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What is the best fixation and preparation for fluorescence microscopy of GFP tagged bacteria in bone?
I am trying to visualize bacteria transfected with GFP tagged proteins growing in bone tissue. The bone has a high level of auto-fluorescence so I was thinking of demineralizing it, but I am not sure of the effect it would have on the bacteria and GFP. Has anyone had experience with fixing GFP bacteria in tissue?
Unfortunately I have no clue whether demineralizing the bone tissue will help but maybe you could try the following things to enhance the signal from the bacteria:
- get an emission filter for GFP if you don't have one. If you take a bandpass centered at the emission of GFP you should cut off part of the autofluorescence spectrum (hoping it is not overlapping too much)
- illuminating with a 488nm laser (if you are not already doing it) will sharpen the emission. If you are using a lamp, then its spectrum will be broader and you will get undesired signal from everywhere in the sample.
about fixation, I usually use 30 minutes 4% paraformaldehyde at room temperature which has been proven to be one of the best fixation protocols.
Also 2.5% glutaraldehyde or 10% formalin preserve well the cell integrity.
Hope this can help!
Is it possible to perform dielectric studies on powder samples?
i have prepared metal oxide nanoparticles in powder form now i want to take dielectric studies on my samples. whether powder sample is enough or make it any pellet form for this studies also mention name of the technique.
I basically agree with K. Sreenivas. With powder the data you get may be unreliable because of the defects at the interface, and the density of the pellet made of them etcFollowing
What are references that provide comparisons among Advanced WRF (ARW) versions?
The versions I am referring to can be found here: http://www2.mmm.ucar.edu/wrf/users/download/get_source.htmlFollowing
How can we draw or get the centerline of a surface using Solidworks?
I would like to learn drawing or getting centerline of a surface in Solidworks. I search on internet but I found limited information. I found a programs like VMTKLab.There is a section for centerline computing.
Ex: http://www.vmtk.org/index.html - computing centerlines.
Probably there is a method to get by using Solidworks.I will work on 3D modelled human leg, I want to get centerline of it to use in analysis as reference. Any ideas? Thanks.
I would like to thank all for your answers.
I would like to have straight line in 3 dimension of a section. Actually the section that I mentioned has been builded by using 2 surfaces.
The model has been downloaded from Grabcad.com (Link has been attached).
I'm trying to get 3d straight centerline of "Pravaia Golen" section.
Is possible to follow Zebrafish develop in diluted E3 medium?
I'm trying to see the development of Zebrafish embryos (4hpf-96hpf) in dilutions of E3 medium in deionized water: 2%, 4%, 6%, 8% and 10% of E3.
I put the 5 embryos in well in a 24 well plate and do static system (did not change the medium during all the time).
All the embryos in the dilutions have normal development until 72hpf after this time they have malformations, pericardial edema, slow heartbeat and they died in 96hpf.
I do some wells as control with 100% E3 medium and the embryos were fine in these wells.
I really do not understand what is happening. If someone can help me with anything about this I would be very grateful!
Thanks for the answers, Namita and Jill.
I'm doing dilutions because I want to expose the embryo to nanoparticles. When I put the nanoparticles in the E3 medium they formed aggregates and precipitated in a few hours. So I need a medium with low ionic strength to prevent ou minimize the aggregation, that why I'm trying the dilutions.
I did some tests with the NPs and 10% is the highest concentration that didn't formed visibles aggregates.
I'm working with embryos dechorinated after 24hpf and no dechorinated.
And all my embryos, controls or not, are from the same clutch.
If I change the medium (E3 10%) everyday, do you think that the embryos have the correct development? Or if I put less embryos per well? Or do you have some other suggestions to help me?
Thanks very much!
What is the minimum no. of images needed to perform Fourier analysis?
Can anyone please help me to know that how many min no. of are required to perform the fourier analysis on time series data?
You can perform Fourier Analysis on a single image. The key is what you want to do to the transformed image in the Fourier Plane. Sometimes it is easier to filter images in the Fourier plane by removing unwanted artefacts and keeping important features.Following
Which membrane does work better to remove quaternary ammonium compounds from an aqueous solution?
I would like to purify an aqueous solution from a quaternary ammonium compound using a membrane. Which is the most suitable one? Could someone recommend also a good company to buy it from? Thanks a lot.
Try Oasis® WCX (Weak mixed-mode Cation eXchange) filter systemsFollowing
Is anyone offer me the program of using Arduino to log the data from PX26-0.005 DV ?
Is anyone offer me the program of using Arduino to log the data from PX26-0.005 DV ？ thanks
There is no "program" for using the PX26. Please read:
for logging the data you can use either the serial output or an SD Card - also described in the Arduino homepage.Following
Could anyone help me ID this species?
Location: Terengganu, Malaysia
It´s a species of Hypomeces, probably Hypomeces squamosusFollowing
Can everyone help me to find a data base to measure diffusion coefficient for elements?
such as this elements is Mn NI and Ti in a dense dislocation or perfect structure of BCC or FCC phases that depended to temperature.Following
Hod do I determine the IC50 for an apoptosis inducing drug?
currently I am trying to determine the IC50 of an apoptosis-inducing drug.
The method we used was a MTT-assay. We treated 4 different breast carcinoma celllines with different concentrations of the drug. And now I have over and over problems with the calculation of the IC50. It is very frustrating.
I tried linear regression (failed of course, because data isn't in a linear shape). Next step was to perform regression via polynom 2. grade, which worked pretty fine I think. But there are the CI missing. Then I tried graph pad prism, but I am very unsure if I am doing it right, so I would like to stick to the probit-analyis.
Here is the main problem that there are some results, where the treated cells have a viability over the control level. Which means that the viability is over 100 %. I know that the probit-analysis can't deal with this. So what can I do?
- Is it "allowed" to simply transform the results ? So over 100 % "equals" 100 %. I can't find any paper for that.
- Should I use another analysis instead of probit?
Thanks a lot in advance.
Exactly :) In our lab. we never use border wells, but only 60 well from 96-well plate to eliminate "edge-effect".
You also can try resazurin reduction test for this experiment - some time it could give better results. Commercial kits (PrestoBlue and AlamarBlue) contain some additives for improvement of stability and speed of investigations, but price very high in comparison to original resazurin (R7017, Sigma).Following
How do I detect and track people in crowded scenes?
Hello every body
I am looking for state-of-the-art methods for detection and tracking multiple individual pedestrians in crowded video scene. Preferably an algorithm able to auto initialize tracking and handling track initiation and termination.Following
How can I draw some beautiful social network mapping efficiently?
Hello, everyone, now I embark on an new research about the relationship on Twitter. However, I was confused how can I map a large amount of links on one map quickly. I have a 250000*250000 0 or 1sparse matrixI which represents the relationship on Twitter. I tried some software like Gephi, but it is too slow to draw the picture on it and it often fail to work. I tried package igraph on R and it runs a whole night and end up with a picture. However, it is very ugly... Can you give me some advice to deal with it with igraph? or can you recommand some powerful software for me? Thank you very much!
Maybe have a try on Mage (although I never tested it with such a huge data set):
Is there a method to decrease the response time(cooling time) of the shape memory alloy wire (Nitinol)/polymer composites ?
As far as I know, the cooling time of the SMA wire (Nitinol)/polymer composite is long ( after they are heated above their transition temperature). I was wondering if there is any method to reduce its cooling time (response time) ?
According to my experience, performance of polymer composite actuators is worse than that of bare wires largely due to the viscoelastic nature and stress relaxation of polymers.Following
Is there any possibility for using home energy management system without wireless communication?
Thanks in advance for your replies.
You could use Power Line Carrier communication. It would depend on how stable is the supply line in your country but for short distances should be no problem for a home monitoring purpose.Following
Can anyone working with RNA extractions for samples containing large amounts of polysaccharides or polyphenols give me some advice?
In particular I would like to know if there are some possible modifications for both protocols, the Takara’s Fruit-mate for RNA Purification kit and the Qiagen for RNA extraction’s kit, and also some information about the concentration and quality that I can expect after use the Takara’s kit.
Thanks Amélie for your answer. We can try that. We are having problems specially with Fruit-mate for RNA Purification kit. We don´t obtain enougth concentration.Following
Y plus and wall functions?
I am grateful that someone explain me about following question and If someone can, please place useful links about this equation.
What difference is between following options in Fluent?
-standard wall functions
-non-equilibrium wall functions
-Enhanced wall treatment
-User-defined wall functions
You can find useful answers for CFD questions/problems in this forum.
How can we best measure the impact of intervention where baseline data are absent?
I want to see the intervention effect of agricultural extension on farmers' livelihoods
I find case studies useful for monitoring change over time if there is no baseline information. Ask the respondents what was your situation before? what changes did you make? what have been the impacts so far? But you have to do this early on in the intervention process so that the first impact change report is akin to a baseline. You can then monitor and measure impacts over time, be they physical, financial, environmental, personal, social, cultural, knowledge based etc,Following
Does anyone have quantitative data proving that learning in outdoor environments is more effective than indoor environments in Early Years Education?
There already exists a wealth of qualitative data suggesting that some children learn more effectively in outdoor environments. Equally, there is physiological evidence that the outdoor environment is better for children than sedentary learning in an indoor setting. Does anyone know of or have comparative data of learning attainment of children indoor and outdoors?
I wrote something about correlation between BMI and outdoor environment. If you think that it can be useful for you, please contact me.Following
LIGHT TO FREQUENCY CONVERTER. Does someone has used it?
Hi, I would like to know if someone has used a light to frequency converter in order to know solar irradiance.
Why? I cannot visual the application. What feqFollowing
Does anyone know literature about well excavated tipi rings?
I'm looking for literature about well excavated tipi rings or other ephemeral dwellings of North American Indians or of Siberian people, preferentially with individually recorded finds.
Not in either location you mention but a tipi type shelter. Page 17 in http://archaeologydataservice.ac.uk/archiveDS/archiveDownload?t=arch-285-1/dissemination/pdf/vol_145/01Butler.pdf
Gives a slightly different perspective.Following
How many annotators for creation of a text classification corpus?
I am searching for a study which examined the number of annotators for creation of a reliable corpus for a text classification task evaluation.
Snow et al  argue that on average 4 non-expert raters are required for annotation tasks but the tasks described are no classification tasks (only the study on affectual data might be considered as classification task). I'm rather searching for a statement for topic-based classifications.
Often, three annotators are used and a majority-voting is done but without real evidence that this a sufficient number...
Thank you very much in advance for your answers!
 Rion Snow, Brendan O'Connor, Daniel Jurafsky, and Andrew Y. Ng. 2008. Cheap and fast---but is it good?: evaluating non-expert annotations for natural language tasks. In Proceedings of the Conference on Empirical Methods in Natural Language Processing (EMNLP '08). Association for Computational Linguistics, Stroudsburg, PA, USA, 254-263.
normally, the number of annotators is not interesting, the most interesting is the correlation between the annotators. if the annotators are so different in their judgements we cannot expect that a machine learning algorithm will do so well.
For example, in SemEval competition, the task of Sentiment Analysis in Twitter or Aspect_based Sentiment Analysis where the annotators are asked to classify a text into three classes. they used Mechanical Turk annotators without a special intrest with the number of annotators.Following
What is the meaning of numerical diffusion?
What is the meaning of numerical diffusion? How to reduce or avoid it?
Numerical diffusion is a difficulty with computer simulations of continua (such as fluids) wherein the simulated medium exhibits a higher diffusivity than the true medium. This phenomenon can be particularly egregious when the system should not be diffusive at all, for example an ideal fluid acquiring some spurious viscosity in a numerical model.Following
Should artists teaching at the university level have to have PhDs in fine art and why do you say this?
University art teaching qualifications
When an artist became an educationist, he should be higher qualification of academic achievement such as Ph.D. degree. in the process of research achievement in academic he is also learn so many academic needs. So It is my perception for academic career an artist must be Ph.D.Following
Different Electromagnetic radiations have different mass ?
different electromagnetic radiations are differentiated on the basis of their energy or frequency of photons.characteristic of Electromagnetic radiations is, all of them travel with a constant speed, that is of light c which is maximum possible velocity in our universe.
Now I have a confusion here ; since E=mc2 and c is a constant whether photon of higher energy is having higher mass in comparison to a lower energy photon..?Following
Can I use ESRI shapefile in ERDAS as training samples for multispectral image supervised classification?
I have the training sample shapefile created in ArcGis. Can I use this ESRI shapefile in ERDAS as training samples for multispectral image supervised classification? If so, what should I do to complete this successfully?
Thanks in advance,
Does death row intend to inflict more pain on the convicted or to give them hope?
Given that some of the convicted remain on death row for many years, the practice may be seen as the one, or the other. I am not sure and invite comments. A penny for your thoughts.
Agree with each and every wordFollowing
Are there some "rules" to validate a rearing technique?
Hello everyone, I’ve just succeed developing a laboratory rearing of a mirid species. I would like to know if there are some “rules” to validate a rearing techniques. That is to say, is it necessary to reach a number of generations, or a special capacity (reproductive capacity,…) ? Does scientist community have a consensus on this subject? And are there some key publications?
Thank in advance
Dear Morguen, IOBC Working group on Mass Rearing and Quality Assurance can give you the answer to your question. Here you are the link to website
Does anyone know about graphene oxide/water simulation?
I recently performed molecular dynamics simulation of mix Graphene oxide in water system using ReaxFF potential. I surprisingly noticed that some hydroxyl groups of graphene oxide were detached and diffused between the water molecules.It make water solution of graphehe oxide basic but it is acidic from experiment. what's going on here?
How did you setup the model (i.e were the initial bond lengths correct etc)? Did you run minimization before starting dynamics?Following