En hyllningspublikation i det bästa av forum

Bloggen får emellanåt tips via mejlen om sidor på nätet att kolla upp,alltså där PHI och deras teknik figurerar.
Men även forskningsartiklar som gått undertecknad förbi (fast jag kollar av nätet regelbundet så missar även mitt öga ibland dessa så värdefulla guldkorn). Så är fallet i detta inlägg. (tack till den som uppmärksammade mig på denna publikation). Det handlar alltså om en artikel som gick upp på nätet för 3 dagar sen.Det är en Early View version som kommer att publiceras i nästa nr av cytometristernas husorgan Cytometry PART A.
Artikelförfattare är ingen annan än "vår" professor Anette Gjörloff-Wingren.

Moving into a new dimension: Tracking migrating cells with digital holographic cytometry in 3D

Anette Gjörloff Wingren

First published: 16 December 2018

Motility, or co‐ordinated movement, of cells is fundamental for the development of many processes, such as formation of the embryo, the immune response, inflammation, wound healing, and metastasis of cancer cells 1.
Cells often migrate in response to specific external signals, but they can also move spontaneously.
Aberrant cell motility can be fatal in cancer diseases when cells start to metastasize 2.
Metastasis is strictly associated with tissue invasion of the primary tumor cells, and occurs as a multistep progress 3. The first phase of cancer cell metastasis involves the physical translocation of the cell to a distant organ, whereas the second phase implicates the ability of the cancer cell to develop into a metastatic lesion at that distant site 4.
For skin cancer melanoma, the main cause of death is widespread metastases 5.
Melanoma cells may travel along external vessel lattices. After settling in the metastatic sites, melanoma cells develop mechanisms that protect them against the attack of the immune system.
It is, therefore, of great interest and importance to investigate, monitor, and understand the principles of tumor cell invasion and migration.
The development of novel therapeutic applications that have the ability to block migratory processes is crucial.

For in vitro studies, the cell culture wound‐closure, and the transwell migration and invasion assays can provide data for an understanding how well a particular cell type can migrate 6.
The wound healing assay is also called a scratch assay because it is performed by making a scratch in the cell monolayer (Fig. 1A). The transwell migration assay is a commonly used test to study the migratory response of endothelial cells to angiogenic inducers or inhibitors (Fig. 1B).
This assay is also known as the Boyden or modified Boyden chamber assay. These assays have been frequently used to study movement of cells. Weckmann et al. developed an interesting system to monitor and quantify neutrophils on single cell level, by utilizing commercially available tissue culture hardware, simple video microscopic equipment, and standardized tracking 7.
However, the interest for novel standardized real‐time analyses using quantitative phase imaging (QPI) for cell tracking is growing (Fig. 2).

(A) The wound healing assay, or scratch assay, is done by making a scratch on a cell monolayer and capturing images at regular intervals by time lapse microscope. The cells that can migrate toward the center of the gap by are monitored over time and quantified. (B) The cells are placed on the upper layer of a cell culture insert with permeable membrane and a solution is placed below the cell permeable membrane. Following an incubation period, the cells that have migrated through the membrane are stained and counted. The membrane is usually coated with some extracellular matrix component (e.g., collagen) which facilitates both adherence and migration. [Color figure can be viewed at wileyonlinelibrary.com]

 The cell tracking data can be displayed in a graph format (left) or directly overlaid on the cell images (right). All data is exported for further analysis (published with permission from Dr Sofia Kamlund). [Color figure can be viewed at wileyonlinelibrary.com]


More than 10 years ago, the digital holographic (DH) microscopy methodology of QPI became known to a larger audience and the technique has since then successfully been applied for label‐free monitoring of live cells undergoing cell division, cell proliferation, cell death, and cell migration over time 8-10. By QPI, studies associated with both cellular thickness and refractive index fluctuations have been performed. The possibilities for utilizing QPI for cancer diagnostics have increased 11. Cellular behavior and phenotypes are other parameters that can be measured 12. For two human osteosarcoma cell types, the motility pattern, but not the proliferative pattern, was different. Early migration studies using DH microscopy showed fibroblast migration 13 and HT‐1080 fibrosarcoma cells embedded in a 3D collagen gel 14. Alm et al. and Persson et al. also presented data on movement of different cell types and cell sizes 15 as well as demonstrated the correlation of cell movement with cell morphology 16.

In the recent article published in this issue of Cytometry Part A by Zhang et al., (page XXX) commercially available DH cytometry platforms equipped with semi‐automated image acquisition, segmentation, and analysis software packages for assessing cell behavior are used and discussed.
The motility data was obtained with QPI/cytometry using “Track cells module” and “Wound healing module,” respectively, and with transwell migration assays determining migration and invasion, respectively.
The different techniques assessing motility were compared. The two melanoma cell lines, WM793 and 1205Lu, used in the study were reported to have different metastatic capacity, resulting in differences in proliferation and motility. The motility measured with the “Track cells module” and “Wound healing module,” using DH cytometry equipment housed in an incubator, produced highly correlated relative motilities, even when compared to the standard transwell migration and invasion assays. Images acquired with DH are from the same position in a cell culture vessel, making it possible to study individual cells. 
This is a strong advantage, since most studies of effects of new drugs on proliferation and migration of cancer cells in culture are performed on a population level.

In a recent study by Kamlund et al., cell images were captured every fifth minute for a total of up to 72 h 17.
For cell movement studies, the semi‐automated algorithm attempts to find each tracked cell in the next frame by selecting the closest cell based on the centroid position.
The advantages of the Track cells and Wound healing modules over the transwell migration and invasion assays in the paper by Zhang et al., included the possibility to use the cells in the experiment for other purposes after completing the measurement. DH microscopy also benefits from the fact that morphology analysis can be performed of each cell.
Indeed, this opens up for almost unlimited possibilities to perform cell morphology analysis using the QPI methodology, since each image is very rich in cellular information. As discussed in a review by El‐Schich et al., automated analysis and machine learning is one approach to handle the large amount of morphological data that can now be generated when analyzing cells with QPI 8.
Only the computer resources and the training speed will set the limitations. 

Artikeln Anette hänvisar till hittas här. Bloggen uppmärksammade den för övrigt i detta inlägg.
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