Re: Enhancing the "blinking" of quantum dots

Posted by Eli Rothenberg-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/LaVision-TriM-multiphoton-microscope-tp4962077p5009527.html

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: [phone] (Office)
>Tel: [phone]
>Fax: [phone]
>
>
>
>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: [phone];
Email: [hidden email]