Officiellt erkännande

I en aktuell studie publicerad 4/2 2024 går forskare ut med ett erkännande till tekniken HoloMonitor bygger på. Forskarna ifråga är de HoloMonitorfrälsta Beatrix Péter,Inna Szekacs och vår gamle vän Robert Horvath. Samtliga bördiga från Ungern. I ärlighetens namn ska sägas att Holotekniken inte är den enda forskarna lyfter fram.Men eftersom dessa forskare är kända HoloMonitorprofiler dristar jag mig till att säga att denna studie bär ett officiellt forskarerkännande av Holotekniken,dvs instrumentet HoloMonitor. Forskarna har undersökt olika mikroskopkoptekniker vid studier ner på molekylstadie. De berättar att traditionella mikroskop som använder infärgning (labeling) inte klarar/duger när objekten är riktigt små,dvs molekyler. Man lyfter fram flera alternativ som bättre klarar sådana studier. Samtliga med icke infärgningsteknik. Men till undersökningen. (Urval)

Label-free biomolecular and cellular methods in small molecule epigallocatechin-gallate research

• Beatrix Péter
• Inna Szekacs
• Robert Horvath

Open AccessPublished:February 04, 2024DOI:https://doi.org/10.1016/j.heliyon.2024.e25603

Abstract

Small molecule natural compounds are gaining popularity in biomedicine due to their easy access to wide structural diversity and their proven health benefits in several case studies. Affinity measurements of small molecules below 100 Da M weight in a label-free and automatized manner using small amounts of samples have now become a possibility and reviewed in the present work. We also highlight novel label-free setups with excellent time resolution, which is important for kinetic measurements of biomolecules and living cells. We summarize how molecular-scale affinity data can be obtained from the in-depth analysis of cellular kinetic signals. Unlike traditional measurements, label-free biosensors have made such measurements possible, even without the isolation of specific cellular receptors of interest. Throughout this review, we consider epigallocatechin gallate (EGCG) as an exemplary compound. EGCG, a catechin found in green tea, is a well-established anti-inflammatory and anti-cancer agent. It has undergone extensive examination in numerous studies, which typically rely on fluorescent-based methods to explore its effects on both healthy and tumor cells. The summarized research topics range from molecular interactions with proteins and biological films to the kinetics of cellular adhesion and movement on novel biomimetic interfaces in the presence of EGCG. While the direct impact of small molecules on living cells and biomolecules is relatively well investigated in the literature using traditional biological measurements, this review also highlights the indirect influence of these molecules on the cells by modifying their nano-environment. Moreover, we underscore the significance of novel high-throughput label-free techniques in small molecular measurements, facilitating the investigation of both molecular-scale interactions and cellular processes in one single experiment. This advancement opens the door to exploring more complex multicomponent models that were previously beyond the reach of traditional assays.

1. Introduction

The need for labeling arises from the limited sensitivity of conventional methods, which struggle to detect biomolecules due to their light elemental composition, small mass, and lack of fluorescence. Techniques such as those based on electrons, X-rays, visible light, and mass spectrometry, which have been effective in physics and chemistry, are much less powerful in biology. Attaching a biomolecule to an artificial entity with a much greater electron density, optical polarizability, or mass, the molecule becomes “visible” to the technique. However, the excitement surrounding this sudden detectability can sometimes overshadow the possibility that the labeling process may interfere with the biological effect under investigation.

New technologies that are sensitive enough to detect biomolecules without labeling them have opened up new possibilities for label-free detection. This is particularly useful for natural compounds, which often have small molecular weights and binding pockets, making labeling difficult or impossible. Label-free biosensors are more and more important in investigating the mode of action of bioactive molecules (even with low molecular weight), and they offer advantages over classical techniques that require labels or dyes, which can disturb the samples. These methods are generally straightforward, cost-effective, and rapid. Moreover, the diversity of natural products is still essential for drug discovery. In the research of active compounds found in traditional (Chinese) medicines, novel label-free methods can be applied to investigate the effects of these compounds on cell behavior without the use of dyes or labels. These methods help to study the biological properties of small molecules, investigate various biological coatings and adhesion assays, and motility tests can be performed in a high-throughput format. These label-free methods, especially optical waveguide-based, highly sensitive biophysical tools, have great potential in drug discovery as they provide a comprehensive view of receptor-ligand binding in living cells. The emergence of these label-free assays has made them increasingly popular in research and development areas focusing on the biological roles of drug candidates.

The in situ monitoring of cellular crawling along the endothelium is an essential process in the action of both cancer and immune cells. However, the techniques of measuring this biological process in real time are limited and have essential drawbacks. The label-free digital holographic microscopy might solve these problems in an elegant way. The technique is perfectly suitable for measuring migration, motility, and morphological changes of living cells in a high-throughput manner. Cellular motility, migration, proliferation rate, and morphological features (optical thickness, volume, area) can be easily measured, even for relatively large cell populations. Digital holographic microscopy is available in a miniaturized format, compatible with a humidified incubator. From the captured images, a continuous movie can be created.

5. Label-free imaging techniques for investigating cellular processes

5.1 Transmission digital holographic microscopy

The development of modern computer technology launched the possibility of quantitative phase imaging, where a computer calculates the object image from a digitally recorded hologram using a numerical reconstruction algorithm. In transmission digital holographic microscopy (TDHM), a laser beam is split into two parts, one passing through the sample and the other serving as a reference beam. The two beams then interfere with each other, producing a hologram that contains information about the phase and amplitude of the transmitted light through the sample. Unlike traditional microscopy techniques, TDHM does not require staining or labeling of the sample. Commercially available digital holographic microscope HoloMonitor M4 (HM, PHI AB, Lund, Sweden) allows the imaging of live cells under physiological conditions in an incubator environment in real- and long time periods. Each image captured contains data on more than 30 cellular parameters in every single cell. HM has essential applications in biology, providing quantitative information on living cells. It has been used to study the morphology and dynamics of biological cells, including the invasiveness of cancer cells, adhesion, proliferation, migration, and death.  

Fig. 3Holographic microscope method and the collection of the results. A. Working principle of the HM4: A laser beam is split into two identical beams (sample and reference), the sample beam transmitting through the study object and interfering with the reference beam creating a hologram. Based on ref 

B. Profile analysis of a single HeLa cell by HM4. This figure is based on the work of ref C. HeLa cells motility analysis when EGCG is added to the HeLa cancer cells. The black lines represent the mean of the values averaged for five typical cells.

Understanding diseases requires studying cell migration and motility, crucial factors in cancer progression. The effect of EGCG on cancer cell motility and migration was investigated using a digital holographic microscope HM4. The software of this system calculates motility as the distance the cell moves from start to end of the analysis and migration as the shortest distance between the two points. Adding EGCG (500 μg/mL) to HeLa cells resulted in decreased migration, as well as motility and motility speed.

HM4 creates sharp 3D images of cells, but due to its limited vertical resolution, very thin parts of the cell lamellipodium may not be detected, leading to an underestimation of cell contact area and volume and an overestimation of cell thickness. A simple method has been proposed to correct this issue.

7. Conclusions

Our review aimed at collecting data on a specific research question, namely the EGCG measurements performed by label-free biosensor methods in cellular adhesion and migration studies, including the characterization of the methods and the compound itself. We have elucidated the main points of interest (adhesion and migration process, cell viability, classical assays with dyes, label-free techniques, EGCG characterization, and findings by label-free methods) with inclusion and exclusion criteria. We have made a careful and systematic search of the literature using the following keywords: epigallocatechin-gallate, green tea, label-free, a natural compound, biosensors, cell adhesion, migration, motility, movement, viability, cytotoxicity, flow cytometry, dyes, oxidation). Approximately 130 relevant papers were selected. Very recent (2023) and old references (articles from 1993) were evaluated. Our report covers the knowledge on this topic from the last 30 years. Our criteria were that the compound must be EGCG measured by label-free methods. Further, recent articles that monitor other natural compounds by label-free techniques were also selected as the outlook for other substances (15–20 % of these references).

In summary, applying label-free methods in small molecule EGCG research is a novel way with advantages; fast and relatively simple methods without using dyes, meanwhile the oxidation process may not affect the results of the experiments. In some instances, the high-throughput format allows many parallel measurements simultaneously to be applied for rapid drug screening on cells. Cell viability can also be estimated from the adhesion kinetic data. It should be however noted that label-free investigations require careful experimental design with adequately chosen controls. This is due to the fact that the specificity of label-free is less compared to that of traditional labeling assays. For example, phenomena like nonspecific binding have the utmost importance and should be avoided by employing engineered surfaces.

Min kommentar
De Ungerska forskarnas studier/undersökning är ytterst noggrann och förmodligen den enda av sitt slag med grundliga tester av alternativen till traditionella mikroskop. Att de vid sin egen forskning använder sig av HoloMonitor bland alternativen talar väl för vilket instrument de skulle rekommendera om de fick frågan. 

PHI bör kika på denna studie/undersökning. Här finns argument och marknadsföringsmaterial att använda. 

Finge man önska (igen),så vore det en höjdare om de kunde knyta nestor Robert Horvath till Bolaget i någon form.

Frågan man ska ställa sig: Är instrumentet HoloMonitor enbart bra eller outstanding? 

Här visar de ungerska forskarna hur användbart det är vid den nya forskningen på molekylstadie,och inte enbart på traditionell cellforskning samt inom Reg Med.

                                            Mvh the99

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