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 :

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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 :






Abstract
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.

Metodik

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.


Conclusions
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.

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