Fluorescence - Science topic

This paper is a good starting point. See section 3.3.

There will be some level of autofluorescence from blank cells without eGFP. This is to be deducted from your samples.

Yes sure

Thank you so much Devin camenares

Hi, the dark probe should also get hydrolyzed, just like a normal probe.

For whom it may be of interest, here is a very helpful answer from Krunalkumar Shah:

There are several software options available for analyzing fluorescent imaging data apart from ImageJ. Here are a few popular ones: 1. **FIJI/ImageJ**: Although you mentioned excluding it, ImageJ's extended version FIJI is worth considering due to its wide user base and extensive plugin library specifically designed for image analysis. 2. **CellProfiler**: This is a free, open-source software designed for high-throughput image analysis. It's particularly useful for tasks like cell counting, object identification, and intensity measurements. 3. **Imaris**: Imaris is a powerful software suite for visualizing, analyzing, and interpreting 3D and 4D microscopy data. It's commonly used for tasks like colocalization analysis, tracking objects over time, and quantifying fluorescence intensity. 4. **Volocity**: Volocity is another software package designed for 3D and 4D image analysis. It offers features for colocalization analysis, object tracking, and measurement of cellular dynamics. 5. **MetaMorph**: MetaMorph is a versatile software platform that supports a wide range of microscopy applications, including fluorescence imaging. It provides tools for image analysis, object tracking, and time-lapse analysis. 6. **CellProfiler Analyst**: This is an extension of CellProfiler designed specifically for machine learning-based analysis of large image datasets. It's useful for tasks like classification, clustering, and data exploration. 7. **Huygens Software**: Huygens is known for its advanced deconvolution algorithms, making it suitable for improving image quality in fluorescence microscopy. It also offers tools for image analysis and visualization. Each software has its strengths and may be better suited to specific types of analysis or workflows. It's often helpful to try out a few options to see which one fits your needs best. Many of these software packages offer free trials or open-source versions, so you can explore them before making a decision.

May consider using Permai fluorescence dye.

May consider using Permai fluorescence dye.

Hi Jhilik Dey

Here's how to measure mean fluorescence intensity in a few simple steps, assuming you're using Fiji (ImageJ) and analyzing a confocal microscopy image:

Hope it helps,

Thanks,

Dear Maryame,

There are plenty of inorganic luminescent materials, which could be used to make enamel of porcelain with fluorescent properties. You can check my web-homepage under PISA, where you will find many datasheets of inorganic luminophores. However, the enamel melt is highly alkaline in character and you can only choose those materials, whish are able to withstand these conditions, e.g. aluminates. Borates and silicates will likely dissolve in the molten glass.

All the best for your research, Thomas

May consider using Permai fluorescence dye.

I would suggest using mouse anti-CD3 and CD19 labeled with fluorescence (FL1) for B and T cells, while using a anti-human Fc labeled with a different FL2 to evaluate the pre-exist anti-lymphocyte antibody. It will be a consecutive gating of first gate is SSC/FSC, second gate is FL1/FL2, while double positive cells are what you want.

Best

Conjugating antibodies to DyLight-NHS ester and carboxylated fluorescent Polystyrene beads involves several key steps:

Hi Anna. Did you find a suitable antibody that works ? If so would you mind sharing which one ?

Thanks !

It is interesting to learn that you can buy fluorescent microplastics. Do they differ from the fluorescent latexes that have been used for years as nano sensors and to study endocytose and phagocytose ? Did you find bibliographic evidence of ROS production in cell against these latexes ,

There is a whole chapter on effects of solvents on florescence emission spectra in Joseph Lakowicz's textbook "Principles of Fluorescence Spectroscopy" (Chapter 7 in the 1st edition). He divides solvent effects into general and specific types. The following is an excerpt from p. 189.

"By general solvent effects we mean those which result from the refractive index (n) and dielectric constant (epsilon)....Specific solvent effects refer to specific chemical interactions between the fluorophore and the solvent molecule, such as hydrogen bonding and complexation."

The rest of the chapter goes into details.

If you are dealing with cells Glass bottom 96 well plate for microscopy is your best fried such as those: https://www.cellvis.com/_96-well-glass-bottom-plates_/products_by_category.php?cat_id=11

You can culture, fix, incubate and wash in the same plate and use PBS instead of mounting media.

If your specimens are actual tissue sections you can keep them wet with PBS using a pap pen but I'm not sure it would be significantly less labor than cover slipping the sections.

May consider using Permai fluorescence dye.

May consider using Permai fluorescence dye.

Hi Shubham,

Is it possible to tell me finally from which company you bought nanoplastics?

May consider using Permai fluorescence dye.

May consider using Permai fluorescence dye.

ICT species can form in nonpolar solvents if the molecule has a suitable donor-acceptor structure, even though they might be more stabilized in polar solvents. The formation of ICT states is mainly determined by the molecule's intrinsic properties rather than the solvent polarity.

DAPI will stain them and make them visible by fluorescence microscopy

Rhodamine B is the most commonly used standard for QY. Whether the standard solution is appropriate for your sample depends on PL spectra and spectrometer calibration. Ideally, the standard should match the spectrum of the sample of interest as closely as possible to minimize spectrometer calibration errors. On the other hand, to minimize QY variations due to handling procedures you can cross-calibrate with another standard solution.

Check the following reference for a detailed discussion about QY standards and the attached file for a practical guide on relative QY measurements using a standard solution.

May consider using Permai fluorescence dye.

I have had success in some cases using polarization/anisotropy to measure binding of Bodipy-labeled ligands to proteins. Other fluorescent dyes can also be useful, such as tetramethylcarboxyrhodamine (TAMRA).

Attaching a large fluorescent dye like Bodipy can alter the ability of the ligand to bind, or its affinity. You should also try other fluorescent probes, if they are available.

Finally, I suggest you analyze the purity of your supply of Bodipy-GDP. Commercially supplied probes are not always of the highest quality. You may be able to find a TLC or HPLC method in the literature. It may be possible to repurify the supply using some sort of chromatography. I did this with Bodipy-penicillin using a column of Sephadex LH-20.

Thank you very much . really grateful to you.

Dear Anjana,

First of all you need the corrected emission spectrum (power in W/nm or counts per second over wavelength in nm) of your sample from 380 to 780 nm typically in 1 or 5 nm steps. Then you can use the TI or other color calculators, while of them you can find under the following website:

All the best,

Thomas

Dear Jiaqi Chen Not sure what your point is but in general fluorescent probes are insensitive to metal ions (at high concentrations ionic strength sometimes might be).

As a matter of fact there are fluorescent probes specifically designed to monitor (certain) metal ions:

Best regards.

hi mate, this link may help you and may arise from high template concentration just dilute samples 10x - 100x and check again

Is there an effect of the dye on the meniscus shape? Buffer alone will have a flat meniscus in a plastic plate. If the dye causes the meniscus to have a concave shape, it could affect the measurement of the Raman fluorescence. Detergent will equalize the meniscus, so that all wells will have the same curvature.

I am not aware of a fluorescence enhancement constant, but I have been in situations where fluorescence enhancement had to be quantified in some way. One approach is to identify the concentration of the substance that produces a certain proportional increase, such as 2-fold or 3-fold. Another approach is to measure the full scope of concentration-dependent fluorescence enhancement, and then to calculate the concentration of the substance than produces 50% of the full change.

The second approach may be more complicated than the first, because each substance tested may enhance the fluorescence by a different amount, or it may not be possible to get to the maximum fluorescence due to insolubility of the substance or interference by the substance with the measurement.

Please check whether substance in question is itself fluorescent by repeating the titration without the DNA. If it is fluorescent, then you have to subtract the background fluorescence from the substance alone at each concentration.

I think this machine tries to do too much, and that eats into cost and practicality...

Personally, the flexibility of having a PC operate the machine is a much better option rather than having an integrated GUI. Sure, you may need a bit more bench real estate for a small PC workstation, but I'd like to highlight a few points on how its better and how Thermo may be losing sight of what costumers need and benefit from. First, configuring an imager to a network or transferring large files via USB is an unnecessary, and possibly tedious step. Relying on a USB stick is likely to be what most occasional users might do and this also increases the chances of cross-contamination with potentially harmful reagents. Secondly, from my personal experience of using the highest-end model (iBright FL1500 Imaging System), I immediately noticed how slow and clunky the interface is. Why charge extra premiums by adding all the extra peripherals to run the imaging and analysis software when it only eats into the cost of the device and the hardware is sluggish? It ultimately makes for a very, VERY unpleasant operating experience. Removing the onboard interface would reduce the price by a large fraction and just a small portion of those savings can afford the user a PC that's going to perform better in every way.

Emptying the stored data to make room for new data? Tedious extra step. Processing units and digital peripherals need a firmware or software update or repair costing you thousands in tech support from Thermo? A tedious (and costly) extra step. Having to wait for a colleague to not only image their gel but also analyze results on the touchscreen? Tedious and time-consuming extra steps; I don't know why anyone would want to stand in front of a machine and play with parameters (that take literal tens of seconds to load) after imaging when it makes a lot more sense to do it at a desk with a faster, more powerful computer... Why Thermo encourages its equipment users to do this and thinks it's convenient is beyond me, now you've got buffer spots all over the touchscreen! Alternatively, a computer hooked up to the machine which only provides the essential hardware to operate is much easier. Run out of space? No problem, swap out a drive, or the whole computer, I dont care and nobody else does, but that's highly unlikely because affordable PCs routinely come equipped with terabytes of storage which would likely last a busy lab years (in comparison to the 256 GB of storage onboard the highest-end model).

Now to the worst part of the entire machine: the automated drawer... When I have a busy day at the lab, the last thing I want to do is wait for a sluggish interface to decide to initiate the drawer opening function when my brain and hand can execute the same task efficiently (I don't actually know if my brain and hand are more efficient, you'd have to run some serious metabolic calculations and compare that to the wattage of the thermo system), oh and it also takes time to initiate the drawer closing function. I just wish we could have a company that realizes that all we want is the same hardware functionality without all the silly bells and whistles that 90% of users won't touch or appreciate and that ruin science for everyone and steal tax money from everyone. Dont buy it

How do you grow them? Is it a dish with agarized solid medium and the separate colonies growing on it? Generally, I would suggest either oxygenation issues (RFP maturation requires strongly aerobic conditions) or contamination of the stock culture. In the latter case, your colonies may have mixed genotype with a pronounced domination of the non-fluorescent phenotype

Fluorescence alone will probably not be sufficient for your analysis because contaminant mixtures will lead to overlapping, indifferentiable results, but in combination with a chemical separation method, e.g. HPLC, you will indeed have a powerful tool at hand:

A distance of 20 bp in DNA (I assume) has a length of 68 Angstrom. This should be close enough for a significant extent of FRET quenching by BHQ-1.

The behavior of FAM in this system could be surprising. It is likely to be at least partially quenched already due to interactions with the DNA, regardless of the BHQ. If the oligo is denatured, its fluorescence intensity could increase. In other words, for certain uses, the BHQ may be unnecessary.

Fluorescent photochroms have been studied in Japan. But I wonder if some are commercially available.

You can buy a premixed solution of PK and LDH in glycerol solution from Sigma (catalog number P0294). I've had success with this reagent in the past.

I think this will be very challenging because there will be a high background of fluorescence from other molecules with similar fluorescence spectra, including most proteins, due to their containing tryptophan residues.

Hi Hoàng Minh, radiative and non-radiative decay rates can be easily obtained using the MOMAP program (http://www.momap.net.cn). Perhaps it would be useful for you to take a look at our recent article: https://doi.org/10.1039/D3CC02534A.

Dear Albrakati,

I would say any image you acquire should store a value in each pixel (eg: from 0-255 for 8-bit images).

In FIJI, you can open these images and select an ROI (region of interest - a rectangle, polygon, or circle where you want to measure).

Then you press M (measure - Menu > Analyze > Measure). This should pop up a Results table in another window.

PS: you can select your desired measurement parameters in Menu > Analyze > Set Measurements...

Cheers from Portugal,

Vítor Yang

I used THP-1 cell line before but I recommend you to doing a time dependent experiment and you can adjust the proper time for your experiment setting.

Visnu Chowdhury you may opt for emission anisotropy changes, steady state as well as time resolved. This can give you an insight into conformational changes, if occurs

The green signal outside the mCherry-expressing cells is most probably from flavins. Its intensity looks a bit higher than I would expect to see considering similar power densities for all fluorescence channels. But we do not know exactly what laser sources were used. If the green laser was an Andor HLE (2 W), then its power density at "50 ms/20%" would be more than 10-fold higher than that from an ILE 637 nm laser.

Concerning the green signal co-localized with the red one from mCherry, I am pretty unsure that it could be attributed to flavins. I'd rather suppose that you observed fluorescence of the mCherry proteins with incomplete chromophore maturation. It is a widespread phenomenon among RFPs (see, for instance, here 10.1007/s43630-021-00060-8) to show some minor green and/or blue populations. Moreover, fixation procedures can likely chemically modify the red chromophore and thus lead to appearance of green fluorescence.

could the plasmid you used to do the recombination be a mixture of wt and recombinant ? (maybe the deletion of Yfp and your GOI happened during the propagation of this plasmid?) did you look whether you have a repeated sequence on each side of the insert (and also in between the yfp and the GOI) DNA polymerase could jump from one repeat to the other during replication.... maybe you could sequence the PCR fragments you got to understand what happened...

Hello, here are some video tutorials for ImageJ analysis https://www.youtube.com/@nrttaye4033/videos

histological image analysis https://www.youtube.com/watch?v=NxNEf1XSPTk

Hello Mohanned Alanber

To study fluorescence and phosphorescence in Gaussian software calculations, you would typically consider the following parameters and steps:

Optimization of Ground State Geometry: Start by optimizing the molecular geometry in the ground electronic state (S0 state).

Excited State Calculations: Once the ground state is optimized, perform excited state calculations using time-dependent density functional theory (TD-DFT) or configuration interaction (CI) methods. This will give you information about the excited states (S1, S2, etc.).

Optimization of Excited State Geometry: Next, optimize the geometry of the first excited singlet state (S1) to find the equilibrium structure in that state, which is important for fluorescence.

Fluorescence Energy: Calculate the vertical emission energy (fluorescence) by taking the difference in energy between the optimized S1 state and the transition to the S0 state.

Spin-Orbit Coupling Calculations: To study phosphorescence, include spin-orbit coupling calculations which are necessary because phosphorescence involves a transition between states of different multiplicity (from a triplet excited state to the singlet ground state).

Optimization of Triplet State Geometry: Similar to step 3, but for the lowest triplet state (T1).

Phosphorescence Energy: Compute the phosphorescence energy as the difference between the optimized T1 state and the S0 state.

Rate Constants: Calculate the rate constants for radiative and non-radiative transitions to predict lifetimes of the excited states.

Spectral Properties: Use the optimized geometries and transition energy differences to calculate the absorption, fluorescence, and phosphorescence spectra.

Quantum Yields: If possible, compare the rate constants for various pathways to estimate the quantum yields of fluorescence and phosphorescence.

Non-adiabatic Couplings: May be necessary if you want to consider the possibility of intersystem crossing, which affects the phosphorescence process.

Vibrational Analysis: Perform a vibrational analysis on the ground and excited state geometries to assess potential energy surfaces.

Note that performing these calculations takes time and requires powerful systems. You can use MolQube.com to prepare the infrastructure. They help you prepare the input, perform calculations and analyze the data. Their email address: [email protected]

Hi Wey,

A great way to practice with flow is to use simple stains like Annexin V and PI (measures of early and late apoptosis). The staining protocol is only about 30 minutes as you don't wash out the stain. You can purposely spike in dead cells to make sure you have positive and negative populations. Another suggestion would be to mix two cell lines with different size/morphology so you can practice gating with FSC/SSC.

For beads BD makes great reagents all the way around. I also like ThermoFishers Ultra comp beads (01-3333-41) which would allow you to use the same colors you'll be using in the future.

Best of luck!

There may be two explanations for this:

1) The concentration of Sytox green is too high (5mM diluted 1000 times = 5µM, which is a lot for this type of agent). It is preferable to work between 10-100nM to hope to see differences

2) Sytox green is a reagent which sticks to nucleic acid, it is impermeant to live cells. Therefore, this reagent also marks any cellular debris that is found in media, this will mask the differences between your different conditions. Flow cytometry makes it possible to overcome this signal which comes from cellular debris.

Below , please find a link for a paper describing the quantification of NETs by flow cytometry,

Best regards

I think it would be OK to compare the fluorescence intensities before and after treatment of the same type of cell, but it would be more difficult to compare different cell types because the cell types could differ in important parameters such as size, volume, and internal composition.

Is the mean intensity of a fluorescent signal a reasonable measure for relative quantification? Yes, but you should also show the distributions, since a heterogeneous distribution could lead to misleading results if only the mean were presented.

Fluorescence intensity is measured in arbitrary units, unlike absorbance. RFU stands for relative fluorescence units. That is the unit of fluorescence intensity. You can compare the intensities of samples to each other as long as the measurements are made under identical conditions on the same instrument, preferably at the same time.

The reason fluorescence intensity is measured in arbitrary units is that the amount of light detected depends on numerous factors, including several instrumental ones and sample geometry.

Dear friend Jiurong Li

Hey there! Well, diving into femtosecond transient absorption (TA) for analyzing the luminescent properties of quantum dots is a fantastic choice. I got your back on this one.

So, TA is like a superhero technique for studying ultrafast processes. With quantum dots, especially those snazzy carbon dots, you Jiurong Li can get some seriously cool insights. Here's the deal:

1. **Time-Resolved Info:** TA lets you Jiurong Li track changes happening on super short timescales, down to femtoseconds. This is crucial for understanding how quantum dots transition between different electronic states during luminescence.

2. **Excited State Dynamics:** You Jiurong Li get a front-row seat to the quantum dots' excited state behavior. It helps you Jiurong Li observe how long they hang out in that high-energy state before emitting light.

3. **Quantum Yield:** TA can give you Jiurong Li the lowdown on the quantum yield of your carbon dots. It tells you Jiurong Li the efficiency of light emission, which is pretty crucial if you're into optimizing luminescence.

4. **Size and Surface Effects:** Quantum dots are finicky about their size and surface characteristics. TA can unveil how these factors influence the luminescence, helping you Jiurong Li fine-tune their properties.

Now, for carbon dots specifically, TA is a beast at shedding light (pun intended) on their fluorescence. It can help unravel the intricate dance of electrons in these dots, giving you Jiurong Li a backstage pass to their luminescent show.

In a nutshell, I recommend going deep into the TA rabbit hole. It's like having X-ray vision for the quantum world. Happy researching!

Nivedya Prabhakar , you should first convert channel values to time (ns). Each fluorimeter has its specification of how many nanoseconds each channel corresponds to. Then, find the channel value at which intensity starts increasing (x0). Make it zero (by subtracting the whole channel column from that x0). Then multiply the new shifted channel column by the conversion value (i.e., 0.027 ns / channel). That'll be your time column.

Having that ready, from the plot of counts vs time (ns), an exponential fit (y = A exp (- t / T); the simplest form) is performed, and the time at which the intensity reaches 1/e of the initial value (T), is called the decay's lifetime.

The use of multiple exponential fit (y = A1*exp(-t/T1) + A2*exp(-t/T2) + ...) is often necessary for good fit convergence, especially when your system contains multiple pathways of excited state relaxation, e.g., when you have a heterogeneous molecular system.

The number of exponentials used to fit your experimental decay data should be physically motivated. Knowing when to truncate the number of free parameters to fit can be tricky, but previous knowledge of your system makes it quite feasible.

I recommend you to read Joseph R. Lakowicz's book "Principles of Fluorescence Spectroscopy" (https://doi.org/10.1007/978-0-387-46312-4) if you need more material.

Dear Ajaya Kumar Singh,

Since you are dealing with a material, it is necessary to measure the fluorescence decay kinetics. There is no other option.

If you read Russian, I recommend my book, it is available on RG

Photoprocesses in biomolecules, carbon nanoparticles and polymer solar cells (Full text)

Book

Full-text available

It is a spectrum view florescent dye that uses wavelengths and laser lines emissions i doubt if it can be fixed or if it will be visible in a fixed tissue. I advise u keep the pictures or recorded charts

At low concentrations of fluorophore (nanomolar), the relationship is direct. There are several reasons why the relationship becomes nonlinear and can even become inverse as the concentration increases.

1. Saturation of the detector

2. Inner filter effect (self-absorption of light by the fluorophore)

3. Self-quenching (either dynamic or static)

To find the concentration of an unknown sample by fluorescence requires a standard curve consisting of measurements of the fluorescence intensities of a set of solutions of the fluorophore of known concentrations. The sample and standard measurements must be done in the same instrument and under identical conditions. If the sample of the substance is impure, the result is likely to be inaccurate, because impurities may also be fluorescent or may quench fluorescence.

Cornelius Krasel Thank you very much for your exhaustive answer.

So if I understand you correctly, to assess desensitization it would be sufficient for me to assess the change in cAMP concentration (via FRET) following agonist stimulation (we are talking about adrenergic receptors) and how this varies following subsequent stimulations over time? Whereas if I wanted to assess its trafficking, I could rely on a BRET assay. Am I right?

The effect of NaOH is probably due to alkaline hydrolysis of the bond linking the fluorophore to the rest of the fluorogenic reagent, generating the fluorescent product.

Well… You could. It's certainly more correct than presenting data that you know is different not because of the experimental conditions but due to a huge confounding error. But it’s not great science.

Cells that are more confluent tend to be happier than cells that are sparsely seeded. If you have cells that are 25%, 50%, 75%, and 100% and you treat them with something that they don't like you will find that in the majority of instances the impact on the less confluent cells is more severe. You have two different populations of cells. One is more confluent, there are more cell-cell adhesions, more cytokines and other factors being released into the media, depending on the cell type more ECM is being laid down etc. The other has less of all these things that are known to have an effect on cell behaviour.

So even if you correct for the cell number it doesn't change the fact that you aren't comparing the same population of cells. The best you could hope for is that the effect of the difference in cell number was less than the effect of your treatment and you could argue that the data is probably, maybe, mostly, kind of right. But this is very bad practice since in the worst-case scenario is that the effect of cell number dwarfs any changes you were expecting to see with your treatment groups to such a significant extent you may as well not have treated them at all.

If you're using Caco-2 cells. A cell line which is known to undergo multiple changes after reaching confluence, and you want to do good science. No, you can't. Caco-2 cells are a good example of a cell line where there can be a really big impact from having different numbers of cells. It's never good science to be treating two populations that you suspect to be different as if they are the same. But if you’re looking at Caco-2 cells you're probably looking at a worst-case scenario.

Even if you have to plate out 100 wells of cells to get 10 that are the same, you'll find in the long run you'll be much better off and be forever thankful towards your past self that you did it until it's done right, rather than take shortcuts, and for what? An extra hour or two of free time? You may well end up spending triple, even quintuple that pouring over largely nonsensical data scrounging for some kind of significance. That is a very slippery slope to be going down.

Yes，we use BODIPY C11 for lipid peroxidation, peroxidized lipids under fluorescence microscopy showed significant fluorescence in the FITC channel.

I'm not familiar with the protocol, but my suggestion is to make additions of BODIPY to the wells at time intervals corresponding to the amount of time it takes to analyze the samples, so that all samples are treated with BODIPY for the same length of time.

What promoter are you using? Some promoters can vary in expression depending on compounds in the media eg. sugars or amino acids.

Dear Emily Fr ,

You will know, that mKeima is a fluorescent Protein to measure the intracellular pH. As you can find here:

Excitation should be around 440 nm and you will have to measure two emission wavelengths i.e. 650 nm and 600 nm, but I have never used mKeima. Standard GFP and mCherry filters will not work, since a GFP filter might be able to excite your protein the band pass filter for the emission will not match the emission of the mKeima. And mCherry Filters will not excite your protein although they should have the right emission filter. If you have the possibility to use a long pass filter for the emission you might be able to check the transmission. Or you can try to use the emission filter of the mCherry Filter set in the GFP Filter set instead of the existing GFP Emission Filter, but that might depend on the dichroic filter.

Best wishes

Soenke

I am fixing them in PFA only, but we used a paraffin embedding system to take sections. Post-fixing in PFA we have to dehydrate the tissue and incorporate wax into it. For sectioning, we can't use PFA fixing alone.

Papiya Saha kindly tell how to perform. with references.

One doesn't usually measure the K_d (equilibrium dissociation constant,

(k_-1/k₊₁) of an enzyme-substrate pair, except in special cases or with special techniques. The usual measurement is the Michaelis constant K_m [(k_-1 + k₊₂)/k_-1]. The main reason for this is that the reaction is occurring during the measurement, so the substrate gets used up, hence it's concentration is decreasing all the time.

The special case is if the value of k₊₂ (the rate constant for conversion of the enzyme-substrate complex to enzyme+product) is much lower than k_-1 (the rate constant for dissociation of the enzyme-substrate complex to enzyme+substrate), in which case the K_m is essentially the same as the K_d. You don't usually have sufficient information to make this assumption, unless it's a very slow enzyme reaction.

The special technique is pre-steady-state kinetics. You need special stop-flow or quench-flow apparatus for this because the measurement is made on a millisecond time scale. You also need some optical (absorbance or fluorescence) readout of substrate binding.

To measure K_m kinetically in the usual manner, you must measure the initial rate of the reaction at several substrate concentrations. Plot the product measurements at multiple evenly-spaced time points at each substrate concentration. Draw a straight line starting from the origin and tangent to the earliest time point measurements, including all the data until the reaction starts to slow down. The slope of the tangent line is the initial rate.

Next plot the initial rate on the y-axis versus the substrate concentration on the x-axis. Use a suitable computer program to perform a nonlinear regression to the Michaelis-Menten equation to get the value of K_m and V_max. The substrate concentrations used for the analysis should cover a range below and above the K_m to show the curvature of the plot.

When dealing with high baseline fluorescence in your control condition during the quantification of relative intensity in images of fluorescently tagged Celegans, there are several alternative methods you can explore apart from ImageJ. One option is to utilize specialized image analysis software designed for this purpose, such as CellProfiler or FIJI. These tools offer advanced algorithms and plugins that can help you handle the high baseline fluorescence more effectively.

Another approach is to consider implementing background subtraction techniques or utilizing normalization methods to adjust for the high baseline fluorescence in your control condition. This could involve subtracting a background measurement or normalizing the data to a reference point, enabling you to better quantify relative intensities between your images of interest.

Additionally, exploring advanced image processing techniques like deconvolution or specialized fluorescence correction algorithms might help you address the issue of high baseline fluorescence more precisely.

By experimenting with these alternative methods and tools, you can improve the accuracy and reliability of your relative intensity measurements, even in the presence of high baseline fluorescence in the control condition.

Can be easily done with Violon plot plugin for FlowJo. you would need to have the R installed on the system also.

Hope it helps.

The particular spectrofluorimeter software will contain a correction file. This is simply a list of correction factors for each wavelength. The correction factors are used to correct for the peculiarities of the instrument for detecting fluorescence at each wavelength. Use the correction feature of the software to correct the spectra with this file.

PBS is a a reasonable choice because it lacks amino groups that could react with the dye. However, it is usually recommended to use fresh 0.1 M sodium bicarbonate which, being a bit more basic than PBS, increases the rate of the labeling reaction.

Dear Adriana PerezGrovas-Saltijeral

I would suggest other alternatives to Manders' coefficients for assessing co-localization instead, because they were found to be very sensitive to the background, noises, imaging resolutions and label densities. I compared different methods for evaluating co-localization of fluorescence signals using simulations and real samples quite some years ago, and the results are found in .

Dear Professor/Researcher,

Book Chapter proposal is invited for the edited book titled “Quantum Machine Learning (QML): Platform, Tools & Applications”.

The main goal of this book is to deliberate upon the various aspects of Quantum Machine Learning in distributed systems, cryptography and security by a galaxy of intellectuals from academia, researcher, professional community and industry. While this book would dwell on the foundations of Quantum Machine Learning as a part of transparency, scalability, integrity, security, it will also focus on contemporary topics for Research and Development on QML.

Topic 4. Quantum Error Mitigation(QEM)

4.1 Introduction to quantum errors and noise

4.2 Quantum error mitigation techniques

4.3 Integrating QEM to the QML framework

Topic 5. Quantum Error Correction(QEC)

5.1. Introduction to quantum error correction

5.2 Quantum error correction techniques

5.3 Fault-tolerant quantum computing

Publisher:

Editors

Presence of oxygen quenches the delayed fluorescence just like phosphorescence. Change of fluorescence intensity in the absence/presence of oxygen also indicate delayed fluorescence. You can simply purge your sample with argon/nitrogen during measurement.