Science topic

Luminescent Measurements - Science topic

Techniques using light resulting from PHYSICAL LUMINESCENCE emitted by LUMINESCENT PROTEINS and LUMINESCENT AGENTS.
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Hey all,
I am doing experiments on cultured cells and need to perform an apoptosis-assay. My supervisor recommended me the Caspase3/7 Glo Assay from Promega and told me to use this (briefly described) protocoll:
- cells were cultured in 6 well lates and lysates in protein lysis Buffer (containing EDTA and phosphatase/proteinase inhibitors) and zentrifuged
-equal amounts of (fresh) cell lysate and Caspase 3/7 reagent would be pipetted in a 96 well plate and incubated for 1-2h
-measure luminescence
As the protocoll provided by the manufacturer is quite diffrent than this (culture cells directly in 96 well plate, measure in cell culture media (living cells) and not in lysate) I wanted to ask if anybody perform this assay as described here or can even recommend me another way to adopt the protocol as I am quite sceptical about this.
Another question would be, if anybody used stored cell lysates (-20 degrees) for this assay as well, as this question arised as well in my discussion with my supervisor. I highly doubt this, but I wanted to ask anyway.
Thank you for your help in advance!
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Just follow the instructions carefully, it is pretty straight forward. Best wishes,
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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)?
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You can check our paper
Synthesis, diffused reflectance and electrical properties of nanocrystalline Fe-doped ZnO via sol–gel calcination technique
Authors
C Aydın, MS Abd El-sadek, Kaibo Zheng, IS Yahia, F Yakuphanoglu
Publication date
2013/6/1
Journal
Optics & Laser Technology
Volume
48
Pages
447-452
Publisher
Elsevier
Description
The nanocrystalline ZnO:Fe semiconductor oxides were successfully synthesized via the sol–gel calcination method. Structural, optical and electrical properties of the investigated samples were characterized by various techniques such as atomic force microscopy (AFM), UV–vis absorption and electrical transport measurements. The optical band gap for undoped ZnO (3.19 eV) decreases (2.75 eV) with increasing Fe-doped ZnO (20%). The temperature dependences of the electrical conductivities of undoped ZnO and Fe-doped ZnO were measured and analyzed by Arrhenius equation. The electrical conductivity of the samples decreases with the increase of Fe doping ratio; hence, the electrical conductivity mechanism is controlled by thermally activated processes. To support the nanostructure of Fe-doped ZnO, AFM micrographs were performed.
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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?
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Dear Michaël,
Thank you. The CCD or CMS camera allows you to measure stress images (see attached paper). In my case, when we measured ML spectra we used a Hamamatsu photonic multichannel analyzer. See for instance:
With best wishes
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I decided to measure luminescence of firefly luciferase in Arabidopsis leaf discs using a Corning 96-well white opaque plate and a CCD (PIXIS 1300B). I chose the white opaque plate to minimize neighbouring well crosstalk.
When taking initial images, areas outside the wells are brighter than the wells themselves, what is causing this increase in background? Could there be fluorescence after exposure to the ambient room light? Why might this be specific to the centre of the plate?
The bottom two rows are blank.
I ended up using a plate reader to measure luminescence, but was initially curious what it would look like underneath a CCD.
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I think it is more likely that the dynamic range of the image is being set by the brightest pixels. Auto-exposure strikes again.
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Hi all,
I have generated two gene expression curves by measuring bioluminescence every 10 mins for 12 hrs. These curves tend to overlap at at some time points, while a difference can be seen at other time points. What is the best way to statistically analyze the curves and differences (if any)?
Thanks,
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Abolfazl Ghoodjani , no you are wrong. It's not that simple.
Note that you checked "Consider each replicate Y value as an individual point" - this is surely NOT correct here. The points within each curve are not "individual", they belong to the same curve, obtained from the same individual/subject/specimen. I don't perfeclty understand the only other option "Only consider the mean value of each point" but I am sure that this is also wrong.
Apart from this, the analysis you proposed compeare slopes of regression lines. I am pretty sure that the curves Kiran was talking about are not just simple straight lines!
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Hi,
I run my experiment with either red or green laser and I use the PPFD meter to measure the intensity before my experiment. Does it make any sense for monochromatic light? What would be the difference if I have 50 micro-mol/m2.s for green laser and 50 micro-mol/m2.s for red laser. Can I consider these two values equally intense or bright?
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Thanks
Christian Conkle
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Hello,
I am using a lentivirus encoding CLuc to transduce target cells. I want to then lyse the cells in the presence of luciferin and measure luminescence. The kit which I had hoped to use is Steady-Glo (Promega). This reagent contains beetle luciferin. Will my CLuc catalyze the conversion of this beetle luciferin? Or do I need a kit which contains Cypridinid luciferin, AKA Vargulin?
Thank you!
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Why did you choose the Cypridina luciferase?
For most purposes, luciferases working with coelenerazine analogs (Gaussia, Nano-Ogluc, Renilla) or with beetle luciferin (beetle luciferases) will be a better and more convenient choice.
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I am using a QuickTiter Retrovirus Quantitation Kit and the protocol says to "read the microplate using a 480/520 nm filter set", but I am unsure as to what this means. Is this suggesting that there should be two excitation wavelengths, or that one wavelength is for background? I am currently using a SpecrtaMax L luminescence microplate reader with Softmax Pro software and I cannot determine the correct settings to use.
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there are filters producing those wavelengths (blue light)
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I need to know the best way to measure Human Chorionic Gonadotropin in culture supernatant and serum. Most of the literature use anelectro chemiluminescence assay. ELISA kits are available from RandD and in vitrogen although published studies utilising these kits are lacking. What has anyone used and if you've used the above methods how does the ECL compare with ELISA ?
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Kou Hayakawa
dear sir i have not mentioned that Insulin, Luteinizing hormone (LH) is present at human chorionic gonadotrophin (hCG). I just mentioned the structure of human chorionic gonadotrophin (hCG) is so similar to that of luteinizing hormone (LH) that mean not present in hCG.
Thank you.
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Hello all,
I would like to know how do the covalency and asymmetry correlate with the measured luminescence lifetime values?
Thank you
Rim
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This is a very interesting question, though difficult to answer simply.
Covalency. When the covalency of the bonds in the first coordination sphere of the RE ions increases (note that is rarely exceeds 5-6%), it means more 4f-orbital mixing with surrounding orbitals and has two consequences. Firstly if energy is transferred from the surroundings of the emitting ion (sensitization process), then this transfer will be more efficient, if the implied mechanism is spin exchange (Dexter mechanism), which is often the case for short distances. Second, the 4f orbitals being less pure, it means that the radiative lifetime will decrease.
Asymmetry. What do you mean? Probably less symmetric environment. When the symmetry in the surrounding of the emitting ion is lowered then more transitions become allowed and overall the luminescence intensity will increase. In turn, since only wavefunctions with the same irreducible representation can mix, less symmetrical environment also induce more orbital mixing, and again this can affect the radiative lifetime.
However quantitative correlations between the observed lifetime and covalency/symmetry are difficult to unravel since the lifetime reflects the balance between radiative processes and non-radiative ones and the latter depends on several other parameters such as phonon energy and density for instance.
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should be necessary in such rigid matrix? solvent could be CH2Cl2
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Oxygen quenching generally occurs by a Dexter (collisional) mechanism, so degassing isn't necessary to see phosphorescence in a frozen solution. You may have issues using CH2Cl2 as your solvent. It doesn't form a transparent optical glass when frozen, and will scatter light. 2-Methyl THF, Diethyl Ether, 4:1 Ethanol:Methanol, and mixtures of propionitrile and butyronitrile are all common glass forming solvents for 77K measurements
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I have to do some bioluminescence assays, in that regard if anyone has a caspase-3 or any other caspase-luciferase construct, kindly let me know.
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I have already checked on Addgene and other such companies such as SinoBiological but unfortunately caspase-3 luciferase construct is nowhere available.
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Hello everyone,
I have been using KRP as a media for PMNs during ROS assays (luminescence based detection of ROS generation upon infection of PMNs with Strep Pneumo).
I couldn't find in the literature why KRP is the buffer of choice for such experiment.
Does anyone knows why KRP is used instead of let's say HBSS-?
Thank you
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If there's no previous literature why use it.... or can trying with this media contain KRB buffer and compare your results with standard media
probably this will be selective media for Strep. Pneumoniae
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Hello all,
I wish to perform a luciferase luminescent measurement of ATP in E. coli.
I have acquired a kit that requires less than 1 µM of KCl (and other monovalent metal salts). Also divalent metals must be in the ~3 mM range, and EDTA inhibits the reaction.
Normally, I would perform serial dilutions of the cells in PBS, but this raises the concentration of salts too much.
I thought about using Tris-HCl 0.01 M, but the only protocols i found are for cell lysis. I want to lyse the cells but not immediately.
Can i use Tris 0.01 M at 7.4 pH for the serial dilution of my cells without them suffering stress?
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Tuning luminescence of platinum complexes is well established by variation of cyclometalating ligand. The most common trend I find in literature is a red shift in the emission upon introducing more electron rich rings to the ligand (for example replacing phenyl with thiophene). I am observing the exact opposite, in which more electron rich ligands will result in blue shifted emission and was wondering what might be the possible reason ?
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There is no simple rule in tuning the emission of Pt(II) complexes. Every ligand system is different. What one should consider is whether that (electron-rich) group is placed on a moiety acting as HOMO or LUMO. Usually F is added to the phenyl ring of a cyclometalating ligand to increase the emission energy of the metal complex. But the following paper shows that addition of F to a phenyl ring can lead to red shift. The degree to which the emission is affected also depends on the substitution pattern.
Apart from this, usually we take it for granted that extending the conjugation of a ligand will lead to redshift in emission. But there are also exceptions:
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How can I convert lux into PPF (μmol m-2 s-1) for Red and Blue LEDs light?
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(1) The conversion of photo-metric units (lux) into radiometric units and after into quantum units is possible in the case that you know detail spectral characteristic of the light source, or in the case of the of monochromatic light.
In the case of LEDs you can use approximation that LED light is pseudo monochromatic source having wavelength same as dominant wavelength for selected LED and related spectral half width, so you can to simplify integral defining photo-metric quantity.
(2) For related theory of unit conversion you can use Handbook of Optics Chapter "Radiometry and Photometry" or some textbook dealing with Photometry.
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Emission starts from within the excitation volume in the material. Looking from the top, this volume can be much bigger than the spatial extent of the point in the CL map for which the intensity is recorded.
Hence, a bright point (pixel) in CL map does not mean that 'higher emission is generated from that particular point' but rather 'it is from the larger excitation volume associated with that smaller point'. Is it correct?
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In general, it is hardly true. Brightness of a point depends on the local chemical composition and crystallinity in a not straightforward way and the increased penetration depth of the electron beam alone does not necessarily lead to more intensive emission.
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We have spurious signal from empty sphere. We use sphere covered with Spectralon ( G8 or Quanta Phi). It seems that this sphere was contaminated. But we never used it before and it is brand new. I would like to communicate with somebody who do such measurements personally. I have some questions regarding measurement procedure.
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It's a shame. I'm affraid I cannot be of more help. I had the luck of having the calibration files. 
All the best.
José
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Currently i am working on alumina film coating for optical application. I have done my Photo luminescence measurement for deposited alumina film. and i have got some of the intense emission peak at 657nm and second at 824nm. can anyone help to distinguish these emission peaks?
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alumina deposited on What kind of substrate? you can check PL measurement for substrate only without alumina film  (un-coating). alumina is a dielectric material, it has band gap energy Eg=9eV, optical transparent in visible light, Why do you use 441 nm for excitation (low energy)? in your case,  PL peaks may be duce to emission from substrate,  impurity in preparation process or defect centers of film material. Check again!
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I get vacuum UV photoluminescence specktums of rare earth doped inorganic compounds. when I check the literature to determine luminescence transitions, I came across emission spectras and excitation spectras. As an chemist, discrimination of these spectras is very hard study for me. Could someone explain this trouble?  
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Hello Gülşah,
Some inorganic compounds do not emit light after absorption of radiation therefore molecules do not have an excitation spectrum. Usually the absorbed energy can scatter in the molecule without emitting light. Moreover, the molecules absorb light at different excited states. The emission usually occurs from the lowest of these excited states known as S1. During the excitation to a higher state from ground state (S0) it often ends up in the lowest excited state S1 and then emits radiation.  In such case the excitation spectrum is same as the absorption spectrum. Nevertheless from a higher excited state a molecule does not have to undergo to the lowest excited state but directly reaches the ground state in that case it does not emit radiation.
Hope that explains your question.
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I am trying to measure change in cellular calcium ion concentration using aequorin expressing plant. I could find many research article and protocols about the measurement using the same system, so I summarized and followed them. However, the signal seems so weak even when I applied twice as much amount of coelenterazin as previous reports. It required at least 30s exposure for our instrument, therefore, not applicable for the purpose of my study. The 30 seconds exposure still does not show any distinctive difference between non-expressing samples and aequorin expressing samples. Even after discharge with CaCl2 and methanol, the signal doesn’t get any stronger. The instrument is NightShade, and sensitivity of the instrument seems fine, based on other luminescence measurement such as luciferase expressing sample treated with luciferin.
If you have an experience in this experiment, please let me know how to solve the problem.
Thanks in advance.
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The best way to measure intracellular calcium is imaging with indicators like Fura-2, Fluo-3, Fluo-4, etc. The signal to noise ratio is usually quite high.   Another approach is to use genetically encoded calcium indicators (GCAMPs), See: Monitoring activity in neural circuits with genetically encoded indicators. Broussard GJ, Liang R, Tian L. Front Mol Neurosci. 2014;7:97
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I working with Carbon nanodots(c-dots) I would like to measure the Quantum yield for prepared samples and also I am very eager to measure the Two Photon Absorption(TPA). In order to realize the above both measurements i need to know the concentration of C-dots in solutions as well as my C-dots are water soluble. How could I do this? I want the details of this. Thanking you in advance all researchers. 
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Concentration is not necessary for quantum yield determine. If you can get the Integrating Spher for absolute QY analysis. you can direct measure it. If not, you can use a reference dye to calculate relative quantum yield.
If you still needs the concentration, here is my approach.
1.First you needs to purify your sample by dialysis to remove salt, precusor, and byproduct.
2. Analysis the sample with TEM to determine the size of C-dot. and calculate the volume of single particle. 
3.Dry a 100 mL C-dot solution to get the powder of C-dot and weight it before and after drying. (determine the density and the weight of C-dot in 100 mL solution)
4. calculate the density of C-dot using information from step 3.
5. dry weight of c-dot / density of c-dot = total volume of c-dot.
6.  total volume/ single particle volume =number of particle, then you can get the concentration.
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I have faced similar problem in different spectrometers and hear a lot also about it from various researchers. Now I am wondering if any option can lit!
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Adjusting slit widths ,filters we can reduce the undesirable wavelengths.....
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The Horiba DAS6 software in my computer is down. If you have the software in your lab, wound you mind helping me to fit the .das data into first order exponential decay and export the outcome to .txt file.
I attached my .das files in the attachement.
You help is very much appreciated!
Thanks!
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i think those answers are enough
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On Belkin et al (1996) and later studies, kanamycin was omitted due to inhibit protein synthesis as it's mechanism of action but on van Dyk et al (1994) kanamycin was included in luminescence reading.
When ampicillin used instead of kanamycin, no luminescence inhibiton was observed. E. coli bioluminescence contain pUCD615 with luxCDABE, recA/grpE/fabA/katG promoter and kanamycin and ampicillin resistance.
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Kanamycin is a potent antibiotic. Even when bacteria are resistant to it they can show significant growth inhibition due to its impacts.
To see this for yourself, grow colonies of this strain or any kanamycin E. coli strain that is stable (such as the KEIO strains) on kanamycin plates and blank agar plates. The kanamycin colonies will be much smaller after growth overnight. This growth "inhibition" can influence the test results as the bioluminescent output is influenced by the cell growth.
BTW, I am a good friend of Belkin and know van Dyk, and even work with those strains in my lab as well. ^^ (Look at my publications for proof if you desire)
When we do these experiments, I also advise my students to grow the bacteria on KAN/AMP plates to prevent any possible contamination but once we inoculate LB media, only ampicillin is used.
Hope that this helps.
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I have added Ag nano particles in Photo luminescent dye solution in order to increase its florescence emission intensity. But unexpectedly its intensity start decreasing. I have use 15ppm to more then 200ppm for finding optimum value. but in all cases intensity decreases. but according to literature, it should increase. can anybody guide me please.. I thought i may be using high concentration which causes aggregation quenching effect but when use low concentrations, results were still opposite to literature.
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Dear Mateen,
A distance between a fluorophore dye and the metal (gold or silver) nanoparticle determines whether the fluorescence is enhanced due to local electromagnetic field produced by the metal nanoparticle or, alternatively, if the energy is efficiently transferred from the fluorophore to the metal nanoparticle, which would occur fluorescence quenching.
I guess your case shall be the later, which a Quenching was resulted in.
Ref:
Pons, T.; Medintz, I.L.; Sapsford, K.E.; Higashiya, S.; Grimes, A.F.; English, D.S.; Mattoussi, H. On the quenching of semiconductor quantum dot photoluminescence by proximal gold nanoparticles. Nanoletters 2007, 7, 3157–3164.
 Dulkeith, E.; Morteani, A.; Niedereichholz, T.; Klar, T.; Feldmann, J.; Levi, S.; van Veggel, F.; Reinhoudt, D.; Möller, M.; Gittins, D. Fluorescence quenching of dye molecules near gold nanoparticles: Radiative and nonradiative effects. Phys. Rev. Lett. 2002, 89, 203002:1–203002:4.
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I am attempting to obtain the fluorescence emission spectra from nanocrystals that I have prepared.  The spectrophotometric equipment utilizes a fiber optic cable (1 meter long) to collect the emitted light and bring it to the spectrophotometer.  This fiber optic cable is of the UV-VIS variety, however, there is a substantial increase in attenuation due to the fiber at wavelengths less than 400 nm (see attached graph from the manufacturer). 
My primary interest is in emissions in the 250-400 nm range.  Is there a way to correct the spectral data to account for the increased attenuation from the fiber at < 400 nm?    
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The attenuation is due to Rayleigh scattering that strongly increases at shorter wavelengths Lambda-4. The correction can be done by considering the attenuation factor based on Rayleigh scattering over the whole UV range.
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I have recently started a project on Luminescent solar contractor. My design is ready to fabricate but I am facing difficulty in measuring the surface emissions, edge emissions, light trapping efficiency of LSC. Most of the researchers have used an Integrated sphere method for these measurements. Can anyone suggest exact the measurement techniques and procedure. Your kind response is highly appreciated. 
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You need to perform 2 PL QY measurnments in integrating sphere: one with clear edges and another with the same sample but with blocked edges (painted by black absorbing paint) http://dx.doi.org/10.1021/nl501627e. Check also these papers: http://dx.doi.org/10.1038/nphoton.2014.54
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In QE measurement by integrating sphere, the spectral irradiance (W/m2) is transformed in to photon irradiance (cps/m2) by dividing the ordinate data by corresponding photon energy at abscissa. The abscissa is also converted to wavenumber scale prior to such transformation, so that the excitation and emission signals could properly be deconvoluted. In this case, we obtain the photon distribution as a function of photon energy in cm-1. The photon distribution for absorption and emission is obtained by taking the difference with reference scattered signal.
Here my understanding is that the total photon count for absorption or emission is the summation of number of photons at each energy. The value of energy is irrelevant (not sure). I am getting difficulty in obtaining the total number of photons for absorption and emission. In literature, the integration is usually taken to get the area under the curve. Also, the abscissa is mostly wavenumber, but sometimes wavelength. My concern is if we take the integration, it also considers the values at abscissa. I tried to integrate same area curve at different wavenumber range (Origin software), which gives different area values. Please suggest if I am making some mathematical mistakes, and also give opinion on how to obtain total photon count and what should be the unit of abscissa for calculations.
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Dear Atul,
The product of the intensity and wavelength is proportional to the number of photons within a given wavelength interval. Wavelength is the unit for the abscissa which you should use.
For more detailed information look to the literature, e.g.:
W. R. Ware et al., Relative fluorescence quantum yields using an integrating sphere. The quantum yield of 9,10-diphenylanthracene in cyclohexane. Chemical Physics Letters 39 (3), (1976), 449-453.
J. C. de Mello et al., An Improved Experimental Determination of External Photoluminescence Quantum Efficiency. Advanced Materials 9 (3), (1997), 230-232.
N. C. Greenham, I. D. W. Samuel, G. R. Hayes, R. T. Phillips, Y. A. R.
R. Kessener. S. C. Moratti, A. B. Holmes. R. H. Friend, Measurement of absolute photoluminescence quantum efficiencies in conjugated polymers. Chem. Phys. Lett. 241, (1995), 89-96.
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Our institute is in search of a new Phosphoimager.  We currently have a Typhoon that no longer works.  Can anyone give me a recommendation on what works well for them, or is the newest technology out there?   We use it mostly for radioactivity detection, and need it to work over a broad dynamic range.  It seems most systems out there require a 32-bit processor or windows XP, which has given us issues using our old system.  Any input would be greatly appreciated!
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Which Typhoon model do you have (e.g. 2000; 5000; 7000; 9500) and what stopped working?  Was it something that couldn't be repaired?  We have a Fuji BAS-5000 and a GE Typhoon FLA 7000 with the extra lasers.  I have briefly used a Perkin Elmer as well.
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I am trying to establish a Gli reporter gene assay in Shh light 2 cells. For that I am using a dual luciferase reporter assay system. However the luminescence values obtained are only in the hundreds. I tried some modifications as well, but nothing helped.
Your suggestions will be of great help to me.
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Hi Mohamed, I checked the firefly luciferase activity after treatment with Gli inhibitor and activator molecules. However in both the cases I get the luminescence values only in hundreds (~700). The Shh light 2 cells are stably transfected with GLI-responsive Firefly luciferase reporter and a constituitive Renilla-luciferase expression vector. I even tried transfecting other cell line with these plasmid but in that case also the luminescence remains the same. I followed many literature available and made the changes in my protocol (cell number, low serum media, incubation time, etc) but nothing helped.   
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I would like to do circular polarizer-photoluminescence measurements, but I need of a standard sample with a well-known polarization degree. Does anyone know if such a standard sample exists?
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Gianfranco,
In a review on CPL:
Frederick Richardson and James Riehl
Circularly Polarized Luminescence Spectroscopy
Chemical Reviews, Vol. 77, No. 6, pp 773-791
Mentions work done by Shindo and Miura. They used optically active sodium 1,3,5-triphenyl-pyrazolinyl sulfate in methanol at room temperature. The g(lum) factor was found to be constant (~ 4 x 10^-4) across the 390-560 nm emission band.
Unfortunately I lost the last page of my copy of the review that contains the direct reference to Shindo's and Miura's work.
Hopefully that is helpful.
Cheers,
John
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Can you suggest some discussions about this?
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Raja,
Fluorescence is a relaxation process from a singlet excited state to a singlet ground state (requiring no change in electron spin of the demoted electron). Whereas, phosphorescence is a relaxation process from an excited triplet state to the ground singlet state, requiring a change in electron spin during an electronic state transition and thus making it a forbidden process (not strictly forbidden). Hence relaxation from a triplet excited state to ground electronic state will usually require more time than relaxation from a singlet excited state. However, with that stated you can have "delayed-fluorescence" which would be on the same time-scale as phosphorescence.
Because of "excange energy", the lowest lying triplet excited state will be of lower energy than the lowest lying singlet excited state. If we assume Kasha's rule holds (it doesn't always), than the emission resulting from phosphorescence will occur at lower energies than fluorescence.
Also, because lifetimes of triplet excited states are longer lived than singlet excited states, phosphorescence lifetimes will be shortened and phosphorescence intensity will be lowered to a greater extent by collisional quenching than that of fluorescence. So removing oxygen from the sample will have a significant impact on phosphorescence lifetimes and intensity.
Hopefully this is of some help.
John
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.
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There are a few very nice reviews written about excitonic (near band edge) luminescence, defects (hence deep luminescence) and theoretical (ab inito) investigations of the electronic band structure of ZnO. I suggest having a look at:
Meyer et al, physica status solidi (2004): excitons
McCluskey and Jokela, Journal of Applied Physics (2009): defects
Janotti and Van de Walle, Reports on Progress in Physics (2009): theory