LaVision TriM multiphoton microscope
Locked
 
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Phillips, Thomas E. |
LaVision TriM multiphoton microscope
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I have been reading about the TriM multiphoton microscope
from LaVision BioTec (http://www.lavisionbiotec.com/en/microscopy-products/trim-scope/).
They use a combination of mirrors and a beam splitting substrate to divide the
incoming beam into 64 beamlets and this lets them scan with 64 lines at once
thereby cutting the scan time. They also use a combination of CCDs and NDDs as
detectors. Can anybody comment on how effective this system is? Tom Thomas
E. Phillips, Ph.D. Professor
of Biological Sciences Director,
Molecular Cytology Core 2
Tucker Hall Biological
Sciences University
of Missouri Columbia,
MO 65211-7400 573-882-4712
(voice) 573-882-0123
(fax) |
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Andrea Latini-2 |
Re: LaVision TriM multiphoton microscope
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There is a system
developed 2 years ago by Light 4 Tech in 64 beams from a diffractive
lens. It works fine and is very fast. You should see the papers
Prof. Pavone have read at Neuroscience or contact him directly. vincenzo via Mattia Battistini 177 00167 Roma phone +39 06 35402933 fax: +39 06 35402879 From: Confocal
Microscopy List [mailto:[hidden email]] On Behalf Of Phillips, Thomas E. I have been reading about the TriM multiphoton microscope from LaVision
BioTec (http://www.lavisionbiotec.com/en/microscopy-products/trim-scope/). They
use a combination of mirrors and a beam splitting substrate to divide the
incoming beam into 64 beamlets and this lets them scan with 64 lines at once
thereby cutting the scan time. They also use a combination of CCDs and NDDs as
detectors. Can anybody comment on how effective this system is? Tom Thomas E. Phillips, Ph.D. Professor of Biological Sciences Director, Molecular Cytology Core 2 Tucker Hall Biological Sciences 573-882-4712 (voice) 573-882-0123 (fax) Internal Virus Database is out of date. |
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Urs Utzinger |
Re: LaVision TriM multiphoton microscope
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In reply to this post by Phillips, Thomas E.
Tom, you might want to contact them by phone (either through the US office at Ragona Scientific or at the German headquarter) and ask for a user reference list if you do not receive enough feedback through this listserv. I have seen their 64 beam system but I do not have enough experience on it to comment on effectiveness. Urs Utzinger, University of Arizona |
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Alison J. North |
Re: LaVision TriM multiphoton microscope
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In reply to this post by Phillips, Thomas E.
The TriM scope is a great system. We demo-ed it here in New York a
couple of years ago. I was particularly impressed by the fact that they shipped it out here from Germany and then had it up and running well on live samples within about 2 hours. You can use it either in single-beam mode if you need to image deep, or in the 64-beam mode if you need to go fast but don't have such a requirement for depth. In the 64-beam mode I seem to remember we were limited to about 100-150 microns in depth, while in the regular mode we could go to around 300 microns or deeper, depending on the tissue. But the 64-beam mode certainly is fast, so although there are several excellent multiphoton options out in the field now - Olympus, Prairie, Zeiss etc. - I do think that the TriM has its own niche. A good thing about the company is that they are extremely knowledgeable and also very flexible about the system design - for example, they have integrated OPO lasers on their systems for some time now. The only downside as far as we were concerned is that it is a multiphoton-only system, and although I can see many advantages to that myself, some of my users were adamant about wanting visible lasers on our system as well. If you contact Sid Ragona, their US rep, (www.RagonaScientific.com) he can give you names of people in the US who are using the system. Best, Alison Phillips, Thomas E. wrote: > > I have been reading about the TriM multiphoton microscope from > LaVision BioTec > (http://www.lavisionbiotec.com/en/microscopy-products/trim-scope/). > They use a combination of mirrors and a beam splitting substrate to > divide the incoming beam into 64 beamlets and this lets them scan with > 64 lines at once thereby cutting the scan time. They also use a > combination of CCDs and NDDs as detectors. Can anybody comment on how > effective this system is? Tom > > > > Thomas E. Phillips, Ph.D. > > Professor of Biological Sciences > > Director, Molecular Cytology Core > > 2 Tucker Hall > > Biological Sciences > > University of Missouri > > Columbia, MO 65211-7400 > > 573-882-4712 (voice) > > 573-882-0123 (fax) > > > -- Alison J. North, Ph.D., Research Assistant Professor and Director of the Bio-Imaging Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065. Tel: office ++ 212 327 7488 Tel: lab ++ 212 327 7486 Fax: ++ 212 327 7489 |
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Urs Utzinger |
Re: LaVision TriM multiphoton microscope
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I have a confocal extension on the "TriM scope I" and it is attached to the OPO port.
However because of this system's working principle, confocal works in single beam mode (original question of this thread).
I am still working on comparing the confocal extension with other commercial system. Urs Utzinger, University of Arizona On Mon, Apr 26, 2010 at 7:17 AM, Alison North <[hidden email]> wrote: The TriM scope is a great system. We demo-ed it here in New York a |
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B. Prabhakar Pandian |
Non Enzymatic Cell Harvesting Buffer
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In reply to this post by Andrea Latini-2
Hello,
We are trying to harvest adherent cells following activation with IL-1 for certain receptor upregulation. However, we feel that the trypsin-EDTA mixture used for harvesting is cleaving the receptors. Can somebody suggest a proven non-enzymatic cell harvesting solution. Thanks, -Prabhakar |
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I think you should test all reagents against your protein of interest - but I use TryplE Express for my receptor tyrosine kinases with success.
Sent from my Verizon Wireless BlackBerry -----Original Message----- From: "B. Prabhakar Pandian" <[hidden email]> Date: Mon, 26 Apr 2010 11:25:48 To: <[hidden email]> Subject: Non Enzymatic Cell Harvesting Buffer Hello, We are trying to harvest adherent cells following activation with IL-1 for certain receptor upregulation. However, we feel that the trypsin-EDTA mixture used for harvesting is cleaving the receptors. Can somebody suggest a proven non-enzymatic cell harvesting solution. Thanks, -Prabhakar |
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F Javier Díez Guerra |
Re: Non Enzymatic Cell Harvesting Buffer
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In reply to this post by B. Prabhakar Pandian
El 26/04/2010 18:25, B. Prabhakar Pandian escribió:
> Hello, > We are trying to harvest adherent cells following activation > with IL-1 for certain receptor upregulation. However, we feel that > the trypsin-EDTA mixture used for harvesting is cleaving the receptors. > Can somebody suggest a proven non-enzymatic cell harvesting solution. > > > Thanks, > > -Prabhakar Hi, Using cold PBS-EDTA (no trypsin), adherent cells detach although more slowly. Javier |
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Dear all,
Can anyone tell me which is the optimal mounting media (composition) for quantum dots labelled samples? Which reactives should be avoided during the immunochemistry with QD? Thank you for your help! Maria Calvo -- Dra. Maria Calvo Microscòpia Confocal. Unitat de Microscòpia Serveis Cientificotècnics-Campus Casanova Facultat de Medicina Universitat de Barcelona. IDIBAPS Casanova 143 Barcelona 08036 Tel: 34 934037159 Fax: 34 934044408 E-mail: [hidden email] |
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Diane G Miller |
Re: Quantum dots mounting media
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As far as I remember only cytoseal can be used. Others will kill the QD's.
Diane -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]]On Behalf Of Maria Calvo Sent: Monday, April 26, 2010 2:30 PM To: [hidden email] Subject: Quantum dots mounting media Dear all, Can anyone tell me which is the optimal mounting media (composition) for quantum dots labelled samples? Which reactives should be avoided during the immunochemistry with QD? Thank you for your help! Maria Calvo -- Dra. Maria Calvo Microscòpia Confocal. Unitat de Microscòpia Serveis Cientificotècnics-Campus Casanova Facultat de Medicina Universitat de Barcelona. IDIBAPS Casanova 143 Barcelona 08036 Tel: 34 934037159 Fax: 34 934044408 E-mail: [hidden email] |
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Kelly Rogers-2 |
Re: Quantum dots mounting media
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This is correct. Some mounting media can not be used with Qdots.
Invitrogen sells a mounting media specifically for Qdots called QMount. Kelly -no commercial interest > As far as I remember only cytoseal can be used. Others will kill the > QD's. > > Diane > > -----Original Message----- > From: Confocal Microscopy List > [mailto:[hidden email]]On Behalf Of Maria Calvo > Sent: Monday, April 26, 2010 2:30 PM > To: [hidden email] > Subject: Quantum dots mounting media > > > Dear all, > > Can anyone tell me which is the optimal mounting media (composition) for > quantum dots labelled samples? Which reactives should be avoided during > the immunochemistry with QD? > > Thank you for your help! > > Maria Calvo > > -- > Dra. Maria Calvo > > Microscòpia Confocal. Unitat de Microscòpia > Serveis Cientificotècnics-Campus Casanova > Facultat de Medicina > Universitat de Barcelona. IDIBAPS > Casanova 143 > Barcelona 08036 > > Tel: 34 934037159 > Fax: 34 934044408 > E-mail: [hidden email] > Kelly Rogers PhD The Walter & Eliza Hall Institute 1G Royal Parade, Parkville Victoria 3052, Australia ph: +61_3_9345 2450 ______________________________________________________________________ The information in this email is confidential and intended solely for the addressee. You must not disclose, forward, print or use it without the permission of the sender. ______________________________________________________________________ |
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Todorov, Ivan |
Re: Quantum dots mounting media
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Hi,
We are always using Vectashield from Vector (no commercial interest) and never had problems with Qdots.
Best
Ivan From: Confocal Microscopy List on behalf of Kelly Rogers Sent: Mon 4/26/2010 8:07 PM To: [hidden email] Subject: Re: Quantum dots mounting media This is correct. Some mounting media can not be used with Qdots.
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Rickman, Colin |
Re: Quantum dots mounting media
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In reply to this post by Diane G Miller
Which mounting solutions should be avoided? I'd like to increase the
time spent in the 'off state' for a QD labelled sample. Colin -- Dr Colin Rickman Department of Chemistry (WP 2.03) School of Engineering and Physical Sciences Heriot-Watt University Edinburgh EH14 4AS Tel: +44 131 4514193 (Office) Tel: +44 131 6511512 Fax: +44 131 650312 On 27/04/2010 02:27, Diane G Miller wrote: > As far as I remember only cytoseal can be used. Others will kill the QD's. > > Diane > > -----Original Message----- > From: Confocal Microscopy List > [mailto:[hidden email]]On Behalf Of Maria Calvo > Sent: Monday, April 26, 2010 2:30 PM > To: [hidden email] > Subject: Quantum dots mounting media > > > Dear all, > > Can anyone tell me which is the optimal mounting media (composition) for > quantum dots labelled samples? Which reactives should be avoided during > the immunochemistry with QD? > > Thank you for your help! > > Maria Calvo > > -- > Dra. Maria Calvo > > Microscòpia Confocal. Unitat de Microscòpia > Serveis Cientificotècnics-Campus Casanova > Facultat de Medicina > Universitat de Barcelona. IDIBAPS > Casanova 143 > Barcelona 08036 > > Tel: 34 934037159 > Fax: 34 934044408 > E-mail: [hidden email] > |
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Haberman, Ann |
Re: LaVision TriM multiphoton microscope
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In reply to this post by Alison J. North
I've also had great experiences with LaVision. They are willing into incorporate custom ordered components, which they can produce with exquisite craftsmanship and elegant design. Very knowledgeable staff and most importantly, a desire to sell and support products that work.
I order a modified version of the TriM a couple of years ago, which now can be ordered as a standard model, so I can't comment on the 64 beam mode. This microscope is dedicated to a single beam of a two-photon laser and has a motorized control of the fill of the back aperture of the objective. It has been a total workhorse in an in vivo imaging facility that scans for hours at a time, day after day. The software doesn't have the same glitz and gloss that the major manufacturers offer, but the plus side is that it is easy to write macros that can be plugged into the software (if you are into that sort of thing). They have a number of microscope in the US, but are a moderate sized company based in Germany. However, they respond quickly to my emails and phone calls and will stop by when they are in the area visiting other installed microscopes or conferences. I have even had a video conference once to assess a potential issue in real time, transcontinental, more quickly than a representative could have arrived. Feel free to call with questions you might have, Ann >The TriM scope is a great system. We demo-ed it here in New York a couple of years ago. I was particularly impressed by the fact that they shipped it out here from Germany and then had it up and running well on live samples within about 2 hours. You can use it either in single-beam mode if you need to image deep, or in the 64-beam mode if you need to go fast but don't have such a requirement for depth. In the 64-beam mode I seem to remember we were limited to about 100-150 microns in depth, while in the regular mode we could go to around 300 microns or deeper, depending on the tissue. But the 64-beam mode certainly is fast, so although there are several excellent multiphoton options out in the field now - Olympus, Prairie, Zeiss etc. - I do think that the TriM has its own niche. >A good thing about the company is that they are extremely knowledgeable and also very flexible about the system design - for example, they have integrated OPO lasers on their systems for some time now. The only downside as far as we were concerned is that it is a multiphoton-only system, and although I can see many advantages to that myself, some of my users were adamant about wanting visible lasers on our system as well. If you contact Sid Ragona, their US rep, (www.RagonaScientific.com) he can give you names of people in the US who are using the system. > >Best, >Alison > >Phillips, Thomas E. wrote: >> >>I have been reading about the TriM multiphoton microscope from LaVision BioTec (http://www.lavisionbiotec.com/en/microscopy-products/trim-scope/). They use a combination of mirrors and a beam splitting substrate to divide the incoming beam into 64 beamlets and this lets them scan with 64 lines at once thereby cutting the scan time. They also use a combination of CCDs and NDDs as detectors. Can anybody comment on how effective this system is? Tom >> >> >>Thomas E. Phillips, Ph.D. >> >>Professor of Biological Sciences >> >>Director, Molecular Cytology Core >> >>2 Tucker Hall >> >>Biological Sciences >> >>University of Missouri >> >>Columbia, MO 65211-7400 >> >>573-882-4712 (voice) >> >>573-882-0123 (fax) >> > Alison J. North, Ph.D., >Research Assistant Professor and Director of the Bio-Imaging Resource Center, >The Rockefeller University, >1230 York Avenue, >New York, >NY 10065. >Tel: office ++ 212 327 7488 >Tel: lab ++ 212 327 7486 >Fax: ++ 212 327 7489 -- Ann Haberman, PhD Department of Laboratory Medicine Yale University School of Medicine 300 Cedar Street TAC S541 New Haven, CT 06510 203-785-7349 203-785-5415 (fax) [hidden email] |
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Maria Calvo-2 |
Re: Quantum dots mounting media
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In reply to this post by Rickman, Colin
Hi,
Answering the question from Colin Rickman, I found in the archives from 2006 an answer from Mike Ignatius to a similar question (he talks about blinking). My question was also related to that mail, what types of mounting media are compatible to Quantum dots? Thank you! Maria Calvo Find below the mail from the archives: Dear Laurent, Caution, Vendor's somewhat torn response. We sell both products and have of course compared them. (I will assume since you call them QDots that you are using our product.) You asked about size and blinking. Size: The QDot nanocrystal conjugates are indeed larger than organic dyes. Depending on the color (the redder the bigger) they can reach 10 to 15 nm's and with full IgGs decorating them (2-4 on average) even larger. So like DPI for printers, larger is not better for high resolution ICC. However, we only see real big differences when we deconvolve the images, since this is all sub-resolution. However, the packing density is less. So while QDot nanocrystals are brighter in the vial, on your prep they are more likely equivalent in terms of brightness to Alexa dyes - less dots per unit area when compared to dyes. A cautionary note: Please do not use Prolong or Prolong Gold or anything but cytoseal or PBS glycerol type mountants, such as our SlowFade gold for mounting QDot Nanocrystals. They will disappear overnight. We have an ever growing "n" value on this behavior. Our PIS stipulates this with the product.) Blinking: The blinking is certainly apparent to the eye and thus to fast cameras. Integrating over time avoids this concern - merely reduce your lamp intensity to help out. We see the blinking with individual dots the most, while ensembles tend to average out. It is worth noting that some have found the blinking a nice confirmation that they are looking at a nanocrystals, not background or organic dyes. When to use Organic dyes when to use Nanocrystals? This is where we are sometimes torn. The narrow full width half maximums of Qdot nanocrystals, as you describe, indeed allow more colors. The photostability, while terrific for live cell work, rare event detection and repeated scanning of confocal stacks, is less of a boon in wide field, single scan experiments. Especially with good dyes and antifades. So when you need photostability or more colors, nanocrystals are worth considering. Mike Ignatius Molecular Probes/Invitrogen. ------------------------------------------------------------------------ *From:* Confocal Microscopy List [mailto:[hidden email]] *On Behalf Of *laurent barbe *Sent:* Thursday, July 27, 2006 12:49 AM *To:* [hidden email] *Subject:* Qdot vs Alexa Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear users, I have a quite broad, general question for users who experienced these 2 dyes. I’m doing colocalization study with 3d stacks, which means quite long exposure. I am doing 4 color imaging, which made me try Qdots to be able to fit all probes trying to reduce overlapping. Obviously Qdots are more stable for long exposure than Alexa dyes but I’d like to have user’s feedbacks from people who compared both. I also read many times that Qdots blink, even thouh I haven’t faced this problem yet, as well as Qdots are more ‘bulky’ after conjugation compared to Alexa. Any thoughts and feedbacks would be appreciated. Thanks Laurent ______________________________________________ Laurent Barbe, Research Fellow Royal University of Technology (KTH) School of Nanobiotechnology - Dpt of Biotechnology Roslagstullsbacken 21, 10601 STOCKHOLM SWEDEN Tel: (+46) 855378832 Fax: (+46) 855378323 -- ___________________________________ Dra. Maria Calvo Unitat de Microscòpia Confocal Serveis Cientificotècnics-C.Casanova Facultat de Medicina Universitat de Barcelona- IDIBAPS C/ Casanova 143 Barcelona 08036 Tel: 34 934037159/39930 Fax: 34 934039946 E-mail: [hidden email] ___________________________________ |
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Rickman, Colin |
Enhancing the "blinking" of quantum dots
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Hi
Thanks for the archive email. My question wasn't so much about observing blinking but enhancing it (hence I have changed the subject line to reflect this). Most recent research has been focused on trying to reduce or eliminate the blinking observed by Quantum dots. I would like to know if anybody has any ideas on how to increase either the frequency of blinking or increase the time the quantum dot spends in the "off" state. Any advice gratefully received Regards Colin -- Dr Colin Rickman Department of Chemistry (WP 2.03) School of Engineering and Physical Sciences Heriot-Watt University Edinburgh EH14 4AS Tel: +44 131 4514193 (Office) Tel: +44 131 6511512 Fax: +44 131 6503128 On 28/04/2010 17:24, Maria Calvo wrote: > Hi, > > Answering the question from Colin Rickman, I found in the archives > from 2006 an answer from Mike Ignatius to a similar question (he talks > about blinking). > > My question was also related to that mail, what types of mounting > media are compatible to Quantum dots? > > Thank you! > Maria Calvo > > Find below the mail from the archives: > > Dear Laurent, > > Caution, Vendor's somewhat torn response. > > We sell both products and have of course compared them. (I will assume > since you call them QDots that you are using our product.) You asked > about size and blinking. > > Size: > > The QDot nanocrystal conjugates are indeed larger than organic dyes. > Depending on the color (the redder the bigger) they can reach 10 to 15 > nm's and with full IgGs decorating them (2-4 on average) even larger. > So like DPI for printers, larger is not better for high resolution > ICC. However, we only see real big differences when we deconvolve the > images, since this is all sub-resolution. > > However, the packing density is less. So while QDot nanocrystals are > brighter in the vial, on your prep they are more likely equivalent in > terms of brightness to Alexa dyes - less dots per unit area when > compared to dyes. > > A cautionary note: Please do not use Prolong or Prolong Gold or > anything but cytoseal or PBS glycerol type mountants, such as our > SlowFade gold for mounting QDot Nanocrystals. They will disappear > overnight. We have an ever growing "n" value on this behavior. Our PIS > stipulates this with the product.) > > Blinking: > > The blinking is certainly apparent to the eye and thus to fast > cameras. Integrating over time avoids this concern - merely reduce > your lamp intensity to help out. We see the blinking with individual > dots the most, while ensembles tend to average out. It is worth noting > that some have found the blinking a nice confirmation that they are > looking at a nanocrystals, not background or organic dyes. > > When to use Organic dyes when to use Nanocrystals? > > This is where we are sometimes torn. The narrow full width half > maximums of Qdot nanocrystals, as you describe, indeed allow more > colors. The photostability, while terrific for live cell work, rare > event detection and repeated scanning of confocal stacks, is less of a > boon in wide field, single scan experiments. Especially with good dyes > and antifades. So when you need photostability or more colors, > nanocrystals are worth considering. > > Mike Ignatius > > Molecular Probes/Invitrogen. |
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Ignatius, Mike-2 |
Re: Quantum dots mounting media *Vendor Comment*
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In reply to this post by Todorov, Ivan
Hi, Sorry for late response here – traveling.
In our studies, Vectashield can initially
dim the dots and by as little as 3 days wipe out the signal entirely. (Prolong
Gold too.) Thus we strongly recommend our QMount mountant for optimal
viewing. See http://probes.invitrogen.com/media/pis/mp10336.pdf
for details. Alternatively, for fixed or live samples
the photostabile Qdot nanocrystals can be imaged directly in solutions, like
PBS or media, though with some loss in initial signal compared to QMount. Coverslips
mounted in holders like our Attofluor chamber, or other coverslip bottomed
devices on inverted scopes work great as do dipping objectives on uprights. Not
great for archiving, but terrific for immediate, easy viewing. Mike Ignatius Molecular Probes/Life Technologies From: Confocal
Microscopy List [mailto:[hidden email]] On Behalf Of Todorov, Ivan Hi, We are always using Vectashield from
Vector (no commercial interest) and never had problems with Qdots. Best Ivan From: Confocal
Microscopy List on behalf of Kelly Rogers This is
correct. Some mounting media can not be used with Qdots.
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Eli Rothenberg-2 |
Re: Enhancing the "blinking" of quantum dots
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In reply to this post by Rickman, Colin
Colin,
We have been doing this with QDs for some time now. But it really depend on what sample you're tryin to image. There are a number of ways to go about this. First you have to realize that generally, aside from size, QDs' fluorescence properties (QY, Blinking) will depend on their surface. Commercial QDs come in different forms, most of the "good ones" come with a well passivated surface. Assuming that these are the ones you're using, you can try to do the following to make them blink more: 1. easiest: max power excitation. Put your laser on max and go. 2. Adding stuff to your imaging buffer- purging your buffer with Oxygen gas, or adding just adding oxidants, chelates, reductants etc might do the job and increase blinking. 3. Diminishing the surface quality of QDs before using them. This could be done by: - spin down your QDs stock, and grab the ones that precipitated, these will have bad surface quality. -using and aged stock. - storing them at RT for a few hrs/day/wks. - storing them at RT + exposing them to light. -Sonicating them.(vary the time) Combining any of the above. You can also combine it with 1 and 2. And again, this will also depend on whatever you're tryin to image in solution, live cells, fixed cells, method of conjugation etc Hope this helps. Eli ---- Original message ---- >Date: Wed, 28 Apr 2010 17:34:33 +0100 >From: Colin Rickman <[hidden email]> >Subject: Enhancing the "blinking" of quantum dots >To: [hidden email] > >Hi > >Thanks for the archive email. My question wasn't so much about observing >blinking but enhancing it (hence I have changed the subject line to >reflect this). Most recent research has been focused on trying to reduce >or eliminate the blinking observed by Quantum dots. I would like to know >if anybody has any ideas on how to increase either the frequency of >blinking or increase the time the quantum dot spends in the "off" state. > >Any advice gratefully received > >Regards > >Colin > >-- >Dr Colin Rickman >Department of Chemistry (WP 2.03) >School of Engineering and Physical Sciences >Heriot-Watt University >Edinburgh >EH14 4AS > >Tel: +44 131 4514193 (Office) >Tel: +44 131 6511512 >Fax: +44 131 6503128 > > > >On 28/04/2010 17:24, Maria Calvo wrote: >> Hi, >> >> Answering the question from Colin Rickman, I found in the archives >> from 2006 an answer from Mike Ignatius to a similar question (he talks >> about blinking). >> >> My question was also related to that mail, what types of mounting >> media are compatible to Quantum dots? >> >> Thank you! >> Maria Calvo >> >> Find below the mail from the archives: >> >> Dear Laurent, >> >> Caution, Vendor's somewhat torn response. >> >> We sell both products and have of course compared them. (I will assume >> since you call them QDots that you are using our product.) You asked >> about size and blinking. >> >> Size: >> >> The QDot nanocrystal conjugates are indeed larger than organic dyes. >> Depending on the color (the redder the bigger) they can reach 10 to 15 >> nm's and with full IgGs decorating them (2-4 on average) even larger. >> So like DPI for printers, larger is not better for high resolution >> ICC. However, we only see real big differences when we deconvolve the >> images, since this is all sub-resolution. >> >> However, the packing density is less. So while QDot nanocrystals are >> brighter in the vial, on your prep they are more likely equivalent in >> terms of brightness to Alexa dyes - less dots per unit area when >> compared to dyes. >> >> A cautionary note: Please do not use Prolong or Prolong Gold or >> anything but cytoseal or PBS glycerol type mountants, such as our >> SlowFade gold for mounting QDot Nanocrystals. They will disappear >> overnight. We have an ever growing "n" value on this behavior. Our PIS >> stipulates this with the product.) >> >> Blinking: >> >> The blinking is certainly apparent to the eye and thus to fast >> cameras. Integrating over time avoids this concern - merely reduce >> your lamp intensity to help out. We see the blinking with individual >> dots the most, while ensembles tend to average out. It is worth noting >> that some have found the blinking a nice confirmation that they are >> looking at a nanocrystals, not background or organic dyes. >> >> When to use Organic dyes when to use Nanocrystals? >> >> This is where we are sometimes torn. The narrow full width half >> maximums of Qdot nanocrystals, as you describe, indeed allow more >> colors. The photostability, while terrific for live cell work, rare >> event detection and repeated scanning of confocal stacks, is less of a >> boon in wide field, single scan experiments. Especially with good dyes >> and antifades. So when you need photostability or more colors, >> nanocrystals are worth considering. >> >> Mike Ignatius >> >> Molecular Probes/Invitrogen. Eli Rothenberg, Ph.D. Postdoctoral Fellow, Department of Physics, NSF Center for the Physics of Living Cells, University of Illinois - Urbana, 1110 W. Green St. Urbana, 61801. Illinois, USA Tel: +217-244-5829; Email: [hidden email] |
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