torsdag 18 oktober 2018

Torsdagskvällsreflektioner + 1 artikel

Tjenixen alla PHI,are. Förmodligen tycker ni som jag att aktiehandeln och kursen är miserabel.
Mig veterligen finns det nada fundamentamässigt som föranlett denna utveckling.
Förmodligen är det (förhoppningsvis) de sista skvättarna ur garanternas innehav som kör ner aktien.
Som ex idag, någon som använder sig av Penser (EPB) avyttrade 4000 aktier till nästan vilket pris som helst. Men men, snart borde dessa garanter vara ute ur aktien så vi får mer normal handel baserad på Bolagets utveckling och förknippade händelser.
Under tiden fortsätter bloggen sitt grävarbete oförtröttligt (nåja).

Här nedan kommer PHI-relaterad info.
Bloggen skrev i augusti om en forskningsrapport som nyligen hade släppts. Inlägget.
Innehållet var som vanligt intressant och visade på HoloMonitorn´s användbarhet i forskning även utanför de traditionella som ex cancerforskning.
Jo, forskarna bakom rapporten har nu sammanställt text som berättar mer i detalj hur och med vilka tekniker de använde för att nå sina resultat och fått en artikel utgiven i branschtidningen Micron.
Volume 113, October 2018, Pages 1-9


The International Research and Review Journal for Microscopy

Micron is an interdisciplinary forum for all work that involves new applications of microscopy or where advanced microscopy plays a central role. The journal will publish on the design, methods, application, practice or theory of microscopy and microanalysis, including reports on optical, electron-beam, X-ray microtomography, and scanning-probe systems. It also aims at the regular publication of review papers, short communications, as well as thematic issues on contemporary developments in microscopy and microanalysis. The journal embraces original research in which microscopy has contributed significantly to knowledge in biology, life science, nanoscience and nanotechnology, materials science and engineering.

I Oktoberutgåvan av tidskriften finns nu mer info om DHM tekniken och fylligare info m exempelbilder från PHI`s HoloMonitor.Jag saxar valda delar ur artikeln:

- The recently developed digital holographic microscopy (DHM) has enabled the quantitative evaluation of cell size and morphology without labeling or destruction.

- Therefore, DHM was validated as a promising device for testing platelet function given that it allows for the quantitative evaluation of activation-dependent morphological changes in platelets. DHM technology will be applicable to the quality assurance of platelet concentrates, as well as diagnosis and drug discovery related to platelet functions.

- Recent developments and subsequent advancements in digital holographic microscopy (DHM) have enabled the continuous or intermittent observation of morphological changes in the same cells with regard to cell height, volume, surface roughness, surface irregularity, and many other indexes without destruction, fixation, or labeling . In general, morphological changes in cells in vitro are microscopically examined using selected views. Consequently, given the limited number of cells subjected to qualitative evaluation, data obtained therefrom may not necessarily represent the overall trend in cell populations. DHM, however, allows the scanning of more than 1000 cells within minutes, as well as the quantitative evaluation of their morphology.
Therefore, it has the advantage of being able to detect small but statistically significant differences among similar cell populations.

- Compared with aggregometry or flow cytometry (FCM), which have typically been used for evaluating platelet activation, we demonstrated the major advantages of DHM in the simultaneous quantitative analysis of adherent single and aggregated platelets without staining or labeling

Data acquisition and DHM image analysis

Fixed platelets on plastic culture wares were directly subjected to imaging without labeling or staining using a digital holographic microscope (HoloMonitor M4; Phase Holographic Imaging AB, Lund, Sweden). As essentially described in a previous study regarding human periosteal cells and mesenchymal stem cells (Kawase et al., 2016), images were captured and analyzed using a specifically designed software (HoloStudio M4; Phase Holographic Imaging AB). In each sample, more than five snapshots of randomly selected views were taken to obtain a total of at least 1500 platelets. All images were binarized, and platelets were segmented under the same conditions (adaptive mean = 128; object size = 8) using the manual mode. Acquired data (N = 1800) were then plotted in histograms using the aforementioned software. Alternatively, quantitative data were exported to Excel (Microsoft, Redmond, WA, USA) for further statistical processing as described in the following section.
Given that the aforementioned software adopts “1.38” as the default setting for the cell refractive index as a default value by the manufacturer (Phase Holographic Imaging AB) (Kawase et al., 2016) based on previously published data (Beuthan et al., 1996; Rappaz et al., 2008; Yu et al., 2009; Persson et al., 2010; Svet, 2013), this value was utilized in the determination of optical thickness and platelet volume. The refractive index of the surrounding medium had been fixed at 1.34 (default) throughout the experiment (Kawase et al., 2016).

- The same samples were then observed by DHM. DHM hologram images for time-course changes in platelet appearance are shown in Fig. 5. These images were further converted to 3D reconstruction images (Fig. 6). As shown in a 2D hologram image (Fig. 5), white small round particles grew in size (i.e., area), whereas those particles decreased, with time of treatment. On the other hand, 3D reconstruction imaging (Fig. 6) has an advantage for the comparison of height (i.e., thickness). The number of tall platelet aggregates apparently increased with time of treatment.

Fig. 5. DHM hologram images for time-course changes in platelet appearance. As described in the legend of Fig. 2, washed platelets were prepared and treated without (A) or with 0.1% CaCl2 for (B) 5 min, (C) 10 min, or (D) 20 min. After treatments, platelets were fixed and subjected to DHM examination. The magnification for all images remained constant (Bar = 100 μm). Similar data were obtained from three additional experiments using different donor samples.

Fig. 6. DHM 3D reconstruction images for time-course changes in platelet appearance. As described in the legend of Fig. 2, washed platelets were prepared and treated without (A) or with 0.1% CaCl2 for (B) 5 min, (C) 10 min, or (D) 20 min. After treatments, platelets were fixed and subjected to DHM examination. The magnification for all images remained constant (Bar = 100 μm). Similar data were obtained from three additional experiments using different donor samples.
The present study tested the feasibility of DHM examination in the quantitative evaluation of morphological changes in activated platelets. Accordingly, DHM is able to examine both living and fixed cells without further labeling or staining. Although fixed cells are suitable for comparing multiple groups at once, the advantage of using living cells is that DHM may be employed to continuously monitor changes in platelet morphology. Considering that the present study needed to simultaneously compare multiple platelet populations obtained from the same blood sample, we opted to use fixed platelets rather than living ones.

During DHM examination, we demonstrated that platelets activated using CaCl2 shifted from the lower left to the upper right region. Moreover, FCM analysis demonstrated that CaCl2 upregulated CD62P expression in single platelets, while SEM examination demonstrated that single platelets aggregated in response to CaCl2 stimulation. Although other platelet function tests had not been performed to further confirm our observations, the amount of data obtained herein is sufficient to suggest that the apparent region shift in the two-dimensional DHM data plots mainly represents the aggregation of activated platelets.
In conclusion, we herein attempted to validate the applicability of DHM in the quantitative evaluation of activated platelets. Data obtained from our feasibility tests implied that DHM could be a promising method for quantitatively examining morphological changes in platelets in vitro.
Regarding clinical relevance, we indicate that this technology will be further applicable during laboratory testing. To date, we have vigorously investigated the quality and potency of platelet concentrates in regenerative dentistry and reached a conclusion that a quality check of individual PRP preparation is indispensable for predictable PRP therapy (Kawase and Okuda, 2018). At present, the methodology regarding a quick and sensitive quality check of platelets is a major matter remaining to be developed. We believe that DHM will be a promising device for this purpose. Furthermore, probably more importantly and influentially in medical fields, DHM will be applied in the diagnosis of platelet disorders and drug discovery for controlling platelet functions as a high throughput system.

Min kommentar
Läser man igenom hela artikeln får man känslan av att de i minsta detalj skärskådat PHI`s teknik (DHM) och letat svagheter o annat som kan vara till teknikens nackdel.
Men och just ett stort MEN, de kommer istället till slutsatsen : 
"Data obtained from our feasibility tests implied that DHM could be a promising method for quantitatively examining morphological changes in platelets in vitro.
Regarding clinical relevance, we indicate that this technology will be further applicable during laboratory testing.
We believe that DHM will be a promising device for this purpose. Furthermore, probably more importantly and influentially in medical fields, DHM will be applied in the diagnosis of platelet disorders and drug discovery for controlling platelet functions as a high throughput system."
Om nu någon aktieägare (baserat på aktiens nuvarande handel) tvivlar på teknikens förträfflighet och kommande kommersiella genombrott, 11 japanska forskare vibbar om dess raka motsats.

                                                                 Mvh the99

tisdag 16 oktober 2018

HoloMonitor i de fina salongerna

En ny forskningsrapport utförd av Zhen Li, Sofia Kamlund, Till Ryser, Mercy Lard, Stina Oredsson  och Christelle Prinz har publicerats i  den högt ansedda skriften Journal of Materials Chemistry B.
 Tidningen (och webbversionen) utges av brittiska Royal Society of Chemistry

Man beskriver sin gärning enligt följande :

-We are the world’s leading chemistry community, advancing excellence in the chemical sciences.

With over 54,000 members and an international publishing and knowledge business we are the UK’s professional body for chemical scientists, supporting and representing our members and bringing together chemical scientists from all over the world.
A not-for-profit organisation with a heritage that spans 175 years, we have an ambitious international vision for the future. Around the world, we invest in educating future generations of scientists.
We raise and maintain standards. We partner with industry and academia, promoting collaboration and innovation. We advise governments on policy. And we promote the talent, information and ideas that lead to great advances in science.
In a complex and changing world, chemistry and the chemical sciences are essential.
They are vital in our everyday lives and will be vital in helping the world respond to some of its biggest challenges.
We are committed to promoting, supporting and celebrating inclusion and diversity. We understand that the success of our community depends on our ability to nurture the talent of the best people regardless of who they are or their background.
We’re working to shape the future of the chemical sciences – for the benefit of science and humanity."

Läs gärna raderna ovanför igen, men den här gången lite långsammare så innebörden att få medverka i detta sammanhang sjunker in. 
Men till forskningsrapporten vår nya PhD Sofia Kamlund medverkar i.(stort grattis till doktorshatten)
Rapporten offentliggjorders i förra veckan, närmare bestämt onsdag 10 Oktober och återfinns i nästkommande upplaga av :

Nanowires are presently investigated in the context of various biological and medical applications.
In general, these studies are population-based, which results in sub-populations being overlooked. Here, we present a single cell analysis of cell cycle and cell movement parameters of cells seeded on nanowires using digital holographic microscopy for time-lapse imaging. MCF10A normal-like human breast epithelial cells and JIMT-1 breast cancer cells were seeded on glass, flat gallium phosphide (GaP), and on vertical GaP nanowire arrays. The cells were monitored individually using digital holographic microscopy for 48 h. The data show that cell division is affected in cells seeded on flat GaP and nanowires compared to glass, with much fewer cells dividing on the former two substrates compared to the later. However, MCF10 cells that are dividing on glass and flat GaP substrates have similar cell cycle time, suggesting that distinct cell subpopulations are affected differently by the substrates. Altogether, the data highlight the importance of performing single cell analysis to increase our understanding of the versatility of cell behavior on different substrates, which is relevant in the design of nanowire applications.

Ur den 20 sidiga rapporten klistrar jag in utvalda delar.


Digital holographic imaging and tracking
HoloMonitor® M4 (Phase Holographic Imaging AB (PHI), Lund, Sweden) with a motorized stage was used for time-lapse imaging. Imaging was started 24 h after seeding. 
Images were acquired using the software Hstudio™ (PHI) at the same position on the substrate (glass, flat GaP, or nanowires) every five minutes for 48 h.
To increase the image quality, the standard lid of the Petri dish was replaced with HoloLid™ 71 110 (PHI) prior to the start of imaging.
The experiments were repeated three times with each substrate and two time-lapse movies in different areas were acquired per replicate.  
The HoloMonitor® M4 is a quantitative imaging system based on digital holographic microscopy.
In digital holography, the image is a computer reconstruction of a hologram.
The hologram is acquired by the interference of two laser beams, of which one is phase-shifted due to passing through the sample and one is the original laser beam.
The hologram is imprinted on a CCD-camera coupled to a computer. 
The hologram contains information about the three-dimensional (3D) sample it is imaging,in this case a 3D-reconstructed cell image.
The HoloMonitor® M4 uses a low power laser (635 nm wavelength, 0.2 mW/cm2) with no associated phototoxicity, making it suitable for extended time imaging. 

After image acquisition, the time-lapses were analyzed by individual cell tracking using Hstudio™.
The tracking is semi-automated, using a frame-by-frame algorithm attempting to find each tracked cell in the next frame based on the centroid position.
The software allows for manual changes when the algorithm fails to predict the correct position.
From Hstudio™, data about cell cycle time and cell movement can be extracted.   
The data acquired from the tracking of cells in the digital holographic images was used to create cell family trees.
The tracking of each cell family started in the first image and the tracked cells were characterized by their fate.
A cell tracked from the first image of the time-lapse until its division is called a mother cell and is marked with a green X in Fig. 1.
For a mother cell, the start of the cell cycle is unknown.
After division, the individual daughter cells were also tracked. If it was possible to track them throughout a full cell cycle, i.e. until the next division, they are marked with a full circle in Fig. 1. 
If it was not possible to track a daughter cell throughout the entire cell cycle, it is marked with a pink X in Fig. 1.
A cell that does not divide at all throughout the tracking period is marked with a blue X in Fig. 1.
Different factors contribute to interrupted tracking before the end of the time-lapse, the most common
ones being that cells migrate out of the field of view, or that cells clump together and can no longer be distinguished.

Using the HoloMonitor® M4 time-lapse data, we have extracted information about cell movement.
The total accumulated cell movement over time is defined as motility and the shortest distance between the first cell position and the point where the cell can be found in each image is defined as migration.
During the time-lapse acquisition, motility constantly increases, while migration can increase or decrease depending on the cell trajectory.

We have tracked individual JIMT-1 breast cancer cells and MCF10A breast epithelial cells on glass, flat GaP, and GaP nanowire substrates using digital holographic microscopy.
We investigated cell proliferation and cell movement using bulk data analysis and single cell analysis.
The two cell lines studied behave differently, both in terms of proliferation and cell movement, on flat GaP and GaP nanowire substrates compared to when seeded on glass.
Compared to JIMT-1 cells, MCF10A cells were more severely affected when cultured on flat GaP or nanowires than on glass.
One may speculate that this is related to the higher adaptability of cancer cells to a foreign environment driven by genomic instability.
Whereas bulk analysis revealed an increase in PDT of MCF-10A cells on flat GaP and nanowire substrates, single cell analysis of MCF-10A cells revealed that this increase in PDT is due to the presence of different sub-populations. 
Therefore, our data suggest that there are sub-populations of cells that react differently to the substrates, which highlights the importance to perform individual cell analysis.
These different populations presumably represent different phenotypes, which are not observed in bulk cell analysis.
In addition, our data clearly show the importance of investigating many cells, although this is a time-consuming process at present.
Here, we choose to analyze all cells in order to avoid excluding relevant data. Future studies will aim at understanding the molecular mechanisms responsible for the different behaviors on nanowire substrates, which is a requirement for using nanowires in cell biological applications.

  Min kommentar
Man förstår varför denna rapport publiceras hos världens äldsta akademedia beträffande forskning och lärande inom kemi.Royal Society of Chemistry "The oldest chemical society in the world".
177 år har denna anrika institution verkat. Få om ens någon annan vetenskaplig sammanslutning med egen förlagsverksamhet når förmodligen det anseende RSoC åtnjuter.
Att då få tillträde till detta fora beror såklart på forskarnas upptäckter.Vilket leder mig in på att försöka förklara anledningen därtill.Denna forskning är vad jag tror ett samarbete mellan Nanoforskare och Cellbiologforskare.
Jag tror även att professor Stina Oredsson leder denna forskning övergripande.För att nå förståelse mellan nanoteknologi och cellforskning har man med en auktoritet inom nano,nämligen universitetslektor och biträdande professor Christelle Printz.
Dessa 2 auktoriteter har samarbetat i tidigare publicerad forskning (Ex på detta) och man kan anta att den nu aktuella rapporten bygger därpå. Vad den berättar är, som jag tror, helt nya fakta som forskarvärlden kommer ha nytta av när forskning bedrivs med nano inom cancerområdet.
Forskarna visar här med 2 cellpopulationer, ena är friska bröstceller och andra är bröstcancerceller, studerade/observerade med 2 olika tekniker hur dessa cellers utveckling skiljde sig åt beroende på vald teknik.Man studerade på glas och på nanotrådar.Resultaten man kom fram till var att celldelning skedde annorlunda vid tillsatt medel (i detta fallet GaP) som tillfördes bägge teknikerna. Den skillnaden (som man tidigare inte var medveten om) kom man fram till genom att  gå från idag standardförfarande att studera celler i s. k. masspopulation till det mer tidskrävande singelcellsobservationer. Deras rek är att forskare idag behöver gå från studier av masspopulation (vilket som sagt är standard idag) till singelcellsanalys för att kunna utveckla och förbättra framtidens nanoanvändning vid cancerbekämpning.
Det som borde glädja oss alla PHI,are (förutom forskarnas upptäckter) är att man enbart använde sig av 1 mikroskopiteknik för att nå dessa insikter. Nämligen HoloMonitor såklart. Förmodligen ligger Stinas tidigare erfarenheter och användande av HoloMonitor`n som grund för att tekniken visade sig vara så lämplig för just singelcellsanalys.
VD borde skaffa klippkort på en blomsterfirma och skicka en fin bukett till Stina. 😉
Marknaden blev just lite större.

                     Som bonus kommer här en film från studierna.Taget med, ja vad tror ni? :-D
Edit. Filmformatet ser inte ut att stödjas av bloggens dator så klicka istället på denna länk för att se den.

                                                                     Mvh the99

Ps.Tycker man ovanstående text är läsvärd finns nu möjlighet att visa det.Kika högst upp till vänster under Intro.