Label-free imaging and biochemical characterization of bovine sperm cells

Abstract

A full label-free morphological and biochemical characterization is desirable to select spermatozoa during preparation for artificial insemination. In order to study these fundamental parameters, we take advantage of two attractive techniques: digital holography (DH) and Raman spectroscopy (RS). DH presents new opportunities for studying morphological aspect of cells and tissues non-invasively, quantitatively and without the need for staining or tagging, while RS is a very specific technique allowing the biochemical analysis of cellular components with a spatial resolution in the sub-micrometer range. In this paper, morphological and biochemical bovine sperm cell alterations were studied using these techniques. In addition, a complementary DH and RS study was performed to identify X- and Y-chromosome-bearing sperm cells. We demonstrate that the two techniques together are a powerful and highly efficient tool elucidating some important criterions for sperm morphological selection and sex-identification, overcoming many of the limitations associated with existing protocols.

Figure 1

Innovative experimental set up that brings together digital holography and Raman spectroscopy for full label-free characterization of biological samples.

Figure 2

Overview of the single sperm cell analysis technology. Single cells can be analyzed by Digital Holography (DH) or Raman spectroscopy (RS). DH microscopy based on morphological parameters measurement is a fast and label-free technology allowing the reconstruction of 3D maps of single selected cells and measurements of cell volumes/thickness. RS detects biomolecule vibrations from a single cell, which serves as a cellular intrinsic “fingerprint”. It is a sensitive and label-free technology allowing the production of pseudo-color images according to the Raman spectral band intensities and the identification of cell phenotype and physiological state. Both technologies can be applied to analyze sperm cell defects or characterize X- and Y-bearing sperm cells.

Figure 3

(A) Acquired hologram, a region is enhanced in order to show the interference pattern (inset); (B) Pseudo 3D representation of the phase map of a bovine spermatozoon obtained by digital holography microscopy.

Figure 4

(A) Profile of the head along the line DD' illustrated in (B).

Figure 5

(A) Raman image (9 × 12 µm²) of a spermatozoon acquired on Xplora inverted Raman microscope of HORIBA Jobin Yvon; (B) Raman spectra (integration time: 10 s) acquired from tail (blue line), nucleus (green line) and acrosomal vesicle (magenta line). The colors of the spectra correspond to the colors in the image.

Figure 6

2D intensity map of each Raman spectrum corresponding to different regions of the spermatozoon: tail, nucleus and acrosomal vesicle.

Figure 7

(A) Phase map; (B) Cut phase map; (C) Otsu’s method; (D) Regionprops maximum area; (E) Boundary; (F) Polygonal interpolation; (G) Filling; (H) Expansion; (I) Product phase-mask.

Figure 8

(A) Histogram of the measured head volume for both 500 X- and 500 Y-bearing sperm cells and the correspondent fit; (B) Mean value of the head volume obtained by the fit.

Figure 9

(A) Average Raman spectra of 300 X- (purple line) and 300 Y-sperm cells (blue line) in the “fingerprint” spectral region; (B) Comparison between the Raman spectra of X- and Y-spermatozoa in the spectral region between 700–850 cm⁻¹ and (C) 1400–1650 cm⁻¹. (D) Measured peak area of the characteristic DNA bands at 726 and 785 cm⁻¹ for X- and Y-spermatozoa.

Figure 10

(A) 3D Principal Component Analysis (PCA) score plot comparing 900 X- and 900 Y-spermatozoa from 3 bulls; (B) Confusion matrix giving the classification for X-and Y-spermatozoa.