Söndagsläsning 1 (Forskningsrapporter)

Oscar har varit ute med grävspaden och hittat 2 nya forskningsrapporter. Den första är en gedigen doktorsavhandling på 200 sidor, författad av Joel Kuhn från Heriot Watt University Edinburgh Scotland.
Ämnet för doktorsavhandlingen är nanopartiklar och hur desssa kan användas som ex läkemedelsbärare  eller som del av diagnostisering via ett antal olika tekniker. Värt att notera är att forskaren ägnar ett helt kapitel åt att berätta om HoloMonitor. Mellan raderna kan man lista ut att han är ganska förtjust i instrumentet.

Surface functionalized iron oxide nanoparticles for applications in biomedical sciences

Abstract
This thesis focuses on the functionalization of iron oxide nanoparticles (Fe3O4) and their  applications in biomedical sciences. Each chapter represents an independent research project that has been conducted within a different collaboration. For each project, magnetic Fe3O4 iron oxide nanoparticles have been functionalized or modified to suit its requirements. 

This thesis aims to show how versatile and most promising Fe3O4 iron oxide nanoparticles are, and how their unique properties in size, shape, and magnetism can be utilized for a broad range of applications in biomedicines.

Chapter 4 presents the cell internalization process of iron oxide nanoparticles, captured using the unique holographic cell imaging technique of a HoloMonitor M4 microscope. 

In most cases where the cell internalization process is monitored, only one or two cells can be visualized and tracked at the same time. 

This is not the case with this technique, where hundreds of cells can be simultaneously visualized, analyzed, and monitored over time. Unlike single-cell observation, the system takes pictures of the cell culture at a high capture rate, allowing to observe and interpret the cell dynamics, cell morphologies, and cell reactions to nanoparticles (e.g., toxicity and apoptosis). Measuring the kurtosis and skewness of MCF-7 cancer cells for 72 hours after nanoparticle exposure, showed that cell splitting and proliferation took place, and no extraordinary damages or cell death was caused by the internalization of the particles. Furthermore, every step of the internalization process was monitored and captured in visible data for the first time.

Ur kapitel 4 väljer jag att enbart ta med delar av texten. Det är tämligen mycket skrivet om HoloMonitor och dess funktionalitet inom olika områden. Ni får gå in på pdf,en och läsa all text.

CHAPTER 4
Monitoring the cell internalization of iron oxide nanoparticles using a holographic imaging system

4.1. Introduction 

The internalization process of PEG-coated iron oxide nanoparticles was monitored continuously using a new holography technique. It was found that the internalization of MNPs completed in 35 minutes, presenting an online study on internalization that has not been found in the literature. The system is based on taking a series of pictures of a selection of cells under a microscope over time and thus generating a time-lapse video. The frequency of captures can be optimized to ensure the desired process is precisely measured and in focus. A long long-term exposure would require a lower frame capture rate (frequency) than for example a short exposure, such as the internalization process presented in this chapter. 

In contrast to a single cell observation, a time-lapse video allows interpreting the cell dynamics, cell morphologies, and cell reactions to nanoparticles (e.g., toxicity and apoptosis). Information about the cell surface and its properties is highly essential to analyze the cell-substrate interactions and possible cellular effects that result from the exposure to the substrates. For example, loading a cell with nanoparticles and monitor the cell proliferation over time helps to identify the cellular uptake of MNPs and their toxicity. 

The HoloMonitor M4 (Phase Holographic Imaging), however, is one of the first microscopes that allows to monitor and average of multiple cells for evaluation. A possible toxic effect and other properties can therefore be identified with higher precision.

One of the most significant cellular processes for drug delivery and contrasting is cellular uptake (internalization). This can occur through cellular activities such as endocytosis, where the particle or substrate is internalized in form of a vesicle. 

Understanding the mechanism and kinetics of the internalization is highly important for the design and optimization of nanoparticles for drug delivery and contrast enhancement. 

Monitoring MCF-7 breast cancer cells with the HoloMonitor M4 microscope did not only present valuable data about the cell proliferation but also allowed study on the roughness of the cell surface, which presented the key factors for this chapter. 

For this experiment, MCF-7 breast cancer cells were loaded with PEG9-12-silanenanoparticles (Chapter 3) and continuously monitored under a HoloMonitor M4 microscope for 72 hours to test the toxicity and cell response. The results not only suggested that the nanoparticles did not cause cell death and improved the cell distribution, but also allowed for a novel way of monitoring each step of the internalization process of nanoparticles into the cell (adhesion, engulfment, and fission).

4.2.2. Loading the cell line with nanoparticles and monitoring the cell internalization process using the HoloMonitor M4 microscope
First MCF-7 cancer cells were seeded on a petri dish (Ø 25 mm) at a density of 1 x 105 cells per dish with 2 mL medium I. Two different media were used during this exposure experiment; medium (I) was prepared as the standard culture medium (4.2.1. Cell culturing); medium (II) is of the same composition as medium (I) except for any fetal bovine serum (FBS). The missing amount of FBS in medium (II) was compensated by adding an equivalent amount of DMEM. The serum-free medium (II) was used during particle loading to avoid any possible interactions between proteins and nanoparticles. 
After 48 h of incubation (37ºC, 5% CO2 atm.), the medium was changed to medium (II), which included 150 µL of a sterile filtrated 5 µg/mL PEG9-12-silane nanoparticle solution. 
The serum-free medium II, used during monitoring of the MNP-loaded cells, was changed to medium I (containing proteins) after 48h to provide fresh nourishment for the cells.
The viability of cells was then digitally monitored using a HoloMonitor M4 time lap microscope (Phase Holographic Imaging, Sweden). Cells loaded with PEG9-12-silane MNPs were incubated at 37 ºC and 5% CO2 atmosphere. The frame capture rate was set to take an image every minute, with a total imaged area of 1.558 mm2. The captured frames generated a time lap video showing the cell proliferation and recorded the surface roughness in skewness and kurtosis. 

Figure 4. 2: MCF-7 breast cancer cells (green) on the first day of MNP internalization (a) and 72 hours after internalization (b). The highlights (peaks circled in white) point out that cell proliferation took place and was not significantly affected by nanoparticle exposure (arrows)

The toxicity of these MNPs will be monitored over MCF-7 cancer cells using a HoloMonitor M4 microscope. This digital imaging method will generate further information about the cell internalization process of nanoparticles and therefore help to optimize the cell uptake of functionalized nanoparticles into cell and tissue cultures and improve their intended purposes such as drug delivery and contrast enhancement.

Min kommentar
Månader (år?) av djuplodande studier inom ämnet nanopartiklar komprimerat ner till 200 sidor låter sig inte förklaras av en enkel lekman som undertecknad.Man kan bara bli oerhört imponerad av att det finns forskare som ägnar så mycket tid,kraft,resurser åt ett sådant mastodontprojekt.Det jag kan addera är dock att det inte undgår mitt skarpa öga att forskaren fått ut massvis med information vid användande av HoloMonitor. Att han med illa dold förtjusning gillar detta instrument skarpt.Och det är gott nog.

Som service till alla ev HoloMonitornyfikna forskare : HoloMonitor Demo

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Nästa forskningsrapport kommer från Italien och är utförd av 6 forskare bördiga från olika institut i Italien och Skottland.Ämnet är naturligtvis cellforskning med betoning på nanoteknik och stamceller.

The Impact of Experimental Conditions on Cell Mechanics as Measured with Nanoindentation

Abstract

The evaluation of cell elasticity is becoming increasingly significant, since it is now known that it impacts physiological mechanisms, such as stem cell differentiation and embryogenesis, as well as pathological processes, such as cancer invasiveness and endothelial senescence. However, the results of single-cell mechanical measurements vary considerably, not only due to systematic instrumental errors but also due to the dynamic and non-homogenous nature of the sample. In this work, relying on Chiaro nanoindenter (Optics11Life), we characterized in depth the nanoindentation experimental procedure, in order to highlight whether and how experimental conditions could affect measurements of living cell stiffness. We demonstrated that the procedure can be quite insensitive to technical replicates and that several biological conditions, such as cell confluency, starvation and passage, significantly impact the results. Experiments should be designed to maximally avoid inhomogeneous scenarios to avoid divergences in the measured phenotype.

1. Introduction

In the last few decades, the mechanical properties of single cells have emerged as an important phenotypic trait to understand key physiological mechanisms, such as stem cell differentiation [1] and embryogenesis [2], as well as pathological processes such as cancer invasiveness [3] and endothelial senescence [4]. The viscoelastic properties of the cytoskeleton and the nucleus are intimately linked to mechanotransduction [5], influencing the ability of cells to sense their microenvironment and adapt to the extracellular matrix [6]. The mechanical properties of cells are a direct proxy of the biological state and constitute a very promising physio-pathological biomarker [7].

The de facto standard for measuring single-cell mechanics is nanoindentation [8], either using an Atomic Force Microscope (AFM) [9], eventually with a colloidal probe [10], or more dedicated devices with different deflection detection methods [11]. The experimental procedure to perform nanoindentation and analyze the data is well established [12]. Nevertheless, obtaining robust and reproducible mechanical characterization of single cells is still a challenging task. Living cells are complex systems and the description of their mechanical properties in terms of a single modulus largely depends on the methods used to probe them, with results that can vary by up to 1000 times depending on the cell type and experimental technique [13]. Even when the same methodology is used, the results still vary and are poorly comparable across different groups due to calibration issues [14] and the impact of the analysis pipeline on the final results [15]. For this reason, many groups have suggested, wherever possible, evaluating relative changes in the mechanical properties, in an attempt to cancel out all major sources of instrumental errors [16,17]. However, the variability of the results of single-cell mechanical measurements not only depends on systematic instrumental errors but is also impacted by the experimental design and sample conditions (sample replication, cell passage, shape, etc.). In this paper, we explored the impact of several experimental parameters associated with the indentation of living cells to highlight the most critical aspects to be taken into account to improve the repeatability and reliability of nanoindentation-based single cell mechanical characterization.

Materials and Methods (urval)

2.3. Cell Indentation and Data Analysis

Single-cell stiffness measurements were performed using a Chiaro system (Optics11Life, Amsterdam, The Netherlands), a nanoindentation device based on a ferrule-top with interferometric read-out that enables high-resolution force measurements in a liquid environment [11]. The experimental protocol to operate the Chiaro nanoindenter is very similar to that required for the more widely adopted atomic force microscope (AFM), and the data can be analysed using the same approach [11,12]. In brief, the force F is measured while moving the tip towards the sample (along the vertical axis, Z) and the corresponding F(Z) curve is recorded. The Chiaro device is mounted on a holographic microscope (HoloMonitor 3, Phase Holographic Imaging, PHI AB, Lund, Sweden) with phase-contrast mode that allows us to precisely target individual cells with the tip (Figure 2). Experiments were carried out with a constant approach speed of 2.5 µm/s using a soft cantilever (stiffness 0.025 N/m) and a spherical tip with a radius R of 3 µm to avoid cell damage and to guarantee a definite contact area. Under these conditions, the indentation process is minimally invasive for the cell, causing no significant alteration in its morphology. The mechanical properties of the cells were calculated by fitting the indentation curve with the Hertz model ([18,19]), considering most often an indentation depth of 300 nm, selected to be smaller than 10% of the thickness of the cell [20]. When higher indentation depths are considered, this is reported in the text, always in the validity condition of Hertz model applicability (500 nm and 800 nm for cells measuring up to 15 µm of thickness). To calculate the contact point we used a threshold method [12], keeping the same parameters for all datasets. The analysis was performed using dedicated python software (https://github.com/CellMechLab/nanoindentation (accessed on 2 March 2023), software version: ee04b82) available under an open-source license [12]. At least 50 force-distance curves were acquired for each condition, to achieve statistical relevance.

Figure 2. Indentation setup. (A) Alignment of the microscope objective with the nanoindentation probe. (B) Sample on the objective, during an indentation experiment. (C) Phase contrast image of the cantilever approaching a cell layer.

2.4. Cell Morphology Analysis

Digital holography microscopy (DHM) and phase-contrast techniques were used to acquire images of fibroblast cells. DHM is a quantitative phase imaging, label-free technique, used to evaluate single-cell 2D and 3D morphological features [21]. It is based on the interference phenomenon: the sample is illuminated with coherent light and the final image is reconstructed from the interference between a reference beam (unmodified) and the one passing through the sample, which introduces a phase delay. The refractive index of the medium is measured in the setup phase, while the value of the cell refractive index was considered to be 1.38 [22]. Data were acquired using a HoloMonitor M3 digital holography microscope (Phase Holographic Imaging, PHI AB, Lund, Sweden) and analyzed using HoloStudio software (Phase Holographic Imaging PHI AB, Sweden). This allowed us to retrieve the value of the cell thickness. The HoloMonitor M3 is equipped with phase contrast objectives, which were used to distinguish flat and elongated cells.

Slutsatser
Under rubriken Conclusions sammanfattar forskarna sina resultat. Och med googles hjälp översätts inledningen enligt följande : De mekaniska egenskaperna hos enstaka celler är direkt kopplade till deras fysiologiska tillstånd eftersom förändringen av egenskaper såsom organiseringen av cytoskelettet, mognad av adhesionsstrukturer och formen på cellen direkt påverkar dess viskoelastiska egenskaper. Det finns en enorm förväntning att cellmekanik kommer att användas, tillsammans med mer traditionella biokemiska markörer, för att ge förmågan att diagnostisera och iscensätta livshotande tillstånd, såsom cancer, och för att erbjuda en holistisk syn på cellfenotyp som kan möjliggöra spännande upptäckter i preklinisk forskning." Klockrent och lättförståeligt va? 😎

Hursom, jag tolkar det som att aktuell stamcellsforskning har fått forskare att mer grundligt analysera celler och dess omgivning,hur de korrelerar och vilken betydelse det har för studier om stamceller. Man kan tycka att forskarna borde äska pengar till att uppdatera sitt HoloMonitorinstrument till en senare version. Men aktuell mjukvara (HoloStudio) ser ut att vara kompatibel även med tidiga versioner av instrumentet.

Et voi´la. Det var första delen av Söndagsläsning. Del 2 kommer produceras efter en välbehövlig kaffepaus.En bulle till det skulle smaka gott 😎

Som service till alla ev HoloMonitornyfikna forskare : HoloMonitor Demo

Bonusmaterial. En forskare (Stephenie Chinwe Alaribe) bördig från Nigeria undervisar en student hur HoloMonitor ger "a great experience with live cells". Klippet är troligtvis från ett engelskt universitet kopplat till Royal Society of Chemistry: London, GB.

                                               Mvh the99