Alexa Fluor 532 with 543nm laser

[Scott Wong]

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Hi everyone,
I was wondering if anyone has successfully used the Alexa Fluor 532
secondary to view tyrosine hydroxylase in dopaminergic neurons. I'll be
imaging it using a 543nm laser with a Nikon confocal microscope. Based on its
excitation wavelength it seems to be an ideal match but I don't any
experience with it.

If not, can anyone comment on its anti-quenching properties? I'm currently
using the Alexa Fluor 546 for the same purpose but it quenches almost
unusably fast with every mounting media I've tried.

Thanks for the help,
Scott Wong

[Craig Brideau]

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Have you checked out the Invitrogen spectra viewer?
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Analysis/Labeling-Chemistry/Fluorescence-SpectraViewer.html

According to that you should be ok, but you will only be catching the tail
of the dye with any practical filter set so you will suffer significant
signal loss. You might still have enough to work with depending on
concentration, laser power, etc.

Craig
On 2013-07-03 12:38 PM, "Scott Wong" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi everyone,
> I was wondering if anyone has successfully used the Alexa Fluor 532
> secondary to view tyrosine hydroxylase in dopaminergic neurons. I'll be
> imaging it using a 543nm laser with a Nikon confocal microscope. Based on
> its
> excitation wavelength it seems to be an ideal match but I don't any
> experience with it.
>
> If not, can anyone comment on its anti-quenching properties? I'm currently
> using the Alexa Fluor 546 for the same purpose but it quenches almost
> unusably fast with every mounting media I've tried.
>
> Thanks for the help,
> Scott Wong
>

[Xuejun Sun]

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Hi, Scott,

I have not tried Alexa 532. However, we did have a lot of experiences with
Alexa 546. Put a long story short, I was told (by then a Mol. Probe person)
that it is a "bad" dye to work with as it is almost incompatible with any
antifading agent and it works best without any antifading product. So we
switched to Alexa 555 or 568 and are much happier. Maybe you could try a
mounting media without antifade?

Good luck,

Xuejun

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Scott Wong
Sent: Tuesday, July 02, 2013 5:08 PM
To: [hidden email]
Subject: Alexa Fluor 532 with 543nm laser

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Hi everyone,
I was wondering if anyone has successfully used the Alexa Fluor 532
secondary to view tyrosine hydroxylase in dopaminergic neurons. I'll be
imaging it using a 543nm laser with a Nikon confocal microscope. Based on
its
excitation wavelength it seems to be an ideal match but I don't any
experience with it.

If not, can anyone comment on its anti-quenching properties? I'm currently
using the Alexa Fluor 546 for the same purpose but it quenches almost
unusably fast with every mounting media I've tried.

Thanks for the help,
Scott Wong



This message and any attached documents are only for the use of the intended recipient(s), are confidential and may contain privileged information. Any unauthorized review, use, retransmission, or other disclosure is strictly prohibited. If you have received this message in error, please notify the sender immediately, and then delete the original message. Thank you.

[Scott Wong]

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Hi Craig,
Thanks for the link. The Alexa Fluor 532 and 555 both seem better suited
for the laser I'm using according the the spectraviewer. A few people have
commented that the 532 also quenches quite fast though so I'll most likely
end up getting the 555.
Cheers,
Scott

>
> Have you checked out the Invitrogen spectra viewer?
> http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Analysis/Labeling-Chemistry/Fluorescence-SpectraViewer.html
>
> According to that you should be ok, but you will only be catching the tail
> of the dye with any practical filter set so you will suffer significant
> signal loss. You might still have enough to work with depending on
> concentration, laser power, etc.
>
> Craig
> On 2013-07-03 12:38 PM, "Scott Wong" <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi everyone,
>> I was wondering if anyone has successfully used the Alexa Fluor 532
>> secondary to view tyrosine hydroxylase in dopaminergic neurons. I'll be
>> imaging it using a 543nm laser with a Nikon confocal microscope. Based
>> on
>> its
>> excitation wavelength it seems to be an ideal match but I don't any
>> experience with it.
>>
>> If not, can anyone comment on its anti-quenching properties? I'm
>> currently
>> using the Alexa Fluor 546 for the same purpose but it quenches almost
>> unusably fast with every mounting media I've tried.
>>
>> Thanks for the help,
>> Scott Wong
>>
>

[Julio Vazquez]

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Scott,

Exciting near the peak is good, but detection efficiency and properties of the dye are also important. Under Alexa dyes in Wikipedia (or the Molecular Probes web site), the quantum yield of Alexa 555 is listed as 0.1, while Alexa 568 is listed as 0.69, so even considering the lower excitation efficiency (about 50%) and the somewhat lower extinction coefficient, Alexa 568 should still be about 2-2.5 times brighter than A555. Or put another way, you could get the same signal with about half the laser intensity (and therefore less photobleaching).

http://en.wikipedia.org/wiki/Alexa_Fluor 

JulioVazquez
Fred Hutchinson Cancer Research Center
Seattle, WA

----- Original Message -----
From: "Scott Wong" <[hidden email]>
To: [hidden email]
Sent: Thursday, July 4, 2013 12:57:32 PM
Subject: Re: Alexa Fluor 532 with 543nm laser

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Hi Craig,
Thanks for the link. The Alexa Fluor 532 and 555 both seem better suited
for the laser I'm using according the the spectraviewer. A few people have
commented that the 532 also quenches quite fast though so I'll most likely
end up getting the 555.
Cheers,
Scott

>
> Have you checked out the Invitrogen spectra viewer?
> http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Analysis/Labeling-Chemistry/Fluorescence-SpectraViewer.html
>
> According to that you should be ok, but you will only be catching the tail
> of the dye with any practical filter set so you will suffer significant
> signal loss. You might still have enough to work with depending on
> concentration, laser power, etc.
>
> Craig
> On 2013-07-03 12:38 PM, "Scott Wong" <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi everyone,
>> I was wondering if anyone has successfully used the Alexa Fluor 532
>> secondary to view tyrosine hydroxylase in dopaminergic neurons. I'll be
>> imaging it using a 543nm laser with a Nikon confocal microscope. Based
>> on
>> its
>> excitation wavelength it seems to be an ideal match but I don't any
>> experience with it.
>>
>> If not, can anyone comment on its anti-quenching properties? I'm
>> currently
>> using the Alexa Fluor 546 for the same purpose but it quenches almost
>> unusably fast with every mounting media I've tried.
>>
>> Thanks for the help,
>> Scott Wong
>>
>

[mcammer]

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Some people in our lab use Alexa 546 because they want to use the same antibody they use for FACS, but in general we recommend Alexa 568 which we use successfully with diode laser excitation at 532 (TIRF and dSTORM/PALM/GSD), 543 (confocal) and 561 nm (widefield and TIRF).
________________________________________________________
Michael Cammer, Assistant Research Scientist
Microscopy Core, NYU Langone Medical Center & Skirball Institute of Biomolecular Medicine
Cell: [phone]   Microscopy Lab: [phone]   Dustin Lab: [phone]  



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Xuejun SUN
Sent: Thursday, July 04, 2013 11:17 AM
To: [hidden email]
Subject: Re: Alexa Fluor 532 with 543nm laser

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Hi, Scott,

I have not tried Alexa 532. However, we did have a lot of experiences with Alexa 546. Put a long story short, I was told (by then a Mol. Probe person) that it is a "bad" dye to work with as it is almost incompatible with any antifading agent and it works best without any antifading product. So we switched to Alexa 555 or 568 and are much happier. Maybe you could try a mounting media without antifade?

Good luck,

Xuejun

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Scott Wong
Sent: Tuesday, July 02, 2013 5:08 PM
To: [hidden email]
Subject: Alexa Fluor 532 with 543nm laser

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi everyone,
I was wondering if anyone has successfully used the Alexa Fluor 532 secondary to view tyrosine hydroxylase in dopaminergic neurons. I'll be imaging it using a 543nm laser with a Nikon confocal microscope. Based on its excitation wavelength it seems to be an ideal match but I don't any experience with it.

If not, can anyone comment on its anti-quenching properties? I'm currently using the Alexa Fluor 546 for the same purpose but it quenches almost unusably fast with every mounting media I've tried.

Thanks for the help,
Scott Wong



This message and any attached documents are only for the use of the intended recipient(s), are confidential and may contain privileged information. Any unauthorized review, use, retransmission, or other disclosure is strictly prohibited. If you have received this message in error, please notify the sender immediately, and then delete the original message. Thank you.

[lechristophe]

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From my parallel tests (but I don't have quantified data), Alexa 555
coupled secondary antibodies give a brighter signal than 546 and 568 ones
with both 561 and 532 nm laser lines on our confocals.

Christophe
Le 5 juil. 2013 18:04, "Cammer, Michael" <[hidden email]> a
écrit :

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Some people in our lab use Alexa 546 because they want to use the same
> antibody they use for FACS, but in general we recommend Alexa 568 which we
> use successfully with diode laser excitation at 532 (TIRF and
> dSTORM/PALM/GSD), 543 (confocal) and 561 nm (widefield and TIRF).
> ________________________________________________________
> Michael Cammer, Assistant Research Scientist
> Microscopy Core, NYU Langone Medical Center & Skirball Institute of
> Biomolecular Medicine
> Cell: [phone]   Microscopy Lab: [phone]   Dustin Lab: (212)
> 263-3208
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Xuejun SUN
> Sent: Thursday, July 04, 2013 11:17 AM
> To: [hidden email]
> Subject: Re: Alexa Fluor 532 with 543nm laser
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi, Scott,
>
> I have not tried Alexa 532. However, we did have a lot of experiences with
> Alexa 546. Put a long story short, I was told (by then a Mol. Probe person)
> that it is a "bad" dye to work with as it is almost incompatible with any
> antifading agent and it works best without any antifading product. So we
> switched to Alexa 555 or 568 and are much happier. Maybe you could try a
> mounting media without antifade?
>
> Good luck,
>
> Xuejun
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Scott Wong
> Sent: Tuesday, July 02, 2013 5:08 PM
> To: [hidden email]
> Subject: Alexa Fluor 532 with 543nm laser
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi everyone,
> I was wondering if anyone has successfully used the Alexa Fluor 532
> secondary to view tyrosine hydroxylase in dopaminergic neurons. I'll be
> imaging it using a 543nm laser with a Nikon confocal microscope. Based on
> its excitation wavelength it seems to be an ideal match but I don't any
> experience with it.
>
> If not, can anyone comment on its anti-quenching properties? I'm currently
> using the Alexa Fluor 546 for the same purpose but it quenches almost
> unusably fast with every mounting media I've tried.
>
> Thanks for the help,
> Scott Wong
>
>
>
> This message and any attached documents are only for the use of the
> intended recipient(s), are confidential and may contain privileged
> information. Any unauthorized review, use, retransmission, or other
> disclosure is strictly prohibited. If you have received this message in
> error, please notify the sender immediately, and then delete the original
> message. Thank you.
>

[lechristophe]

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Just to correct my mistake : the laser lines I tested are 532 nm (Leica
SPE) and 543 nm (Zeiss LSM 780), not 561 nm.

Christophe
Le 5 juil. 2013 23:11, "Christophe Leterrier" <
[hidden email]> a écrit :

> From my parallel tests (but I don't have quantified data), Alexa 555
> coupled secondary antibodies give a brighter signal than 546 and 568 ones
> with both 561 and 532 nm laser lines on our confocals.
>
> Christophe
> Le 5 juil. 2013 18:04, "Cammer, Michael" <[hidden email]> a
> écrit :
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Some people in our lab use Alexa 546 because they want to use the same
>> antibody they use for FACS, but in general we recommend Alexa 568 which we
>> use successfully with diode laser excitation at 532 (TIRF and
>> dSTORM/PALM/GSD), 543 (confocal) and 561 nm (widefield and TIRF).
>> ________________________________________________________
>> Michael Cammer, Assistant Research Scientist
>> Microscopy Core, NYU Langone Medical Center & Skirball Institute of
>> Biomolecular Medicine
>> Cell: [phone]   Microscopy Lab: [phone]   Dustin Lab: (212)
>> 263-3208
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of Xuejun SUN
>> Sent: Thursday, July 04, 2013 11:17 AM
>> To: [hidden email]
>> Subject: Re: Alexa Fluor 532 with 543nm laser
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi, Scott,
>>
>> I have not tried Alexa 532. However, we did have a lot of experiences
>> with Alexa 546. Put a long story short, I was told (by then a Mol. Probe
>> person) that it is a "bad" dye to work with as it is almost incompatible
>> with any antifading agent and it works best without any antifading product.
>> So we switched to Alexa 555 or 568 and are much happier. Maybe you could
>> try a mounting media without antifade?
>>
>> Good luck,
>>
>> Xuejun
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of Scott Wong
>> Sent: Tuesday, July 02, 2013 5:08 PM
>> To: [hidden email]
>> Subject: Alexa Fluor 532 with 543nm laser
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi everyone,
>> I was wondering if anyone has successfully used the Alexa Fluor 532
>> secondary to view tyrosine hydroxylase in dopaminergic neurons. I'll be
>> imaging it using a 543nm laser with a Nikon confocal microscope. Based on
>> its excitation wavelength it seems to be an ideal match but I don't any
>> experience with it.
>>
>> If not, can anyone comment on its anti-quenching properties? I'm
>> currently using the Alexa Fluor 546 for the same purpose but it quenches
>> almost unusably fast with every mounting media I've tried.
>>
>> Thanks for the help,
>> Scott Wong
>>
>>
>>
>> This message and any attached documents are only for the use of the
>> intended recipient(s), are confidential and may contain privileged
>> information. Any unauthorized review, use, retransmission, or other
>> disclosure is strictly prohibited. If you have received this message in
>> error, please notify the sender immediately, and then delete the original
>> message. Thank you.
>>
>

[Scott Wong]

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*****

>Thanks everyone for the help with my antibody question. It's definitely
appreciated and the information I got will save me from some inevitable
headaches later down the road.
Cheers,
Scott

 Hi everyone,

> I was wondering if anyone has successfully used the Alexa Fluor 532
> secondary to view tyrosine hydroxylase in dopaminergic neurons. I'll be
> imaging it using a 543nm laser with a Nikon confocal microscope. Based on
> its
> excitation wavelength it seems to be an ideal match but I don't any
> experience with it.
>
> If not, can anyone comment on its anti-quenching properties? I'm currently
> using the Alexa Fluor 546 for the same purpose but it quenches almost
> unusably fast with every mounting media I've tried.
>
> Thanks for the help,
> Scott Wong
>
>