole) and the CMOS image sensor are both powered through a USB connection from the side. This compact on-chip microscope can provide both amplitude and phase images of the sperms (loaded within a sliding tray) over a field-of-view of 24 mm² with an effective numerical aperture of ~0.2. (b) A schematic diagram of the lensfree holographic microscope shown in (a) depicts the relative positions of the light source, the semen sample and the sensor chip. This schematic diagram is not drawn to scale.

Figure 2

(a) A digitally cropped lensfree hologram of an immobilized semen sample (6.05 million sperms per mL) that is acquired with the unit in Figure 1(a) is shown. (b) For comparison purposes, a bright-field microscope image of the same FOV as in (a) is acquired with a 40× objective-lens (NA = 0.65). (c) The amplitude image reconstructed from the raw hologram shown in (a) for the same FOV indicates the locations of the heads of the sperms. (d) The phase image reconstructed from the raw hologram shown in (a) for the same FOV illustrates both the heads and the tails of the sperms. (e) Automatic characterization results that are generated based on the reconstructed phase image in (d) are illustrated. The elliptical areas corresponding to sperm heads are enclosed by red circles while the tails are labeled with green lines. Defective sperms with missing or unusually curved tails (marked with the white arrows in (e)) are not reported toward positive sperm counts.

Figure 3

(a) A digitally subtracted lensfree hologram of three moving sperms is generated from two successive frames (500 ms apart) (b) A microscopic image, that is digitally reconstructed from the lensfree differential hologram shown in (a), illustrates the positions of three sperms in two successive frames (white spots show the sperms’ end positions and black spots show their starting positions). The displacement vectors of these sperms are labeled as S1, S2, and S3.

Figure 4

(a) Dynamic trajectories of 221 sperms within a field-of-view of ~24 mm² are automatically tracked over a time-span of 10 s. The blue spots mark the end positions of the tracked sperms, while the green lines refer to their trajectories. (b) The speed histogram of these motile sperms is calculated using the information in (a) by summing the sperm displacements from all the consecutive frames and then dividing this sum by the total image acquisition duration.

Figure 5

Counting accuracy of the presented automated semen analysis platform for (a) immotile sperms; (b) motile sperms; and (c) both the motile and immotile sperms is illustrated at various sperm concentrations up to 12.5 × 10⁶/mL. The x-axes are the sperm concentrations that are manually counted using a conventional bright-field microscope. The y-axes are the sperm concentrations that are automatically counted for the same semen samples using lensfree holographic images acquired with the on-chip microscope shown in Figure 1(a). The total counts in (c) are the summed up concentrations of the immotile sperms in (a) and the motile sperms in (b). Correlation coefficients (r) of these characterization results shown in (a), (b), and (c) (0.98, 0.99, and 0.98, respectively) further validate the accuracy of this compact and lightweight holographic lensfree microscope as a semen analysis platform.