Kvällsfundering angående Avidin

Cell Biology And Screening

Avidin has recently set up a label-free screening laboratory, where three cutting edge technologies were established, the Enspire plate reader from Perkin Elmer, the xCelligence system from Roche/Acea and the holographic microscopy from Phiab.
Besides using these technologies for in-house screening programmes, Avidin provides the following SERVICES:

1. Real-time measurements of the cytotoxic effects of small molecules/mixtures/extracts on primary cells or cancer cells in a medium high throughput manner.
2. Determination of G-protein coupled receptor interaction of small molecules/mixtures/extracts in a medium highthroughput manner.
3. Providing hit molecules (Kd about 1 uM) (binders, inhibitors, inducers) for drug targets in a medium highthroughput manner.

PROVIDE THE NAME OF THE TARGET, AND WE GIVE YOU PATENTABLE NCEs AND CORE STRUCTURES WITHIN 1-2 MONTHS (inquire more details at hackler@avidinbiotech.com).

Denna text är från Avidins hemsida.
Jag funderar över om jag missar värdefull info när jag läser den.
Nån klipsk bloggläsare som ser det jag inte ser?
Återkommer i detta inlägg senare om jag hittar "det" intressanta.

Forts.
Svaret kom av Tobias i kommentarsfältet.
Uttrycket att "inte se skogen för alla träd var i vägen" vet jag nu andemeningen av.
Självklart var det kombon Aceas xCelligence system och Holomonitorn som jag egentligen VISSTE trivdes alldeles förträffligt tillsammans.
I många av de forskningsrapporter jag ögnat igenom har just denna kombo kommit upp som instrument vid redovisningen av forskningsresultaten.
Här hos Avidin var det extra tydligt att de ser hur bra dessa 2 instrument lirar tillsammans.
De visade med hela handen det genom att sätta samman ett "minilabb" bestående av i deras ögon de 3 viktigaste komponenterna.
Ok,vad kan vi dra för slutsatser då?
Jo då kommer spekulationsaspekten in.
xCelligence system + Holomonitor = ? Acea + PHI = ? Acea + Roche + PHI = ?
Här hittar jag ur ett rent spekulativt perspektiv samarbeten först mellan Acea och PHI.
Samma marknad,samma högteknologiska produkter,bägge tillsammans stärker de varandra än separat var och en för sig.Det bör forskningsrapporterna vibba ganska bra om.
Ok, i förlängningen då?
Acea har koppling till Roche då det finns ett gammalt samarbetsavtal dem emellan.
Ev att Roche finns med i ägarbilden i Acea.
Sen vet vi att Life Science marknaden till stor del består i att växa genom uppköp.
Jag spekulerar friskt här nu, men Roche kliver fram ganska starkt som en intressant aktör.
Som ett ledande forskningsföretag har de behov av acess/tillgång till de bästa instrumenten.
Det visar samarbetet med Acea då Roche fanns med i bakgrunden som finansiär? vid framtagandet av xCelligence system. Sen hur deras samarbete eller ägarskap ser ut idag vet jag inte.
Är Roche den hemliga Big Pharma aktören som tidigt införskaffade 3 Holomonitrar?
Om ja, såg de kombomöjligheten xCelligence + Holomonitor? Vad kan de då leda fram till?
Min lekmannabedömning är att vi är slutphasen av PHI,s uttalade mål.
Med alla dessa forskningsrapporter som sista tiden drösat in, med alla dessa nya insikter Holomonitorn bidragit till, med en Nobelpristagare som vouchar för teknikens förträfflighet, med Holomonitorns nu bevisade förmåga att ingå som delkomponent tillsammans med redan etablerade instrument.
Nu i det här exemplet xCelligence, men även med Raman som jag tidigare sett som en bra polare och funderat kring.
Vad ger det för signaler tycker ni?

2 nya rapporter ( andra kommer här )

Denna rapport kom även den ut i fredags 26/5, med uppdatering igår 29/5.
Den är enligt undertecknad speciellt intressant för oss PHI,are.
Inte enbart för dess innehåll och de forskningsresultat man åstadkommit.
Nix, det intressanta momentet är vilka som står bakom den.
I vanliga fall får vi rapporter från välrenomerade Universitet och Institutioner.
Men här har vi en rapport från ett kommersiellt företag. (tänk Big Pharma men i mindre storlek)
Det rör sig om det Ungerska företaget Avidin Biotechnology som hittas på PHI,s sida över Users.

14 av deras forskare med László Puskás, PhD, DSc, Chief executive officer i ledningen har fått nedanstående rapport godkänd.

Mannich Curcuminoids as Potent Anticancer Agents

Abstract

A series of novel curcuminoids were synthesised for the first time via a Mannich-3CR/organocatalysed Claisen–Schmidt condensation sequence.
Structure–activity relationship (SAR) studies were performed by applying viability assays and holographic microscopic imaging to these curcumin analogues for anti-proliferative activity against A549 and H1975 lung adenocarcinoma cells.
The TNFα-induced NF-κB inhibition and autophagy induction effects correlated strongly with the cytotoxic potential of the analogues.
Significant inhibition of tumour growth was observed when the most potent analogue 44 was added in liposomes at one-sixth of the maximally tolerated dose in the A549 xenograft model.
The novel spectrum of activity of these Mannich curcuminoids warrants further preclinical investigations.

Jag berör inte rapporten denna gång utan vänder blicken mot avsändaren istället.
Företaget beskriver sin verksamhet såhär:
"Avidin Ltd. was established in 2002.
The major focus of Avidin is to identify novel small molecules and further develop into drug or clinical candidates in cancer and CNS disease indications.
Avidin has widespread international scientific and industrial partnerships in drug discovery (medicinal chemistry, screen development, HTS genomics, animal models).
Besides in-house drug development programmes, the company provides services and products in the segment of pharmaceutical research, in the preclinical phase of analyzing different drug candidates (target and phenotypic-based hit identification, medicinal chemistry and lead optimization, proof of concept studies in vitro and in animal models, preclinical non-GLP toxicity and ADME/Tox studies) in cancer, inflammation and CNS indications.
Avidin has a Medicinal Chemistry Department where new drug-like molecules, and novel drug-like and drug candidate molecules can be synthesized. Avidin runs an SPF-like animal house and capable of working with different cancer models in immune deficient animals. Avidin can also conduct toxicology measurements in rodents in acute and chronic studies.

The company develops molecular biology-based novel diagnostic solutions as well.
Among these, one is exceptionally successful the high-density nanoliter volume real-time PCR technology (100.000 QRT-PCR/week capacity). This technology was applied to screen the expression of toxicology-related genes and genes coding for cancer stem cell and activated stromal cell related genes. In 2011 Avidin is specialized in single cell genomics, where the company enables digital measurements of mRNA, miRNA expression of individual cells with superior sensitivity and reproducibility."

Företaget framstår som en underleverantör till de större tillverkarna av läkemedel.
Med egna forskare som fått fram delkompononenter till färdiga läkemedel, men även komponenter behövda för vidare forskning.Se under fliken Products.
Att de rönt framgång kan man konstatera här.
Vi får även lite info om deras användande av PHI,s Holomonitor.

Label-free 3D Cell analysis by using holographic microscopy

A new innovative microscopic technology, HoloMonitor™ M3 (Phiab) has been purchased and the technology has been optimized for different screening projects. Phase Holographic Imaging´s HoloMonitor™ M3 is the first totally non-invasive live cell imaging microscope/cell analyzer.
Avidin is using the technology for evaluating/screening in primary cells, cancer cells and for testing immune functions, toxicity of different drug candidates.

Min kommentar
Med denna rapport får vi ett kvitto på att kommersiella företag inom läkemedelsindustrin ser behov och fördelar med att använda sig av PHI,s unika teknik. Den låsta rapporten kommer när den släpps fri förmodligen ge oss mer fakta hur betydelsefull Holomonitorn var för rapportens resultat.
Ska vi gissa att andra aktörer som Big Pharma kommer kika lite extra på dess innehåll?

2 nya artiklar ( första kommer här )

I dagarna har 2 vetenskapliga rapporter efter granskning och bedömning blivit godkända och kommer göras offentliga. För tillfället är bägge låsta och vi får hålla tillgodo med utdrag från resp rapport.
Först har vi forskare från PHI,s användare i Italien och deras rapport som kom ut i fredags 26/5.

A biophysical approach to quantify skeletal stem cells trans-differentiation as a model for the study of osteoporosis

L. Petecchia, F. Viti, F. Sbrana, M. Vassalli, P. Gavazzo
Institute of Biophysics, National Research Council, Via De Marini 6, 16149 Genova, Italy
Received 12 April 2017, Revised 9 May 2017, Accepted 22 May 2017, Available online 26 May 2017

Det är alltså forskare från The Biophysics Institute (IBF), Genoa som man kan läsa om här.
Rapporten berör benskörhet och hur forskning med stamceller från benmärg kan påverka synen på hur benskörhet uppstår och därigenom ge svar på hur bota,reparera detta tillstånd.

Abstract

The stroma of human bone marrow contains a population of skeletal stem cells (hBM-MSC) which are common ancestors, among the others, of osteoblasts and adipocytes. It has been proposed that the imbalance between hBM-MSC osteogenesis and adipogenesis, which naturally accompanies bone marrow senescence, may contribute to the development of bone-associated diseases, like osteoporosis. 

The possibility to reproduce this mechanism in vitro has been demonstrated, providing a good model to disclose the details of the complex bone-fat generation homeostasis. 

Nevertheless, the lack of a simple approach to quantitatively assess the actual stage of a cellular population hindered the adoption of this in vitro model.

In this work, the direct differentiation of hBM-MSCs towards a single (osteo or adipo) lineage was characterized using quantitative biophysical and biological approaches, together with the parallel process of trans-differentiation from one lineage to the other. 

The results confirm that the original plasticity of hBM-MSCs is maintained along the initial stages of the differentiation, showing that in vitro conversion of pre-osteoblasts into adipocytes and, vice versa, of pre-adipocytes into osteoblasts is extremely efficient, comparable with the direct differentiation.  

Moreover, a method based on digital holography is proposed, providing a quantitative indication of the phenotype stage along differentiation.

 In the present study, image acquisition has been performed through HoloMonitor M3
(Phase Holographic Imaging PHI AB Sweden).
Denna text framkommer vid sökning på själva rapporten.
Nästa rapport lägger jag i ett eget inlägg då den är av speciellt intresse.

Breaking News

Tidskriften Cytometry gör en specialutgåva som innehåller allmän info,fakta och forskningsrapporter baserade på Digital Holografisk Mikroskopering. Den kom ut i fredags 19/5 och finns idag tillgänglig på nätet.

                        Special Issue: Quantitative Phase Imaging for Label-Free Cytometry 

Man gör ett undantag och viker hela upplagan åt att berätta om denna "nya spännande teknik".
Innehållsförteckning hittar man här.

I utgåvan florerar namn vi PHI,are är bekanta med.
Ed Luther, Vladimir Torchilin, Birgit Janicke, Kersti Alm mfl..
Även Björn Kemper jag skrivit om tidigare.
Och.. "trumvirvel"..Peter Egelberg hittar vi även där.
Det lär väcka förvåning och uppmärksamhet för alla Cytometriare världen över, att deras "husorgan" valt att avvika från gängse rutiner att ha aktuell info och artiklar som annars täcker hela spektrat inom Cytometrin.
Men nu som sagt gör man ett undantag.
Redan i inledningen skriver redaktionsledningen, där för övrigt Elena Holden gästspelar, anledningen till denna avvikelse.

Introduction

In order to understand complex biological processes, scientists must gain insights into the functioning of individual live cells. Unlike fixed cell imaging, where a single snapshot of the cell's life is retrieved, live-cell imaging allows investigation of the dynamic processes underlying the cell's function. Label-free imaging avoids the limitations inherent to fluorescent probes (phototoxicity, photobleaching) and maintains an appropriate environment for normal cellular behavior.

This special issue is focused on introducing to our readership the subject of Quantitative Phase Imaging (QPI) and its benefits to cytometry. QPI is a valuable method for studying live cell dynamics, as it provides a noninvasive analysis over a wide range of time scales. This type of analysis is gaining traction very rapidly because it is performed with little to no phototoxicity and requires minimal sample preparation. There are no effects of biological and chemical labels or genetic modification, which would alter cellular behavior. QPI offers the benefit of repeated observations and quantitative analysis of cell cultures over time providing minute-by-minute insight into cell proliferation, cell death, and transient events. Quantitative measurements are based on direct phase image analysis of cell structure. QPI yields optical path difference maps associated with the specimen of interest and, as such, it is sensitive to both local thickness and the refractive index of the sample. Several QPI related publications have previously appeared in Cytometry Part A, paving the way for this new field of applications. A collection of manuscripts in this special issue attests to the fact that QPI is becoming a prominent technique complementary to traditional cytometry technologies and indispensable in dynamic label-free live-cell analysis applications.

Och som slutkläm skriver man:
We hope you will find the articles published in this special volume thought provoking. QPI field spans a broad area of interests and biological applications. It is a relatively new member of the cytometry instrumentation techniques and developing very rapidly. Multiple, commercially available QPI systems are now available.
We anticipate that the subjects of performance metrics (sensitivity, specificity, reproducibility, and reliability of QPI measurements) will be addressed in systematic ways compliant with the principles of cytometry, especially in applications proposed for clinical use.  
A pressing need has already emerged in developing optimal analysis strategies and intelligent machine learning algorithms of large multi-dimensional data sets.
Finally, we wish to thank the dedicated scientists and technology developers for their contributions to the QPI field and making this special issue possible.

Bland de artiklar man har med i utgåvan finns PHI,s senaste forskningsrapport :

Label-free high temporal resolution assessment of cell proliferation using digital holographic microscopy

 Figure 1. (A) Technical principle of digital holography as used in the HoloMonitor M4. A low-power laser beam is split into two, one illuminating the sample and the other providing a reference beam. Once combined, the two beams create an interference pattern which is recorded by a digital image sensor. The recorded interference pattern—the hologram—is then processed computationally to produce a quantitative phase shift image. (B) HoloMonitor M4 inside a cell incubator. The motorized stage allows time-lapse image sequences in multiple wells to be acquired automatically. (C) DHM images showing L929, Jimt-1, and SK-MEL-5 cells after 36 h of etoposide treatment. The cells are color-coded to more clearly visualize cell thickness as shown with the color bar. Images are representative of three independent experiments with 10–12 images per sample.

Min kommentar
Denna specialutgåva talar nästan för sig själv. 
Betydelsen för DHM i allmänhet och PHI i synnerhet...kan den bli större?
ALLA inom Cytrometrin (forskare,tillverkare,återförsäljare mfl..) kommer ta del av den.
Magiskt.
                                        

Kompletterat & Bekräftat (DHM i klinisk verksamhet)

I anslutning till mitt tidigare inlägg När 2 blir 1 tänkte jag komplettera den texten.
Angående fertilitetsproblematik hade jag inte behövt gå så långt med grävspaden.
"Våra" svenska forskare Zahra El-Schich, Sofia Kamlund, Birgit Janicke, Kersti Alm & Anette Gjörloff Wingren har berört detta område samt att de även levererar en brasklapp som heter duga i sammanhanget.
På PHI,s hemsida hittar vi under Centers/Malmoe publikationen

Holography: The Usefulness of Digital Holographic Microscopy for Clinical Diagnostics

Där hittar vi under kapitel 4 :

Clinical applications

Lately, DH microscopy is being developed for clinical applications in widely different areas of medicine such as transmembrane water flux, cancer screening, sperm motility, blood cell analysis, and inflammation.

4.3. Sperm analysis

DH has been used to characterize sperm cells, supplying data for both morphology, motility, and the concentration of the sperm cells, without affecting the sperm reviewed in Ref. [43]. The morphology of the sperm head has often been correlated with the outcome of in vitro fertilization and has been shown to be the sole parameter in semen of value in predicting the success of intracytoplasmic sperm injection and intracytoplasmic morphologically selected sperm injection [44, 45]. 

Indeed, DH microscopy generates useful information on the dimensions and structure of human sperm, not revealed by conventional phase-contrast microscopy, in particular the volume of vacuoles. This suggests its use as an additional prognostic tool in assisted reproduction technology to better underline the differences between normal and abnormal sperm morphology.

Och för att understryka mitt tidigare inlägg angående de italienska forskarnas upptäckter har ett samarbete mellan Harvard,USA och italienska institut mynnat ut i denna publication :

Unlabeled Semen Analysis by Means of the Holographic Imaging

Published: March 22, 2017

Ur den kan vi läsa

1. Introduction

Following the advent of human in vitro fertilization, much attention has been given to understand both the spermatozoa morphological alterations and the kinematics/dynamics of the swimming spermatozoa. In fact, semen analysis is commonly employed both in human and in the zoo-technic field. In the first case, the analysis is mainly applied to study the couple’s infertility or to confirm success of male sterilization procedures. Moreover, several studies have shown that for infertile men, the risk for developing a testicular cancer is slightly higher-than-average. So, independently of the will to have children, male fertility is a good indicator for general health. On the other hand, in the zoo-technic field, animal semen analysis is commonly used in animal production laboratories and reproductive toxicology.

The main requirements for the development of techniques used for an accurate semen analysis are the following:

• avoid any alteration of the health of the spermatozoa under test;
• use a label-free approach to eliminate all adverse effects of the probe labeling;
• obtain results independent on the technician’s experience and/or the laboratory environmental conditions (such as temperature, humidity, and duration).

In particular, over the last few years, holographic imaging in microscopy has been established as a valid noninvasive, quantitative, label-free, high-resolution, and phase-contrast imaging technique. 

So this chapter tries to summarize the state-of-art on the semen analysis and recent achievements obtained by a holographic imaging. 

We will show that the unique potentialities of the holographic imaging have been used to provide structural information on both the morphology and the motility of sperm cells. 

Moreover, the combination of the holographic technique with others approaches, such as the Raman spectroscopy, will be described, too. In fact, spermatozoa from infertile men could present a variety of alterations (such as alterations of chromatin organization, aneuploidy, and DNA fragmentation) that can decrease reproductive capacity of men. 

Current methods of DNA assessment are mainly based on fluorescence microscopy, and thus samples are unusable after the analysis. Therefore, the ability to simultaneously analyze, in a nondestructive and noninvasive way, both the morphology and biochemical functionalities of the spermatozoa could bring greater understandings. Thus, the chapter will allow a bird’s-eye view into the potentiality of the semen analysis performed by means of the holographic imaging, showing that this approach is extremely important for the intracytoplasmic sperm injection (ICSI) procedure, where it is highly required the development of a method that allows characterizing and directly select the best spermatozoon to inject into the oocytes.

 

Figure 3.

Pseudo 3D representation of the thickness of a spermatozoon with: (a) a cytoplasmatic droplet along the tail; (b) a bent tail; (c) an acrosome broken. Ref. [33] (by permission of IEEE Society).

6. Conclusion

In this chapter, an overview of the recent achievements in holography imaging applied to both morphological and motility characterizations of sperm cells has been reported. 

Results obtained by means of digital holography have demonstrated the possibility to provide 3D information on both the morphology and motility of sperm cells; this information could be used to better emphasize the differences between normal and abnormal sperm morphology. 

Moreover, the DH approach is a noninvasive technique, allowing the analysis of live spermatozoa, such as 3D tracking of the spatial motion, in order to select normal sperm cells. In particular, the possibility offered by digital holography to add the third dimension in the sperm analysis will give information useful both to relate the sperm anomalies with male infertility and to enable differentiation of the spermatozoa in good health. Finally, DH can be easily combined with other techniques allowing different simultaneous characterization. Indeed, it was demonstrated that a promising optical approach, based on digital holography and Raman spectroscopy technologies can be used for the quality assessment of spermatozoa. Applying this combined approach for analyzing the sperm cells, high-resolution images, and Raman spectra have been obtained, clearly highlighting some morphological and biochemical alterations. In particular, DH and Raman spectroscopy simultaneous approach was used for studying the photodamage induced by visible green light in the spermatozoa structure.

Min kommentar

Våra svenska forskare bekräftar de facto att Digitala Holografiska Mikroskop "is being developed" för kliniskt användande.Denna info är av allra högsta magnitud.

Vi får förhandsinformation som kommer påverka PHI,s värdering i hög grad.

Nu öppnas en marknad vi tidigare inte räknat in.Många har utgått ifrån att PHI,s Holomonitor enbart är riktad mot forskarmarknaden, 120 000 labb + större forskningsbolags egna labb.

Med denna nya info är det dax att börja räkna in hur många kliniker som kan vara addresserbara för tekniken. 

"Våra" forskare nämner 5 olika områden där Holomonitorn är lämplig för kliniskt användande.

5 x hur många kliniker världen runt är det vi ska ta sikte på.

Ska vi gissa och spekulera i att marknaden plötsligt dubblerades? Eller räcker det?

Jag lägger för övrigt till en 6e kliniktyp, veterinärmedicinska.

När 2 blir 1

Här kommer en uppdatering rörande tidigare inlägg. 2 olika områden, men bägge hör ihop i detta sammanhang. Först användningsområden där vi är bortskämda med forskningsrapporter en masse.
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)