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Cell Imaging - Science method
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Hi! I am trying to prepare hydroxyapatite scaffold samples for SEM imaging of cell growth. I have the Karnovsky's fixative kit but the procedure provided in the tech sheet (attached) is not sufficient for my applications. First, does anyone have a standard protocol for this SEM fixation using Karnovsky's fixative kit? Second, do I need to do the post-fix using OsO4 or is there an alternative method to the post-fix mentioned in the tech sheet? Can I do the fixation procedure without it, followed by the graded ethanol dehydration or will it have a negative impact on my sample preparation?
I would really appreciate any help answering this question. Thanks!
I'm newly working with MDA-MB 231 cells. I have sub-cultured cells using 4. 0 mL L-15 media with 10% FBS in a T-25 culture flask. The cells have incubated them incubator at 37°C, with (0 % CO2, recommended company for L-15 media). After the 48 incubations, I checked the cells under the microscope, and the cells were dead. I checked the flask also, and some of the white precipitated parts were attached to the flask. For reference, I have attached the cells Images. This is the 4th time I face this issue and I cannot figure out why. I would appreciate any suggestions/tips on what I might be doing wrong. Thanks in advance!
We have Zoe cell imager from Bio-Rad that we are mostly happy with, but most of the time it refuses to save files to USB sticks unless they are completely empty (complaining that they are full even if they're not). As long as all files are exported in one go it works fine, but adding an extra file in a second export doesn't work.
We have been in contact with Bio-Rad, and they claim nothing is wrong with it, so now I'm just curious if anyone else is having this problem with the Zoe?
I have been trying to take images of my cells using 40X lens. I focus my cells first with the 4X then 20X lenses; but whenever I switch to the 40X I can't see the cells, not even out of focus. All I see is blurred image of the plate. I tried zooming in till the lens almost touched the plate but to no use.
Firstly, I want to say Thank You All to see my questions
I have a fundamental questions to see Fl imaging after DNA transfection.
I did DNA transfection into Neuro2A cell on 6-well. when i see FL image of each cell, FL signal of control seems whole cell body because it is just iRFP protein and FL signal of Target cells seems it is related specific subcellular organelle. (only some spots in the cell)
Before that, I wanted to check transfection efficiency and this is my experimental design.
i) Trasfect cell with DNA plasmid on 6-well (control and target dna plasmid each)
ii) after 1 day, transfer cells on slide glass for confocal imaging
iii) DAPI staining and check (# of transfected cell/# of whole cell) x 100 to check the efficiency
and the questions are
i) do i need to fix the cell to see confocal imaging?
ii) is it okay transfer cell from 6-well to cover glass after cell transfection
If there is better protocol or opinion, Please tell me, it would be very thankful.
Does anyone know a simply way (i.e. using free image softwares) to quantify chromatin condensation? Cells were labeled with DAPI and analyzed by confocal microscopy.
I am using Matlab for image segmentation watershed algorithm has been done successfully , i want to ask how do i further segment each cell image and segment each blood cells and label them in different article i need Matlab complete code
I have been using Fluo-8AM for imaging calcium transients in ATDC5 cells following drug administration. I have had no problem using it in prior experiments and even the first samples with a newly generated stable cell line. Recently, following staining/incubation/washing, my cells no longer keep their normal morphology and begin to ball up , increase their spontaneous signaling, and eventually become unresponsive to the drug I use to induce signaling.
I've tested incubation and media temperature, the use of pluronic f-127 in the calcium staining media, and different dishes (96, 24, and 6-well [MatTek] all glass bottom, some TC some not). The cells are healthy before the staining but the staining process now seems to 'kill' them. I'm quite careful during the handling of the cells during media removal/washing, so I'm not sure if the dye is causing any issues.
Has anyone experienced this issue before? Thanks for any help.
I need some suggestions about differentiation process of U-937 cell line. It would be about seeding concentration of cell, PMA concentration for treatment or resting step ...
If you have, could you send to me cell image for each step...
I usually use PFA 4%. However I must fix cells (Hs 578Bst) with a not hazard-toxic compound. I thought to use air flow comes of biological cape, but the morphology must be affected. Some suggestions are more than welcome.
Thanks to everybody.
I want to analysis bio availability and integration of liposomal drug in cancer cells. How the cell line sample was prepared for raman analysis to obtain single cell images and respective spectrum? And how the VCA or MCA was done?
Has anyone used MitoSOX™ Red mitochondrial superoxide indicator *for live-cell imaging* (M36008) for suspension cells? I want to analyze fluorescence with a plate reader. I am wondering whether washing step in the protocol wont affect my cells - for adherent cells is not a problem but suspension cells must be centrifuged and I don't know how to make it without affecting cells. Does anyone has experience with this analysis with suspension cells? I will be grateful for some tips!
I have problems to set up the experimental conditions to see endosome maturation in Hela cells expressing GFP-Rab5 and Cherry-Rab7. I am using ZEISS LSM 880 with Airyscan for Life Cell Imaging, with objective 63X/1.4 oil. There are many publications with the method published but I still don't manage. My main problems are:
- To many endosomes that move extremely fast. I cannot track the endosomes as they disappear in the Z position or they mix with other endosomes making it difficult to differentiate the ones being tracked. I tried to do Z-stacks but the focus is lost.
- Some bleaching of the cherry tag as I acquire pictures as fast as possible (every 5 sec) because the endosomes move very fast.
- Cells move so the focus is lost with the time as well.
Has somebody conducted a similar experiment? Which microscope have you used? Can you advice how to improve the experimental setting? Alternative mammalian cell line that I could use (easy to do a KO and easy to transfect/transduce)?
Thank you and kind regards,
We recently bought Nikon eclipse Ti2 inverted fluorescent microscope.
It claims that it can get very high-quality images. In bright field it is OK (not superb!) but when switches in phase contrast, the quality of images are not good at all. The technician from Nikon just introduced the image processing capability of the system.
The instruction of the device is also full of explaining unnecessary data and is not user-friendly. Does anyone have any suggestion to get better quality images using this system?
I am taking photos of cells in culture using a FLoid™ Cell Imaging Station. I would like to add a scale bar on ImageJ. I know I need to go to Analyse->set scale, and input the information regarding pixel size in microns. I know the image resolution is 1296 x 964 pixels. But I am unsure of how many microns are per pixel. What am I missing?
I hope you are doing well.
Is there any free soft that I can measure the distance between cells on the time lapse cell image? I have time lapse images of dissemination of cells from cell body. I want to measure how many cells or how far they move from the cell spheroids compare to the control. I am thinking of checking the images (interval would be 10 or 20 min) and track the cells from spheroid. is there any free program that I can do it, let me know please. Thanks in advance
Have a nice weekend.
I would like to know would it be possible to measure the colocalisation between 2 dyes / stains in the IHC stained tissue using ImageJ colocalisation algorithm. I notice that the image J colocaliation algorithm only used for fluorescence stained tissue. We have another option to use colocalisation algorithm by aperio, but it costs a lot.
KIndly advise me for any software that able to measure dual color / colocalisation in IHC stained tissues.
I truely appreciate all the replies and suggestions.
I am planning for an experiment using MitoTracker. I wish to do time lapse study in vitro. I want to know about the stability of the mitotracker dyes. For how long is the fluorescent intensity maintained? Is it possible to add the dye, then perform the experiment and then visualize the cells. The experiment will last approximately 48 hours. If not, can you suggest some alternative for the same?
I usually use blue DAPI to visualize nuclei, but I have seen articles where they report nuclei with a DAPI staining in purple but they do not report the brand or the catalog number and I have not found it. I think it is due to a combination of red and blue channels, someone could guide me
anybody knows how I can get propidiom iodide stainining image by olympus BX53. I couldn't find defined channel for PI?
In SEM, I heard that "The optimum condition for imaging is when the escape volume of the signal concerned equals to the pixel size", what dose that mean?
I need to isolate diatoms from the various other debris, wat are the possible methods? by using inverted microscope, How can we do ?
I wonder if someone used the imageJ to analyze the puncta-like fluorescence intensity using Image J software? I tried to quantify the intensity using regular method, but the S.D. appear too high, the regular CTCF (corrected total cell fluorescence) method might not be proper for the puncta-like staining. Who knows how to quantify it? the picture i attached is the one i hope to quantify. Thank you.
I'd like to image ROS accumulation in cells with a fluo dye and I'm wondering if anyone have ever performed cell lysis to mesure ATP after ROS imaging ? Does the ATP quantitation is still reliable ?
a) Leica DM4B
b) Zeiss Axioimager 2
c) Nicon Eclipse NiE
d) Olympus BX53
How config is the best performance for FISH staining of chromosome?
Could you give me a detailed config of fluorescence microscope or filter?
Which one can I prefer?
Thanks a lot from now..
I am using gfp tagged chloramphenicol marked staph aureus and normal gfp tagged pseudomonas for my antibiotic studies. I find that there are few rod shaped bacterias in the staph aureus cultures while doing confocal imaging. Can the appearance of rod shape give rise to changes in shape of staph aureus? Or could there be a contamination of pseudomonas? Or what are the chances of e.coli engulfing staph aureus and glows green?
How to measure fluorescence intensity of cell membrane vs cytoplam stain of a 2 ch. confocal img where one is cell mem. and other ch is a antibody.
Hello, I am currently working with human Jurkat T cells, fixing in solution and then acquiring images with a super resolution microscopy that we work with but I found that if I fix cells with PFA 2% cells have lots of autofluorescence, in all wavelength. I cannot found in the bibliography of immunofluorescence techniques using this cells any case, anyone had similar results?
I attached a picture of one cells, illuminated with 647 20% of the laser power.
performed dot blot hybridization using DIG labelling but no result even in positive control.
Mitochondria have been stained using mitotracker dye under different experimental conditions. Is it possible to use software or an imageJ plugin to quantify mitochondria? The mitochondria cannot be easily counted by hand given the resolution of the microscope.
I have taken Z-stacks and timelapse for two fluorophores in HeLa cells. I have the full hyperstack. Can anyone inform me, how to assemble it for a video file?
Thanks in advance,
Does anyone have recommendations for breathable plate seals for 96 well plates that are compatible with fluorescent (high content) imaging, in other words are not autofluorescent/ increase light scatter in the well?
I am planning to label dextran with Texas Red and then study the uptake by different cell types.
Anyone have any idea how to conjugate Texas red to dextran and then any clear protocol to study the uptake by using both plate reader(measuring protein) and confocal microscope?....
I'm now needing to image cell cultures. I am thinking about transiently transfecting a fluorescent DNA marker, but I think it will be too sparsely labeled for my purposes. And throwing a dye on would be simpler and would let me know if more rigorous methods were called for.
Does anyone have a reccomendation for good live nuclear staining dyes?
analyzing the histology of ventricles includes measurement of the ventricle diameter in histological images. Thus I am looking for a method or plugin for either ImageJ or Image-Pro Plus to do so.
There is Fischer's vessel diameter plugin for ImageJ(https://imagej.nih.gov/ij/plugins/diameter/index.html) but it depends on a single line selection.
The diameter measurement of Image-Pro Plus measures the Diameters of objects within the polygon selected area of interest. Both systems offer no solution for my problem.
My idea of how it could work is that the software measures the average length of diameters measured at 2° intervals passing through the centroid of the polygon after drawing a line around the ventricle using the polygon selection tool (implemented in both Imagej and Image-Pro).
Does anyone know such a system or a similar solution?
Any answer or comment is highly appreciated.
Thanks in advance!
I have YFP tagged STIM1, transfected in HEK293 cells and imaged in TIRF plane for formation of punctae/clusters upon ER store depletion with thapsigargin. I have been trying to analyze the number of clusters and area, intensity of each cluster using ImageJ.
However, I have uneven illumination and background in the fluorescent images in TIRF plane. And, the clusters are of different sizes. So, I am facing difficulty to subtract background and set a common threshold for both small and big clusters. I tried FFT filter, background correction, local threshold but nothing helped so far.
Has anyone analyzed such clusters? Any help is much appreciated.
PS: I have attached a sample image with clusters.
I'm looking for a dataset of images (>100 images) of cells under bright field microscopy. They can be from any species, but human or mice cells are preferable. I have found a couple of sources such as the cell image library but it does not seem to contain bright field images in the quantities I need.
Also note, I am looking for images of cell cultures in particular where only one type of cell is in the image. As such images of tissue are not suitable for my application.
The reason I am looking for these image is to test some image recognition and classification software.
Thanks in advance.
the bacillus spore was viewed under microscope and I got difference morphology of the spore.
the first picture is pure spore suspension stained with malachite green 0.5%.
2nd picture is transparent cell looks like spore
3rd picture is a group of green color unknown particle.
may i know are they all spore?
Currently with cellSens Standard version1.15 and below,.vsi file can be opened with ImageJ Olympus plugin, but not for version 1.16. Would like to know if there is a compatibility issue which can be the one causing this problem.
I am trying to use JC-1 to stain the oocyte, however when I put the oocyte on the slides and cover the coverslip, the oocyte will be destroyed. I am the frist one doing this in my lab, so did anyone provide the correct way doing oocyte confocal? That will help me a lot.
I am trying to analyse RNA synthesis and obtain pulse-chase upon silencing of my GOI. I would like to obtain RNA content from the cells under different time courses which will be measured via FACS and Immunofluorescence simultaneously.
Any suggestions will be highly appreciated. Thanks in advance.
We had cultured, fixed, and stained cells directly in the wells of a standard 6-well tissue culture plate. However, we are realizing we need to do higher magnification to see some of the staining features, and because our confocal is inverted it would be best to punch out the wells or cut them out in some way so they can be mounted under a coverslip and imaged. Does anyone have a suggestion for ways to do this?
I am working on a project in which I need to count cells stained with Cy3 and Prodynorphin. I am trying to find the best way to automatically count the double-stained cells. Currently, I am using ImageJ to threshold everything within the desired color range and then removing the other colors. However, I think this method may be unreliable, as the color range for the double-stained cells isn't very clear. Does anyone have any suggestions for a better way to do this?
Are there any methods/assays available to detect or quantify riboflavin inside the cells without using fluorescence spectroscopy/microscopy? Since I am using cultured cells, detection in cell lysate can also be considered.
I used Hoechst dye to stain fixed cells for high content object count. I have read that MTT and resazurin induce morphological changes on NIH3T3 cells, and some papers suggest to use GF-AFC reagent instead of MTT. Could that be the reason?
I was working with Mycobacterium Smegmatis. The cells cluster are not spherical in a 2D plot but appears in the form of ROCKET. I am attaching two figures for kind convenience. The figure shows control M Smegmatis cell without any fluorescence!! the pattern of the cells looks weird!! If this is because of autofluorescence then how do we find out and gate for true fluorescence in positive cells.
I work with phospholipids.
It is necessary to quantify phosphatidylserine on the outer and inner surface of the membrane. How to do it? What kind of dyes used?
I know, I used annexin V with flow cytometry. I have no flow cytometry. Which method is suitable for Fluorescent microscope?
I want to do SIM with three different proteins in living cells. Therefore, I need three different fluorescence proteins with high photostability and high brightness? Could anyone suggest a combination (blue, green and red)? Especially I need a good advice for choosing a blue and red FP.
Thanks for your help and your suggestions! Best Andreas
Is there any way to visualize a fluorescent drug loaded inside the nanoparticles? I think confocal microscopy won't help due to the small size of the nanoparticles and TEM won't be useful too.
We want to do super resolution microscopy of stromal vascular cells isolated from adipose tissue.
As these cells are extremely sensitive, we can't culture them on glass microscopic slides (which would be ideal for high resolution microscopy, due to its minimal auto-fluorescence).
Does anyone know a coating procedure for glass slides that would suit cells of the stromal vascular fraction?
Or plastic microscopy slides with a thickness lower than 0.17mm?
I found a lot of fluorescence pictures of primary cilium stained with alpha-tubulin antibody in the literature, but I am not sure if they made by confocal or epifluorescence microscope? Will I see it in epifluorescence mic. or do I need confocal? thanks
how to merge acridine orange and ethidium bromide staining pictures?
I am having difficulty in observing the karyotype of Canna lilies under a compound light microscope. can anyone please suggest the right type of microscope to use in dealing with minute chromosomes?
Histologically stained slide how to choose area for selection of adipocyte?& how to mesure the size?
we would like to grow cells of one cell line in a way that it sticks to the top of cells of another cell line like it is depicted schematically in the attached file. The easiest way would probably be to just add cell on top of a confluent layer, but we would like to avoid that. Instead, we would like to have single cells of one line with cells of the other cell line attached mainly not to the coverslip, but to the top of the first cell line.
Does anybody have a method how to do that?
I am trying to measure Young's modulus on plant cell walls. I am usin atomic force microscopy to do it. I tried fixing my cells with poly-l-lysine and it does attach some cells, except that cells keep moving on one side or slide whenever the cantilever is scanning them. Anyone has an idea about how to fix the whole cell file ad keeping it from moving while scanning?
I am working with stromal cells that have been fixed- I am wondering if it is possible to do hoescht and nile red stains on them like I did with stromal cells in culture
I am working with Quantum dots for some therapeutical applications. As a part of it, I am using versadoc 500 fluorescence imaging instrument for quantum dots imaging studies. As a result, I am getting emission in all the region (blue, green, yellow and red). I want to know that how to filter the particular emission for example: my quantum dots has only red fluorescence. Is there any separate filter to proceed with this or I have to made changes in settings while calibration.
I understand that if you have 3 mice and are generating data from microscopy images, you might want to average the data in such a way that you enter in 3 numbers to Prism for example.
Should you handle in vitro microscopy data in the same way? For example let's say you are interested in the intensity of a fluorescently stained protein on cells. You do the experiment 3 independent times (n = 3). For each experiment you have cells seeded on 4 coverslips. For each coverslip you take images from 5 fields. Within each field you might have 10-15 cells.
How many numbers do you ultimately report (i.e. how many numbers go into Prism). Do you put in every cell you analyze? Every coverslip? Or 3 numbers for each experiment? If you average them, should you take an average of the coverslip and then average the coverslips? Or just average all the cells in the experiment?
I am trying to quantify green (viral protein) and red (apoptotic marker) fluorescence. How do I use Image J to perform this function. FYI, my picture has multiple R/G dots/puncta. I have attached two sample images for your perusal.
So I have a microarray on a glass coverslip (1.5 mm thick). I would like to create incubation chambers on it so I could use one array for several incubation targets. I did some lookup and it seems like there were only those designed for microscope glass slides.
Any ideas are greatly appreciated.
A431 cells observed with an Olympus BX-51, 100x magnification.
I noticed aggregates indicated by arrows, randomly diffuse in the cytoplasm; someone is visible also in the nucleus.
I can not tell if they are aggregates or some kind of contamination.
I am analyzing 3D-reconstructed images taken of embryo vasculature. In these images, I am trying to understand the behavior of endothelial cells; comparing controls with mutants. So far I managed to have the X,Y, Z coordinates of these cells. I am trying to figure out how clumpy they are. And compare that between controls and mutants.
So basically I am trying to analyze the data using these coordinates. I am sure that there some sort of formula or plot that can show me how clumped these cells are.
I am wondering if anyone has a good background with this.
Besides Promega, is furimazine, the substrate for Nano Luciferase, available from other sources? I found Promega sells it in kits, which is expensive.
Does anyone have experience staining B cells for confocal imaging? I am trying to image B cells under confocal for 3 colors. In the past I tried IgM and IgD and the images did not turn out very well. What surface markers/colors do you recommend, and at what antibody concentrations? Also what do you recommend fixing with?
I have a sequence of 150 phase contrast time lapse images. I want to zoom and make a movie keeping single cell in a video. Has anybody tried in imageJ?
I am able to make and save the video of original file and not of zoomed images. Can anybody suggest wayout?
I'm doing an invadopodia assay, and from what I found on the literature, people use x63 magnification of the confocal microscope to find a specific area, image it and use ImageJ to analyse the degradation area over the area covered by cells. But one thing that I found is that x63 when image only cover a very small fraction of the observing field when the software snaps the image. So this makes the quantification quite biased and nonrepresentative in my opinion.
Do you think I can use the smaller magnification (like x40) to take the image to analyse? I asked some people and they said x smaller than 63 doesn't give enough resolution so the quantification will not be accurate. But I would argue that this applied to the whole image so there shouldnt be any change? And also can ImageJ still distinguish the degradation area from the surrounding with lower magnification than 63?
Thank you very much
Attached is a sample picture of Leishmania parasites that are stained with mitotracker. This dye gets accumulated into the mitochondria in a potential dependent manner. So, accumulation of the dye in the mitochondria is a measure of the mitochondrial membrane potential. Each parasite has a single mitochondrion which is pretty big in size compared to other eukaryotes. I have such images for wild type parasites as well as from different mutants and I am interested to see if there is any difference in fluorescence intensity.
Particularly, I would like to use ImageJ for this puropose. However, I am confused about some technical aspects. I would feel great if anybody could explain those to me!
So, my plan is to measure the fluorescence intensity from individual cells and then take the average of them. As the size of the mitochondria may vary cell to cell, how would I maintain the same area of interest across different cells? Is it necessary to maintain the same area or for each cell I will have to draw the line around it's own mitochondrion?
So basically you have a coverslip on a microscope slide and you want to make an unbiased observation of certain representative areas of the coverslip, so that you can later generalise the result to the whole coverslip (similar to random sampling from a population). I know one way to do it is to image 5 photos in an X manner, so 4 images at 4 corners and 1 at the the centre. Data from each image can later be used to generalise to the whole coverslip.
But I'm wondering is there any other method to do this?
Also, sometimes the cells just clump together or distributed unevenly which make this X method a bit inaccurate. Is there any way to troubleshoot this apart from evenly seeding your cells next time?
Thank you very much
What is the simple method to calculate FP value with parallel and perpendicular intensities? Any one using Cytation 3 for FP experiment?
BioSig3D is a 3D cell image segmentation software that was recently developed at Lawrence Berkeley National Lab., and runs using a Virtual Machine. I've got the VM working, but to upload images for analysis, there's a Java based Image Transfer app. My Java Console blocks the app since it's linked to an http rather than an https site, and using the exceptions list doesn't help either. I was wondering whether anyone else here had managed to get the software working.
I need to collect GFP labelled cells using micromanipulator but cannot see capillary tube for cell collection under fluroscent light. Is there anyway to coat the tip of capillary with some dye to make it visible even under fluorescent lamp in order to facilitate collection.
Thanks in Advance!
I am working on in vitro co-culture intestinal models, and when I try to seed Caco-2 and HT29-MTX cells, I observe changes in the cell surface morphology of HT29-MTX cells. They appear like holes on the surface of the cells when observed with Confocal microscopy (image attached). I have tried monocultures already to confirm that the problem is with HT29-MTX cells and not with Caco-2 cells. I have already tested multiple batches of HT29-MTX, and multiple batches of media, but these problems just continue. Thanks in advance for any help and suggestions.