Alexa Fluor 488 / solubility problem
Locked
 
|
Norman Watts |
Alexa Fluor 488 / solubility problem
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
I'm seeking advice on how to dissolve the Alexa Fluor 488 phenylmercury dye from Molecular Probes? I am finding it impossible. I have tried three different solvents: DMSO, DMF, and 100 mM HEPES pH 7.5. I have tried adding from 100-1000 ul of solvent to the vials (which contain 1 mg dye according to the manufacturer). I have vortexed, warmed, and waited while trying with a total of six different vials from two lots. In all cases the majority of the dye remains as solids at the bottom of the vial. In addition, the supernatant does not appear to be dissolved dye. I am unable to remove it with either a spin column, a 3000 MWCO, or even a 10000 MWCO dialysis lasting many days. I have heard that the dye molecules associate by stacking, making for a seemingly high molecular weight. Molecular Probes Tech Support claims ignorance of any problems. Ideally I would like to be able to add 100 ul of solvent to 1 mg of dye (to obtain an ~11 mM solution, which I can then add in 1/10 volume to my sample, to have dye in ~10-fold molar excess over my ~100 uM protein). I would like to keep the solvent (if its DMF or DMSO) below 10% final concentration to minimize denaturation. I have seen reports in the literature where other Alexa 488 series dyes have supposedly been prepared as 10 mg/ml stock solutions in DMF. Norman
__________________________________________________ Norman Watts, Ph. D. National Institutes of Health 50 South Drive, Rm. 1509 Bethesda, MD 20892-8025 Phone: (301) 402-3418 Fax: (301) 480-7629 |
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi, Anybody know which company
produces high-quality Avalanche photodiode (APD)? I am planning to use it
to detect 800nm signal. I prefer high sensitivity, low noise, analog one used
in confocal reflective devices. Thank you for your advice! Best regards, |
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Ciao Is there anyone working with 2PE and mCherry? In case, do you have excitation spectrum, a real one? All the best Alby ---------------------------------------------------- "Water slowly flowed under the sky" (Cesare Pavese) ----------------------------------------------------- Alberto Diaspro, EBSA President-Elect, MicroScoBIO LAMBS-IFOM, Department of Physics, University of Genoa, Via Dodecaneso 33, 16146 Genoa, Italy - fax +39-010314218 - tel +39 0103536426/309; URLs: www.lambs.it ; EBSA is Biophysics in Europe - European Biophysical Societies' Association www.ebsa.org ---------------------------------------------- |
|
|
In reply to this post by Peng Xi-2
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Have a look at hamamatsu? I'm sure they are high quality. http://sales.hamamatsu.com/en/products/solid-state-division/si-photodiode-series/si-apd.php I have no connection with Hamamatsu. Dale Peng Xi wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi, > > Anybody know which company produces high-quality Avalanche > photodiode (APD)? I am planning to use it to detect 800nm signal. I > prefer high sensitivity, low noise, analog one used in confocal > reflective devices. Thank you for your advice! > > > > Best regards, > Peng Xi > Associate Professor > Institute for Laser Medicine and Biophotonics > Shanghai Jiao Tong University > 800 Dongchuan Rd. > Shanghai 200240, China > Tel: (86) 21-3420-4076 > http://biophotonics.sjtu.edu.cn/ > > > |
|
|
Dale Callaham |
Re: Detector question -- APD - a bit more info
|
|
In reply to this post by Peng Xi-2
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I also noticed that Hamamatsu has some applications literature that might be of interest: for APDs: http://sales.hamamatsu.com/assets/applications/SSD/Characteristics_and_use_of_SI_APD.pdf and also other detectors: http://sales.hamamatsu.com/en/support/technical-notes.php Again, I have no connection with Hamamatsu, but thank them for making good technical information available! Dale Peng Xi wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi, > > Anybody know which company produces high-quality Avalanche > photodiode (APD)? I am planning to use it to detect 800nm signal. I > prefer high sensitivity, low noise, analog one used in confocal > reflective devices. Thank you for your advice! > > > > Best regards, > Peng Xi > Associate Professor > Institute for Laser Medicine and Biophotonics > Shanghai Jiao Tong University > 800 Dongchuan Rd. > Shanghai 200240, China > Tel: (86) 21-3420-4076 > http://biophotonics.sjtu.edu.cn/ > > > |
|
|
In reply to this post by Alberto Diaspro
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Alby, check the Svoboda paper: PLOS Biology in 2006 vol. 4 (11) pp. e370 (Rapid redistribution of synaptic PSD-95 in the neocortex in vivo.). They used a fixed wavelength 1030 nm laser for mCherry (110 mW in bfp). We tried ~1050 nm on Spectraphysics MaiTai laser, but power was too low (while still getting nice images with 559 nm single photon). An Ultra II should give a bit more power in this range, however, still not much (200 mW at laser head). So either the mentioned laser from Amplitude Systems, or maybe this OPO unit. Michael > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Ciao > Is there anyone working with 2PE and mCherry? In case, do you have > excitation spectrum, a real one? > All the best > Alby > > ---------------------------------------------------- > "Water slowly flowed under the sky" (Cesare Pavese) > ----------------------------------------------------- > Alberto Diaspro, EBSA President-Elect, MicroScoBIO LAMBS-IFOM, > Department of Physics, University of Genoa, Via Dodecaneso 33, 16146 > Genoa, Italy - fax +39-010314218 - tel +39 0103536426/309; URLs: > www.lambs.it > ; > EBSA is Biophysics in Europe - European Biophysical Societies' > Association www.ebsa.org > ---------------------------------------------- |
|
|
In reply to this post by Dale Callaham
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Dale, Yes, I contacted them in the first place, as they have the best PMT (in my opinion). But the APD they recommend me is really not good (C5460) -- 2-3 times weaker signal than the PMT. I think APD should perform better than PMT in 800nm region, and that's why many reflective confocal setup uses APD (well, also APD should be cheaper). Best regards, Peng Xi Associate Professor Institute for Laser Medicine and Biophotonics Shanghai Jiao Tong University 800 Dongchuan Rd. Shanghai 200240, China Tel: (86) 21-3420-4076 http://biophotonics.sjtu.edu.cn/ Dale Callaham wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Have a look at hamamatsu? I'm sure they are high quality. > http://sales.hamamatsu.com/en/products/solid-state-division/si-photodiode-series/si-apd.php > > > I have no connection with Hamamatsu. > > Dale > > Peng Xi wrote: >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Hi, >> >> Anybody know which company produces high-quality Avalanche >> photodiode (APD)? I am planning to use it to detect 800nm signal. I >> prefer high sensitivity, low noise, analog one used in confocal >> reflective devices. Thank you for your advice! >> >> >> >> Best regards, >> Peng Xi >> Associate Professor >> Institute for Laser Medicine and Biophotonics >> Shanghai Jiao Tong University >> 800 Dongchuan Rd. >> Shanghai 200240, China >> Tel: (86) 21-3420-4076 >> http://biophotonics.sjtu.edu.cn/ >> >> >> |
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Professor Xi, I think that the main reason APDs are used in that application is that they are fast detectors, not affected by the potential high ambient light exposure and there are plenty of photons available so sensitivity is less an issue. Below I am pasting in some text from a Hamamatsu paper: http://sales.hamamatsu.com/assets/applications/Combined/lightlevelnoise.pdf I don't know if this is any help. I think that ~0.5A/W is all that silicon is capable of yielding, and then it is a matter of signal, speed, or noise level, or damage by high ambient light. But I have to admit I do not work with this in detail and there may be other issues. Dale The avalanche photodiode (Figure 3) has features of both the photodiode and the photomultiplier tube. It is a solid-state device that generates electron-hole pairs upon exposure to light. A reverse bias of 100 to 1000 V is placed on the PN junction, creating an internal electric field large enough to accelerate the electrons. When the electrons collide with the crystal lattice, they generate electron-hole pairs. This process cascades, amplifying the initial signal by a factor of 50 or more. The statistical fluctuations in the number of collisions and the yield of electron-hole pairs create more noise than the dynodes in a photomultiplier tube but less noise than a photodiode’s external amplifier. Thus, avalanche photodiodes are often used when light levels are too high for photomultiplier tubes but not high enough for photodiodes. Avalanche photodiodes are inadequate for some applications, such as those in which the lifetime of the photon source is short, a measurement must be made rapidly, few photons arrive at the detector or the photons are spread over a large area. These situations will usually require a photomultiplier tube. Peng Xi wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi Dale, Yes, I contacted them in the first place, as they have the > best PMT (in my opinion). But the APD they recommend me is really not > good (C5460) -- 2-3 times weaker signal than the PMT. I think APD > should perform better than PMT in 800nm region, and that's why many > reflective confocal setup uses APD (well, also APD should be > cheaper). > > Best regards, Peng Xi Associate Professor Institute for Laser > Medicine and Biophotonics Shanghai Jiao Tong University 800 Dongchuan > Rd. Shanghai 200240, China Tel: (86) 21-3420-4076 > http://biophotonics.sjtu.edu.cn/ > > > > Dale Callaham wrote: >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Have a look at hamamatsu? I'm sure they are high quality. >> http://sales.hamamatsu.com/en/products/solid-state-division/si-photodiode-series/si-apd.php >> >> >> >> I have no connection with Hamamatsu. >> >> Dale >> >> Peng Xi wrote: >>> Search the CONFOCAL archive at >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>> Hi, >>> >>> Anybody know which company produces high-quality Avalanche >>> photodiode (APD)? I am planning to use it to detect 800nm signal. >>> I prefer high sensitivity, low noise, analog one used in confocal >>> reflective devices. Thank you for your advice! >>> >>> >>> >>> Best regards, Peng Xi Associate Professor Institute for Laser >>> Medicine and Biophotonics Shanghai Jiao Tong University 800 >>> Dongchuan Rd. Shanghai 200240, China Tel: (86) 21-3420-4076 >>> http://biophotonics.sjtu.edu.cn/ >>> >>> >>> |
|
|
In reply to this post by Peng Xi-2
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi all, I confess that I know nothing of the specific APDs in question. However some years ago, I was very interested in APDs in general and, at that time, those that worked in the analog mode, had to operate at low gain (10- 100x) so that they did not run away. In this mode they had very high multipicative noise* and in addition, at this low field strength, the most likely amplification for an individual photoelectron (PE) was zero. (i.e., Although the photon was absorbed and the PE produced, the local field was insufficiently large to make the PE initiate a cascade). All the PE with a gain of zero essentially reduced the effective QE to a level much below that of the silicon itself. If you increased the acceleration field enough that most PE did initiate a cascade, this produced a gain of maybe 10 million (+/- a lot, depending on where in the depletion zone the PE was produced.). However, you then had to quench this process (either passively, by putting a resistor in series with the diode, so that the current flow of the discharge reduced the applied voltage and terminated the discharge, or by using active circuits to do the same thing but with a shorter time-constant.) to prevent the diode from overheating. This meant that you could only operate in a pulse-counting mode. If the maximum throughput of the pulse-counting circuit was 30 MHz, then even counting 3 photons in a 1 microsecond pixel time would lead to a 10% chance of having missed one more pulse because of pulse pileup: a 30% error that would become WORSE as the count-rate increased. Not very useful for normal confocal. At that time, you could only get a high effective QE if you used a high field strength, and is you did this you had to use pulse counting. It would be nice to know away around this problem but I have yet to hear of one. Jim P. *The extreme end of this trend is a single stage of the gain register in an EM-CCD with a gain of 1.01, a situation that produces a multiplicative noise almost exactly as large as the Poisson Noise from the same signal and effectively halves the QE. ********************************************** Prof. James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-265-5315 1117 Johnson Ave., Madison, WI, 53706 [hidden email] 3D Microscopy of Living Cells Course, June 14-26, 2008, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ Applications still being accepted "If it ain't diffraction, it must be statistics." Anon. >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Hi Dale, > Yes, I contacted them in the first place, as they have the best >PMT (in my opinion). But the APD they recommend me is really not >good (C5460) -- 2-3 times weaker signal than the PMT. I think APD >should perform better than PMT in 800nm region, and that's why many >reflective confocal setup uses APD (well, also APD should be >cheaper). > >Best regards, >Peng Xi Associate Professor >Institute for Laser Medicine and Biophotonics >Shanghai Jiao Tong University >800 Dongchuan Rd. >Shanghai 200240, China >Tel: (86) 21-3420-4076 >http://biophotonics.sjtu.edu.cn/ > > > >Dale Callaham wrote: >>Search the CONFOCAL archive at >>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >>Have a look at hamamatsu? I'm sure they are high quality. >>http://sales.hamamatsu.com/en/products/solid-state-division/si-photodiode-series/si-apd.php >> >>I have no connection with Hamamatsu. >> >>Dale >> >>Peng Xi wrote: >>>Search the CONFOCAL archive at >>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>>Hi, >>> >>> Anybody know which company produces high-quality Avalanche >>>photodiode (APD)? I am planning to use it to detect 800nm signal. >>>I prefer high sensitivity, low noise, analog one used in confocal >>>reflective devices. Thank you for your advice! >>> >>> >>>Best regards, >>>Peng Xi >>>Associate Professor >>>Institute for Laser Medicine and Biophotonics >>>Shanghai Jiao Tong University >>>800 Dongchuan Rd. >>>Shanghai 200240, China >>>Tel: (86) 21-3420-4076 >>>http://biophotonics.sjtu.edu.cn/ >>> >>> -- |
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
I am surprised that Leica haven't replied to this thread since (to my
knowledge) they are the only major manufacturer using APD detectors.
I have used their APD-equipped STED system and can comment that
the APD is much more sensitive than PMT detection (in the wavelength
range 650-730nm) but is very sensitive to excess light - with a bright
sample the software can shut down the detector to prevent damage
halfway through collecting an image, which can be annoying. But in
the end you live with this since you need the sensitivity. As I understand
it they are working in photon-counting mode, but I must assume that
it's at a much higher rate than Jim suggests.
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net From: Confocal Microscopy List on behalf of James Pawley Sent: Fri 08/04/04 8:42 AM To: [hidden email] Subject: Re: Detector question -- APD Search the CONFOCAL archive at |
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Guy, Just to make it more precise: You can do APD-imaging also with a Zeiss-confocal if you have the ConfoCor3 FCS/FCCS setup. IT works very nicely and gives you indeed a huge signal increase. Cheers Gabor -- Gabor Csucs Light Microscopy Centre, ETH Zurich Schafmattstrasse 18, HPM F16 CH-8093, Zurich, Switzerland Web: www.lmc.ethz.ch Phone: +41 44 633 6221 Fax: +41 44 632 1298 e-mail: [hidden email] |
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Dear Guy, > >Just to make it more precise: You can do APD-imaging also with a >Zeiss-confocal if you have the ConfoCor3 FCS/FCCS setup. IT works >very nicely and gives you indeed a huge signal increase. > >Cheers Gabor > >-- >Gabor Csucs Light Microscopy Centre, ETH Zurich >Schafmattstrasse 18, HPM F16 CH-8093, Zurich, Switzerland > >Web: www.lmc.ethz.ch >Phone: +41 44 633 6221 >Fax: +41 44 632 1298 >e-mail: [hidden email] I agree that ConFoCOr seems an appropriate use: the signal level is very low indeed. And the contrast is basically the presence or absence of a single fluorophor in the spot. The STED also is noted for low signal levels as most of the excitations are "entrained" by stimulated emission to go away from the detector. On the other hand, even with a 30MHz counter you can conceivably count up to about 20 pulses in a microsecond, before the pileup losses become too obvious to the eye, while a faster circuit or a longer pixel-dwell time allow you to count proportionally more. Cheers, Jim P. -- ********************************************** Prof. James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-265-5315 1117 Johnson Ave., Madison, WI, 53706 [hidden email] 3D Microscopy of Living Cells Course, June 14-26, 2008, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ Applications still being accepted "If it ain't diffraction, it must be statistics." Anon. |
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear all, Does anyone know of a stand-alone multiphase pixel integrator system suitable for custom-built laser scanning scopes? Sometime ago, Cairn Research in the UK was marketing such a system but it seems to have been discontinued. Thanks. Quoc Thang NGUYEN, Ph.D. Assistant Project Scientist Physics Department, UCSD e-mail: [hidden email] Cell Phone: (949) 246-8143 Fax: (858) 534-7697 |
| Free forum by Nabble | Edit this page |