Introduction

For various biological applications that monitor cell numbers, reproduction, health, fusion, and cytotoxicity, it is necessary to conductnoneMarking cellsdetectionThese applicationsneedUse effective and reliable transmitted light(TL)Imaging and analysis capabilities, which provide precise segmentation to quantify cells and evaluate various cellular responses and morphologies. In addition, the combination of high contrast transmitted light imaging and fluorescent labeling imaging is crucial for many cell-based detectionsanalyzeIt is very important.

MetaXpress 6.5Custom Module EditorTLCell segmentation algorithm is a method of imaging cells using transmitted lightandQuantitative optimization and reliable analysis algorithms. This application manual demonstratesTLThe practicality and accuracy of cell segmentation.Analyze and quantify by comparing the total cell count in transmitted light with the cell nuclear count stained with fluorescenceCHOandHeLaThe algorithm of cells (Figure) 1）.

method

Translate into EnglishCHOandHeLaInoculate the cell diluent onto the black, bottom layertransparent96In the orifice plate, row from the top of the plateStart laying the board (in the top row)per well20，000A cell）. Press each type of cell according to1：2The proportion isDownward movement in the microplatedilute，6A compound hole.cell culture24hourafter, in37°CThe5% CO2 Used in the incubatorHoechst 33342cellNuclear dyes stain cells30minute. then use4%Fix cells with polyformaldehyde and useDPBSWash twice. passImageXpress® MicroHigh connotation imaging system andMetaXpressSoftware images cells. use10x Plan FluorThe objective lens images a field of view and can accurately capture up to1500A cell. Using transmitted light andDAPIFluorescence channel captures images with exposure times of15 msand35 ms.

advantage

use
Transmitting light imagingtarget objectPerform segmentation and counting monitoringUnder transmitted lightcell proliferation

Using multiple analytical methodsevaluateTLThe Effect of Cell Segmentation Algorithm

Understand various cell types and sizesQuantitative tools
Quantitative and analytical tools

image 1：Set up for analyzing transmitted lightshootofHeLaandCHOcellimageCustom modules. makeuseHoechst 33342cellNuclear dyes stain cells. useDAPIChannel andtransmitted light(TL)conductCell imaging,SettingsCustomize modules to analyze and quantify cells based on transmitted light imaging and nuclear fluorescence imaging.

In the custom module editorSettingsTLCell segmentation algorithm

Accurate cell segmentation is crucial for quantifying cells captured through transmitted light.forTLcellsplitSelected cellsalgorithmAcanAccurately segmentingandrecognizeHeLaandCHOcell （image 2）. There are other settings available for selection. cellalgorithmBOr cellsalgorithmCcanrespectivelyUsed for analyzing small or large cells.in the analysisIn the moduleSettings can be madeTo filter the total arealess than150 pM2The cells (Figure) 3）. thisA settingcanwithEnsure to avoid surrounding small dead cellsregard asCounting and analyzing live cells.In addition,Pre-set cellsThe calculation algorithm canwithaccuratequantitativeHeLaandCHOofCell nucleus (Figure) 4）.

combineCell counting under transmitted light and fluorescent nucleus counting

useTLCell segmentation algorithmSettingsThe custom module can accurately segmentHeLaandCHOCells (Figure) 6）.There was no statistically significant difference in the total cell count and cell nucleus count in the transmitted light,indicateTLThe accuracy of cell segmentation algorithm (Figure) 7）. In addition to cell counting,TLCell segmentation algorithms can also generate multipledataTo evaluate cellular responses andcellDifferent characteristics of form (Figure) 5）. For example, it can generate cells and intracellular cellstarget objectTotal and average counts, area, intensity, shape, height、Width and variousotherDifferent readings. useFilter optionsRemoving trapped dead cells can further enhanceoptimizeenhanceimproverefinethisProcess and reducefewThe necessity of using live cells or whole cell staining. In addition, nuclear staining and countingcellcheck

elephant 2. The use of algorithms demonstrates the ability to analyze transmitted light imaging.imageTranslate into EnglishTLCell segmentation algorithm applied to selectedHeLaCell transmission light image.In the custom module editor, you canwithpassSettingsSelect source wavelength and representative cell type pairsTLConfigure the cell segmentation algorithm. cellalgorithmAcanwitheffectivelyIdentify imagesofHeLa

andCHOCells.image3. TL Cell segmentation overlay andFilter settings after filtering150 Overlayed comparative images.Reduce the minimum area2set toMmwithfilter out

死 4. cell.imageuseCell verificationelephantsplitModule forFluorescent cell nucleus imageconductCounting.Quantify cells based on cell nucleus,makeuseHoechst 33342nucleusconductStaining. Optimize the estimation and intensity of minimum and maximum widths that exceed local background parameters for precisionrecognizeallfineNucleus.fine

The nucleus is blue (left image), analysiscoverIt is yellow (as shown in the image on the right).image5. HeLaRepresentative images of cells,Settings Custom module image analysismethod.bluecoverThe representative passedTLCell segmentation algorithm usageTLfiltercoverScreening and maskingDiscovered cells, yellow represents checked by countingelephantThe nucleus discovered by the algorithm. The required measurement items for custom modules can be conducted in the Measurement tabSettings.needThe object to be measured isdefinitionforcanpassTLCell segmentationalgorithmObjects to be obscured, select count checkelephantcoverCover up

Measure eachtarget objectThe characteristics.image6. HeLaandCHOImages of cells, andapplicationTLCustom module for cell segmentationoptimizedImage Analysisresult.DAPIThe nucleus is displayed in blue(Left)，In the imageanalyzeChinesemaskFor yellow(Right)Cells in transmitted lightLight blue mask. Through10x Plan FluorObjective lens inImageXpressMicro

Capture images on the system and use new ones 7. TLThe custom module for configuring cell segmentation algorithms analyzes images.imagebased onTLCell segmentation and countingNumber of objectsModule comparisonHeLa and1CHO2Total cell count of cells.20press000：

Dilute cells in proportion, from each well

，The concentration of individual cells begins.ConclusionMetaXpressCustom Module EditorTLCell segmentation algorithms canwithEffectively quantify and analyze multiple cell types and sizes.ImageXpress®Micro ConfocalThe optimized segmentation function supported by the turntable confocal high content imaging analysis system can analyze fluorophore independent datacellMorphology. Quantify cells using transmitted light imagescanMake researchers' detection capabilities more flexible. thisApplication Instructions