Denna nyligen publicerade rapport (2017-04) jag har grävt fram berör både forskning som praktiskt användande.
Italienska forskare med kompetensområde inom reproducering har kikat närmare på vilka tekniker inom mikroskopin som är bra/bäst att använda sig av inom fertilitetsområdet. Alltså både för forskare som medicinare. Här ser jag ett kommersiellt intressant område för PHI,s Holomonitorteknik.
Tänk par med svårigheter att skaffa barn,där mannens spermier är under lupp.
Tänk assisterad befruktning,där spermakvaliteten är avgörande för ett lyckat resultat.
Tänk djuravel, där spermakvaliteten skiljer en medel mot en champion.
Combined Raman Spectroscopy and Digital Holographic Microscopy for Sperm Cell Quality Analysis
Abstract
The diagnosis of male infertility is vastly complex. To date, morphology, motility, and concentration have been used as key parameters to establish the sperm normality and achieve pregnancy both in natural and in assisted fecundation. However, spermatozoa from infertile men could present a variety of alterations, such as DNA fragmentation, alterations of chromatin structure, and aneuploidy, which have been demonstrated to decrease reproductive capacity of men. Therefore, the ability to see detailed relationships between morphology and physiology in selected spermatozoa with submicrometric resolution in a nondestructive and noninvasive way and within a functional correlated context could be extremely important for the intracytoplasmic sperm injection procedure. In this review, we describe label-free optical spectroscopy and imaging techniques, based on the combination of Raman spectroscopy/imaging with holographic imaging, which are able to noninvasively measure the (bio)chemistry and morphology of sperm cells. We discuss the benefits and limitation of the proposed photonic techniques, with particular emphasis on applications in detection/characterization of sperm cell morphological defects and photodamage, and the identification/sorting of X- and Y-bearing bovine spermatozoa.
1. Introduction
Sperm cell analysis is an imperfect tool but remains the cornerstone of the investigation of male infertility.
The sperm selection is one of the most crucial aspects of the assisted reproductive techniques and an effective procedure for selecting normal sperm is greatly needed .
Routine semen analysis is based on the analysis of sperm cell morphology, motility, and concentration .
Although this assay reveals useful information for the initial evaluation of the infertile male, it is not a test of fertility.
The standard selection protocols are not discriminatory with respect to the identification of spermatozoa with normal haploid chromosome or intact chromatin or DNA. Sperm DNA integrity is assessed by destructive methods such as terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, comet assay, sperm chromatin dispersion (SCD) test, or sperm chromatin structure assay (SCSA), which are assays for the detection of damaged DNA or altered protein in sperm nuclei by using specific stains.
Indeed, current sperm physiology tests are of limited clinical utility as they render the sample unusable.
Recently, spectroscopy has emerged as one of the major tools for biomedical applications and has made significant progress in the field of clinical evaluation. Research has been carried out on a number of human cells using spectroscopic techniques, including Raman spectroscopy. This vibrational spectroscopic technique does not require sample labelling, getting biochemical information directly from the inelastic light scattering induced by its molecular vibrations, and only small amounts of material (micrograms to nanograms) with a minimum sample preparation are required (water or culture medium can be used as solvent). This makes Raman spectroscopy a relatively simple, noninvasive, and nondestructive technique providing molecular-level information, allowing investigation of functional groups, bonding types, and molecular conformations. Interestingly, due to the cited characteristics, it has been successfully employed for the study of several living/fixed cells, including sperm cells. Huser et al., in a recent paper, studied membranous human sperm cells by Raman spectroscopy and correlated the nuclear shape (normal versus abnormal) with protein content and DNA packaging. The Raman approach has been used to demonstrate that spectra could identify UV-induced or oxidative nDNA damage, localize and map DNA damage, assess the mitochondrial status in human spermatozoa, and separate sperm cells that are bound to the human zona pellucida from unbound sperm cells. In 2015, Edengeiser et al. showed that Raman spectroscopy allows to chemically assess single, living human spermatozoa in near-physiological conditions.
Additionally, Raman spectroscopy can be easily combined with complementary optical approaches, such as holographic microscopy. Based on the refractive index difference between the cell and the surrounding medium, holographic microscopy analyses the phase of the light transmitted by the sample and allows 3D quantitative sample image reconstruction.
Compared with the established morphological approaches used in biology such as fluorescence microscopy or more sophisticated techniques for instance atomic force microscopy (AFM), holographic imaging has three potential advantages:
(i) The entire volume information can be acquired in one shot, avoiding mechanical movements.
(ii) The reconstructed images can be exploited for quantitative microtopology (such as volume measurements).
(iii) The sample can be analysed in a physiological state.
Therefore, holographic microscopy enables the high contrast characterization of live specimen.
Figure 3: (a) Pseudo-3D representation of the phase map of the investigated spermatozoon obtained by digital holography microscopy. The arrow indicates the observed protuberance in the region connecting the tail to the head of the spermatozoon. (b) Phase intensity profile of the spermatozoon along the line DD′ stressed in (c)
Due to the holographic microscopy ability for quantitatively monitoring cell structure and dynamics, it has been used to address specific questions in the field of andrology research. Indeed, several configurations have been implemented to track the principal features of sperm morphology and navigation in a 3D chemical landscape enabling accurate analysis of cell parameters such as 3D tomography, biovolume, curvilinear velocity, and straight-line velocity.
The combination of both holographic microscopy and Raman measurements could further improve the sperm cell analysis monitoring simultaneously morphological and physiological parameters.
A combined approach based on sequential measurements with the same laser probe has been proposed by Kang et al. for the investigation of blood disorders. By using two separate laser probes, Pavillon et al. demonstrated for the first time the possibility to simultaneously measure the morphological live cell (HeLa cell) characteristics in real time during the Raman acquisition .
Thus, this combined imaging approach could be the perfect candidate for noninvasively and nondestructively selecting single, live spermatozoa for the intracytoplasmic sperm injection procedure.
4. Discussion and Conclusions
In this work, a promising optical approach, based on digital holography and Raman spectroscopy technologies, has been proposed for the quality assessment of sperm cells.
The great advantage of digital holography is the possibility to retrieve 3D quantitative imaging of the sample under investigation with a single-shot acquisition and directly in its native environment.
Raman spectroscopy, based on the inherent molecular vibration excitation/response, allows label-free, non-destructive (taking into account the laser wavelength and power) biochemical investigations.
Applying the holographic approach for analysing the sperm cells, high-resolution images have been obtained, clearly highlighting some morphological alterations. In particular, a sort of “protuberance” was observed in the postacrosomal region of few investigated spermatozoa and was correlated, by using the Raman imaging approach, to the increased protein concentration (probably due to the presence of centrioles).
Therefore, with the proposed combined approach, it is possible nondestructively delineating the distribution of DNA and protein in the head, acrosome, and tail but also detecting morphologically and physiologically small discrepancies such as the presence of defects in a correlative manner. It provides information at the molecular level, allowing investigation of functional groups, bonding types, and molecular conformations and correlating them to the cell structural properties.
In conclusion, the proposed Raman/holography imaging approach could be a very promising method to test the physiology and morphology of preselected sperm cells due its noninvasive and nondestructive nature and its high-specific identification capabilities.
This would be crucial for the investigation of human sperm before assisted fecundation, where 100% selection success is mandatory.
Min kommentar
I denna rapport beskriver man kombon Rama/DHM som ett nytt ypperligt instrument både för forskare men även för medicinare. Tänk alla fertilitetskliniker världen över, dit par som singelkvinnor vänder sig för att skaffa barn. Betänk även sekundärt veterinärsektorn där avel på hög nivå bedrivs där lyckade resultat står i proportion till den gissningsvis höga kostnadsbilden.
(forts)
Känns som att marknaden inte är 120 000 labb längre utan avsevärt större.
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