Luminescence - Science topic
Emission of LIGHT when ELECTRONS return to the electronic ground state from an excited state and lose the energy as PHOTONS. It is sometimes called cool light in contrast to INCANDESCENCE. LUMINESCENT MEASUREMENTS take advantage of this type of light emitted from LUMINESCENT AGENTS.
Questions related to Luminescence
The context: The measurement of ATP using firefly luciferase is the most commonly applied method for estimating the number of viable cells in HTS applications. ATP has been widely accepted as a valid marker of viable cells. When cells lose membrane integrity, they lose the ability to synthesize ATP and endogenous ATPases rapidly deplete any remaining ATP from the cytoplasm. Although luciferase has been used to measure ATP for decades (1), recent advances in assay design have resulted in a single reagent addition homogeneous protocol that results in a luminescent signal that glows for hours.
In my case I use the 3M™ Clean-Trace™ ATP Monitoring System for ATP detection of surfaces in a very delicate environment: a hospital. As you can imagine the quality of hygiene in a hospital is a very delicate and important job, as a complement to the daily task of cleaning, the work of hygiene detection is a control to check the efficiency of disinfection, and a good way to ensure that nosocomial diseases will not proliferate in high-risk environments such as ICU, operating room, etc..
BUT, in my company's R&D group teams there are multiple opinions that this ATP detection mechanism does not detect LIVING or VIABLE cells, they think it only detects ORGANIC MATTER, and it does not matter if it is alive or not to contain ATP. In my opinion, only living matter produces ATP, and in dead cells there is a rapid decomposition or hydrolysis of ATP to ADP, which is naturally spontaneous and energetically favored (It is a very exergonic reaction) (2).
The question: Who is right? Does the detection of ATP on surfaces determine the existence of VIABLE CELLS or ORGANIC MATTER (including dead and/or living cells)?
Equipment: 3M™ Clean-Trace™ ATP Monitoring System (3M™ Clean-Trace™ Luminometer LX25) https://www.3m.com/3M/en_US/p/d/v000236906/
1) Hall MP, Gruber MG, Hannah RR, Jennens-Clough ML, Wood KV. 1998. Stabilization of firefly luciferase using directed evolution. In: Bioluminescence and Chemiluminescence—Perspectives for the 21st Century. Roda A, Pazzagli M, Kricka LJ, Stanley PE (eds.), pp. 392–395. John Wiley & Sons, Chichester, UK.
2) Gajewski, E.; Steckler, D.; Goldberg, R. (1986). "Thermodynamics of the hydrolysis of adenosine 5′-triphosphate to adenosine 5′-diphosphate" (PDF).
In case of the chemiluminescence signal representing the quantitated value of the target expression. Does an increase of the exposure time to 64 folds of A to reach a similar signal as B; does this mean that B is 64 folds enriched than A?
I have generated cell proliferation luminescences OD value of breast cancer cells. Now, I am trying to calculate CI value with compusyn software. Can anyone help me to convert OD into effect as compusyn software only takes a value of 0-1 but my OD values are in hundred to thousand range.
I often run many Southern Blots at the same time, but on the last day I am having trouble getting good images because the chemiluminescence imaging machine (FluorChem) begins to overheat and artifact forms over the lens, so I would like to find a last day step (ex. blocking, washing, detection buffer) that can be extended overnight so I can image some membranes the following day. Does anyone have experience letting any of these steps extend? I have seen literature suggesting blocking overnight at 4 degC; does anyone know the upper limit of time that membranes can be left blocking at 4 degC, and if the membrane should be on a Nutator/shaking?
I am currently performing dual-luciferase assays, to assess the impact of two mutations in the promoter of a gene in the expression of Luciferase.
As it is recommended I inserted my sequence, that includes ~ 1kb of the 5' UTR region and a bit of the first exon, in a pGL3 vector. Instead of using the Basic, I used the pGL3-promoter vector, in which I cutted off the SV40 promoter and inserted my sequence. I then inserted my mutations by site-directed mutagenesis.
However, when I read the luminescence, I am having very low Firefly readings, while the Renilla seems to be ok.
I was looking to my sequence and I have no stop codon, and the sequences from the FOXE1 promoter and Luciferase are in frame. But in my promotor sequence I have an ATG start codon before the Luciferase ATG, therefore, the transcription of luciferase may not be starting correctly since I have this "tail" of aminoacids in frame with the Luciferase starting site. Do you think that this might be the reason for the low RLU values that I am having? Or do you think it's something else?
If so, do you have any suggestions to correct this problem?
I am working on my Ph.D., Synthesis of lanthanide complex, i am confused about ligand selection !
Can Anyone Suggest a ligand? which heterocyclic Compound is best for synthesis
Is measuring Bst Polymerase activity (by comparing it with NEB Bst Polymerase) using SYBR and FLUOstar Omega machine (because our RT-PCR machine has been broken and I'm not sure when it will get repaired).
FLUOstar Omega is a microplate reader that have function for incubation with temperature up to 65 degree C (LAMP reaction need 60).
Here is the list of detection modes this thing have:
Fluorescence intensity - including FRET
Luminescence (fl ash and glow) - including BRET
Time-resolved fl uorescence - including TR-FRET
does anyone have experience like this?
any opinion/suggestion would help alot.
Thanks in advance! :D
im trying to compare CHD4 (about 250kDa) protein-expression in different cells. As u see on the picture, there are bands where the marker is and u see also the same bands on every single lane. I tried another protein with the same protocol and it worked fine.
What could cause this problem? Any solutions? Maybe primary AB?
Loading control GAPDH works fine.
Scattering and absorption of bio-tissue could attenuate luminescence intensity. And strong luminescence intensity is the prerequisite for an accurate lifetime computation. So Could the absorption and scattering of bio-tissue make the luminescence lifetime different from the one without traversing tissue.
Please help me to answer this question?
I plan to check TB growth and viability using Bac-titer Glo assay. This is a luminescence assay. Instead of recommended 96 well white walled, clear bottom plates can we use complete white plates?
One of my students asked me today what is the difference between light and chemiluminesce (ALNOUR)? I told her that light can be called chemiluminesce, but chemiluminesce cannot be called light. The light is the last edge of the fire or the coldest area far from the center of the fire, while the chemiluminesce comes from cold and the end is touched by peace. Light can carry heat, but chemiluminesce carries a soft touch, coolness and peace. The chemiluminesce is self-luminous, as is the light of olive oil. As for the heat, it needs a plug and a flammable substance. The chemiluminesce comes from eternity. I ask physicists to talk to me to complete this discussion. Perhaps the word “ALNOUR” has no equivalent in English, but I chose the closest synonyms that come close to the meaning of the word in the Arabic language.
Hello researchers !
Could you please explain me or help me get the references or books about the basics and fundamentals of time decay/ PL life time/ decay time processes of PL downconversion ?
kindly help me , get some references!
Is there any standard mechanism for time decay?
How are increasing and decreasing time decay measured with a fixed excitation for a particular emission related to concentration of activator ions?
please help me understand this!
Thank you all !
Visible to UV Up-conversion luminescence for inorganic phosphors can be achieved only when Laser is used as excitation source but if we want some practical application, Vis to UV up-conversion should be obtained by Xenon discharge lamp excitation. So is there any way to achieve Visible to UV Up-conversion luminescence without using Laser as excitation source?
To investigate the carrier dynamics in solar cells (CdTe) by simulating what free software do you suggest ? Thanks in advance.
Hello, I am using the Multiplex ELISA Kit For Mouse Cytokine Panel 2 (4-Plex) purchased from Boster Bio to measure the four cytokines IL-6, IL-1B, TNF-a, and INF-y. In this plate, all four biomarkers for each cytokine are present in each well.
Boster Bio and its distributor Quansys Biosciences claim that I need an imager, a microplate reader that will actually capture an image of my plate, to be able to analyze the luminescence emitted by each cytokines' antigen.
I currently do not have access to an imager of this sort, and I am wondering if anyone has been able to circumvent this issue and was still able to use a normal microplate reader? Or, does anyone have any idea for how I should go about measuring each cytokines' luminescent response with a normal ELISA reader?
PL of Water Samples were recorded at SAIF-SPIHER by using PerkinElmer LS 45 Photo Luminescence spectrometer.
considering the transmembrane proteins on exosomes (such as CD9, CD63, and CD81) how do you conduct Western Blot for an optimal detection?
- lysis: yes/no
- stripping incubation for serial analyses of membranes: duration and temperature
- re-use of milk+antibody: yes/no (eventually, 4°C / –20°C / –80°C)
- chemiluminescence: best setting?
I am checking the Reduced GSH form on the HT-22 cell line for, 24hours of seeding (0.5x 10^4 cell/ well) /3hours of treatment, by Promega GSH-GLO kit. Whether it was because of HT-22 proliferation rate is fast GSH sample Luminescence range (149109 - 82356) is out from Standard curve (4736 - 84792). Would you help me with how could I fit my GSH sample fits into the Standard curve? Would it be better to decrease the cell seeding range as 0.25 x 10^4 cell /well, or dilute reaction buffer (1:100)?
Thank you so much!
What is the meaning of diffuse and specular reflectance in the case of UV VIS when we are recording in the form of powders (glasses)?
Is it possible to view/record luminescence from luciferase on a standard confocal microscope? Some articles seem to be saying about needing a microscope with a CCD camera, is this the only requirement to view luminescence?
Dear Experts, In order to apply carbon dots (CDs) for the removal of analytes, why do we need to encapsulate these nanodots with other materials? Is it possible to use CDs as a lone adsorbent? Thanks in advance for your valuable comments.
Dear all, I have found white precipitate in my transcription buffer which is expired 6 months ago (Promega ribomax sp6 kit). I used the buffer to synthesis mRNA and run a luciferase assay. Unfortunately, I didn't get any luminescence signals.is it because of my transcription buffer or any other issues?
I am using the Tecan Infinite 200 Pro M Plex multimode reader for luminescence assays. Before beginning the assay, I did a test run using just nuclease free water in the 96-well plate. The average luminescence reading I got was 20. I also did a reading with empty wells (absolutely nothing in the wells) and my readings were ranging from 5 to 3805.
My parameters were:
Plate definition: Tecan 96 Flat White (TEC96fw)
Integration time: 10000 ms
Settle time: 0 ms
Shouldn't the luminescence for nuclease free water as well as empty wells be zero? Is there a way to calibrate the microplate reader for blank reads?
the doped rare-earth（sensitive ion and luminescence ion）under low concentration in host shows abnormal high luminescence efficiency , i suspect that the rare earth ions distribute within linear arrangement to each other. Can i affirm this phenomenon with any simple measuring ways? Thank you
can we justify the continuous decrease in upconversion luminescence with increasing time of UV/laser irradiation without finding the luminescence lifetime (decay curves)?
We use R&D Proteome Profiler Rat Cytokine Array for detection of multiple cytokins in tissue samples. The standard protocol utilizes streptavidin-HRP with a chemiluminescent detection reagent that is not specified in the product documents. Does anyone know, what is the two reagents provided to the kit (chemi reagent 1, chemi reagent 2)?
Have anyone tried another detection system for visualizing (either chemiluminescent or other) the dots?
The lab I am working in has always done chemiluminescent western blots, but we are looking to start incorporating fluorescence in as well. We have the new fluorescent secondary antibodies, but we want to confirm that the same primary antibodies can be used for both protocols, or if we need to purchase any new ones. Thank you!
I am facing difficulty to show separate bands for LC3A/B bands. I find only one band. Here are my procedures; I cultivated THP-1 (monocyte, macrophage both tried) cells with 2% FBS Media 24 hours,. I used M-PER and Cocktail Protease inhibitor to make lysate
1. Lysate (20 ug) Load on 4-12% NuPage Bis-Tris gel with MES buffer. Run 100 V, 90 min
(I also tried Bolt 12% gel at the same condition)
2. Transfer to PDVF membrane (0.2 um) at 35V, 2H
3. Blocked 5% Milk with TBST
4. Blotted LC3A/B, Cell Signaling (1:1000), overnight
5. Washed 3x5min
6. Blotted Secondary Ab (1:2000)
7. Washed 3x5min
8. Developed 2 min with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermofisher)
9. I found always one band.
Can you suggest some journals, published article in 1 or 2 months in Luminescence field. Its just a small project work on dosimeter so I want to publish it. Thank You.
I am doing work on rare-earth doped metal oxide for luminescent, photo detection and optoelectronic devices
I am finding a suitable research papers using which I could vary thickness to study the UCL and DCL. anyone can guide me about it how I can cover this one. Unfortunately, I am not finding suitable research paper.
NaYF4: Yb, Er / Nd @ SiO2 / TiO2 @ Ag/Au
SiO2 / TiO2 is spacer which I want to vary the thickness.
I have had this issue for a while now. When I've completed all my probing and am going to develop my Western Blot using the chemiluminescence solutions from Bio-Rad, I find that after about a minute or two post-treatment, right around the time I'm imaging them, the blots develop brown bands where the probe is. This causes issues both with quantifying the bands present and with reprobing, as the stains appear to be permanently adhered to the membrane. In the attached image the bands are starting to become visible, and will continue to darken until they're a solid brown.
I suspect it's an issue with the chemiluminescence solutions. This has happened with multiple membranes, multiple antibodies and antibody concentrations, and multiple blots. Does anyone have any experience with this issue? Is there anything to be done about this?
How I can vary the thickness of of Spacer SiO2 / TiO2 in lab ? I am preparaing the UCNPs hexagonal phase-NaYF4: Yb3+, Er3+/Nd3+ @ SiO2 / TiO2 @ Au / Ag. I am not finding sutiable way to vary the spacer thickness. kindly guide , suggest easy and suitable way for completion.
What basic information is obtained through the RL signal that is not obtained by using OSL/ TL technique? some literature says, RL helps in identify the presence of impurity but how ???
Is there any relation between the luminescence and optical band gap? I means to say can we explain the change in optical band gap (calculated from tauc plot) by taking cathodoluminescence into consideration.
Need all your suggestion. Kindly tell me.
I have been trying to get the luminescence of my desired cell line which is transfected with luciferase gene and GFP via Varioskan Lux Plate reader. I get a GFP reading but when I use the D-luciferin (1mg/ml in DMSO), working 100ug/ml) on live cells luminescence value is the same as wells which are empty. My queries are:
1. Is it right to dissolve the D-luciferin in DMSO
2. Why there is luminescence in empty wells, is there any way to put it at zero in software or luminescence should is enough high and then subtract the base level lumnisence.
Please suggest the troubleshoot and protocol.
In any OSL phosphor we require optical energy more that the thermal trap depth of that trap for optical stimulation. For example in case of Al2O3:C we require 2.6 eV photon to detrap the electron from the trap having 1.12 eV thermal trap depth. How are they related to each other?
for some quick tests I am looking for nanoscopic sources of circularly or elliptically polarized light, ideally single photon sources. I am aware of publications about specially synthesized twisted fluoresence molecules which emit elliptical polarized light after optical pumping (e.g. Kumar, J.; Nakashima, T.; Kawai, T., Circularly Polarized Luminescence in Chiral Molecules and Supramolecular Assemblies. The Journal of Physical Chemistry Letters 2015, 6, 3445-3452.)
In contrast to time-consuming fabrication, are there also circular/elliptical light sources, which are commercially available (e.g. fluorescent organic molecules, colloidal quantum dots, 2Dmaterials, defects in 2Dmaterials)?
I synthesized CdSe and CsPbBr3 QDs in the same amorphous host. Intriguingly, the center emission wavelength of CdSe QDs redshifts through varying excitation wavelengths in the range of 325-425 nm. Excitation wavelength-dependent emission spectra are attached.
I have 2 cell lines that I want to use for a drug treatment and my readout would be cell death that I want to quantify using the CellTiter-!Glo kit so it would be luminescence. My only problem is that one of the cell lines expresses RFP very strongly (so much so that the cells and even the lysates are very very pink). Do you think that would cause some issues for the reading in terms of background?
I have recently added a technical history of illumination by incandescent metal oxides to my RG page:
Dear Scientists and Scholars,
Luminescent bacteria, (Vibrio harveyi), a gram-negative luminescent bacterium is a major constraint for the successful operation of the black tiger prawn (Penaeus monodon) hatchery.
Does anybody the treatment of this infection during the larval stages in the hatcheries?
Or can anybody please direct me for recent research related to luminescent bacteria?
Thank you in advance.
I have come to choose one of ultrafast gating and highly sensitive ICCD camera from either Andor istar 340 or PI-Max4 to be used in standoff stimulated Raman detection experiment. Anyone who has experience on using one of those, or both would be of great help.
In my experiment, I am going to excite stimulated Raman emission from some compounds at long distance (>100m) using multiples laser beams (all have 10 ns pulse, 10 Hz rate, wavelength around 532 nm). The backscattered Raman signal will be collected and detectes under ambient light, and the noise will be minimized via fast gating operation, synchronized with the laser.
I have read that both type of cameras can do the job, but still wonder which one has superior performance?
Thanks inadvance for your help.
Hello, I have been using Chemidoc XRS+ for Western Blotting the last three months (I am new at this), and sometimes I have got a strange issue. After doing all the steps and placing the PVDF membrane into the Chemidoc, the camera does not reveal anything, just as if nothing had been placed inside the device (picture attached). I make use of the next protocol: After electrophoretic separation of my sample proteins, I tranfer them to a PVDF membrane by using Trans-Blot® Turbo™ Transfer System. Then, I block it for 1 hour at room temperature with PBS-Tween 0,5% and incubate it overnight at 4°C with the primary antibody. Next I do is wash 5 times with PBS-Tween 0,05% (10 minutes each time), incubate for 2 hours with the secondary antibody at room temperature, and wash 5 time again with PBS-Tween 0,05%. Finally, I pour the Western Lightning Plus-ECL, Enhanced Chemiluminescence Substrate (PerkinElmer) and, after 3-5 minutes, I drain it and reveal the membrane with the ChemiDoc system. Does anyone know about this? I have been working with this protoco for the last month, and I hadn't had problems until now. Thank you very much.
Li-doped materials such as Ga2O3 show red luminescence which is proposed to result from the Li doping. One such explanation for an emission is the 1s2 3s -> 1s2 8p transition at about 687 nm. See "Accurate Atomic Transition Probabilities for Hydrogen, Helium, and Lithium" for specific details.
My confusion comes from the 3s and 8p levels. Where does the electron in the 3s level come from prior to excitation? Maybe this is a confusion of the notation on my end.
I am growing multiple lentivirus to test the activity of drugs in blocking their entry into cells. The lentiviruses deliver a plasmid encoding luciferase. I would like to compare drug effect between different lentiviruses, pseudotyped with different envelope proteins. What is the best method to normalize the titers of different virus stocks? I have RT-qPCR to determine genome copy number and I have luminescence read-out after infection. While virus genome copy number gives me the number of genomes in the stock, it does not necessarily correlate with infectivity. On the other hand, different virus pseudotypes will have different entry efficiencies, so even with an equal copy number, baseline luminescence will vary. Is it more legitimate to normalize titer based on genome copy number or luminescent read-out? If two viral envelope proteins utilize two totally different cell receptors, can they even be compared in this way? I ask because the number of available receptors may be a limiting variable for certain viruses but not others.
Please any one can suggest me, that how to calculate the EQE of an OLED from the available data of Luminance (cd/m2) and I-V or J-V curve data of an LED with out using an integrating sphere? I went through few previous conversation, but not clear for me. It will be quite helpful, if any one can help me in this regards.......
According to the well-known mechanisms of luminescence, the fluorescence has a nanosecond lifetimes, while the phosphorescence has a microsecond lifetimes. Is there any examples of transition metal complexes which displays a triplet emission with nanosecond lifetimes of luminescence?
Thank you in advance.
I have encountered some studies doing this through some calculations involving FWHM values of PL bands but I couldn't figure that out. I am calculating radii by using the effective mass approximation. EMA predicts radii through bandgap energy, therefore, the output is unrealistically precise and doesn't have any error function. One of the reviewers especially asked for the size distribution calculation from the FWHM value of the PL band.
Currently doing research on mechanoluminescence materials, and I plan to create a measurement set-up. The aim is to measure luminescence (wavelength and intensity) properties while the material is compressed by a universal testing machine. Is there anyone who's familiar with this? If so, I might also need recommendations on measurement tools (spectrometer etc.) that I should buy for measurement.
Aside from the wavelength and intensity, is there any properties that is important for mechanoluminescence materials?
If it is possible to have two luminescence bands of the same peak position in a spectrum, what is the reason behind formation of two separate bands instead of one? This phenomenon is related to the stimulating incident beam or to the nature of the sample?
I am facing two main issues while using this assay:
- The values for luminescence are super low (0-200) compared to what shown in the protocol (in 2 hours they reach 10,000 or more). I use a wavelength of 485nm for detection, but I also tried several others in the range of 285-585. I treated cells with TNF-alpha but it is probably not good for a positive control.
- I saw an increase of luminescence signal from 0 to 6 hours (10 to 280), but a drop of the values after 24h (down to 8 again).
Does anyone have an explanation or can help somehow? That would be very nice!
As part of my master's thesis, we are gasifying biomass in an entrained flow reactor. We are collecting the light emitted by the flame via a collimating lens and an optical fibre in order to analyse the emission spectrum using a spectrometer. Our aim is to resolve emission peaks in the UV-VIS range as these peaks typically correspond to chemiluminescent emissions of excited radicals such as OH*, CH* or C2*. (See joined spectrum).
I was able to identify some of the transitions as these are quite well referenced in gas flame combustion literature (see table). However, there is close to no literature related to biomass gasification chemiluminescence (which appears to have different emission peaks when compared with typical gas flames).
Therefore, I would like to know if there are any collections referencing a large number of molecular state transitions and their chemiluminescent emission spectra in order for me to identify the remaining peaks. I have been trying to find such collection myself, but have not yet succeded.
Thanks in advance for your help.
At the last step for data analysis, we always use a STOP solution to stop the TMB oxidized by HRP. Why do we have to stop the reaction? Couldn't we measure the color wavelength of chemiluminescence directly? Just like Western Blot data visualization, we just add substrates of HRP to see bands.
I'm doing a bunch of Cell Titre Glo assays to assess the effects of several inhibitors on 2 cell lines. Problem is, for some of the wells they seem to be luminescing less than the background control (media only) wells, some of the time. So I end up with negative normalised values. What would cause this? It's not consistent between replicates or repeats.
The radiocarbon ages (say for the Holocene) is calibrated to Before Present (1950) by using calibration curves and with software such as OXCal. If you use other dating methods such as U series or Luminescence, the age is calculated (Before Sampling Year, lets say 2018). What would be the best way to calibrate these two type of ages, simply add 68 years to 14C age? or calibrate U/Th or Lum ages to 1950?
I am working on the luminescence properties of Nd3+ ions doped phosphate glasses for which I have to study the decay time. I measured the decay time experimentally and also calculated the decay time through JO theory. Now i want to calculated the quantum efficiency (%) which is given by (measured decay time/calculated decay time)*100.
In my case the measured decay time is greater than the calculated decay time this situation is quit confusing. kindly anybody can suggest a solution or literature having same situations.
i have got the following photo luminescence spectra data of AU-PD tio2 film for diffrent depotion rates ,can some one help me to analyse it broadly? can we discuss it ??
In a Z-pinch test using Pulsotron-3 fusion reactor, I have seen at high-speed camera that a pyrex glass generated a beautiful green light during some milliseconds after the electromagnetic pulse was finished.
The magnetic field was over 300 kilotesla in the target that was several centimeters from the pyrex glass.
It can be seen under "Project log" here:
The pyrex glass was broken but I think there was not a high-temperature raise in the glass. What could generate the luminescence?
In our lab, we use Corning 3903 White 96 wells plate to culture cells and use Celltiter-Glo (Promega G7570) to test cell viability, I wonder if we could use normal clear 96 wells plate, such as Corning 3599 to do the same test? I know it's better to use white plate to do the luminescent test, but it is also very burdened, thank you!
I would be grateful to my colleagues for the transfer of samples for the purpose of comparison with natural samples in terms of luminescent properties.