Optic fiber for confocal microscope

[Schebique]

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Dear list.

I would like to rebuild our old optic fiber coupling Ar/ArKr laser and
merge module in our Leica TCS SP2 system. I found a company which is
able to do it in reasonable price. Despite of we now know all needed
parameters we do not know a diameter of fiber optic core. My question is
if anybody in this great list know this and if this is somehow important
for a performance - other words, how can a diameter of the fiber cable
affect a light transmission and a confocal spot?

Thank you very much.
Sincerely Ondrej Sebesta


------------------------------------------
Mgr. Ondřej Šebesta
Laboratory of Confocal and Fluorescence Microscopy
Faculty of Science, Charles University in Prague
Vinicna 7
128 44 Prague
Czech Republic

Phone: [phone]
e-mail: [hidden email]

[Pascal Weber]

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You should always use a mono mode fiber. You can find it in the laser dealer.
It is not really expansiv.

Cheers

[Craig Brideau]

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Yes, you need single mode fiber matched to the wavelength of the laser.
 ThorLabs sells patch cords pre-built for this purpose:
http://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=1362
They tend to be a good price.  You might find another vendor in your area
that offers similar.

The only thing to check is whether the fiber is terminated FC/PC (flat
cleave polished connector) or APC (angled polish).  The APC connectors have
a very slight angle on the tip to help prevent back reflections.  The most
common is FC/PC but it's good to check.

One option is to have a stainless steel jacket put on the fiber, which I
usually recommend for anything carrying a laser.  You have to request this
special order though.  The default is just a plastic cable, which will
work, but I prefer the steel just to be extra safe.

Craig



On Mon, Nov 25, 2013 at 2:20 AM, Pascal Weber <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> You should always use a mono mode fiber. You can find it in the laser
> dealer.
> It is not really expansiv.
>
> Cheers
>

[Volodymyr Nechyporuk-Zloy]

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Dear Ondrej,

You can contact Qioptiq - particularly Fiona Evans
([hidden email]), - and ask for an appropriate fiber, as
Qioptiq produced the fibers for SP2; they know all required parameters.
We had already ordered from them the substitute fiber.

Best regards,
Volodymyr

[Feinstein, Timothy]

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For the many of you who have more experience in histology, do you prefer any particular label for immunostaining a challenging protein?  I understand that some proteins cannot be reliably imaged in their native state using confocal.  Nonetheless it seems like there must be better and worse label choices for those close calls.

Years ago I might have warned people away from red and far red labels due to Low PMT semsitivity, but GaAsP detectors in those channels seem sensitive enough to moot that.  Red or far red seem preferable since most tissues have relatively low autofluorescence in that range.  Maybe labels in the 594-647 range are now better, or was that always true?

Any input appreciated.

Cheers,


TF

Timothy Feinstein, Ph.D. | Confocal Manager, Van Andel Institute
333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503<x-apple-data-detectors://0/0>
Phone: [phone]<tel:[phone]> | Email: [hidden email]<mailto:[hidden email]>

[George McNamara]

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Hi Timothy,

I am a fan of Alexa Fluor 555 or Alexa Fluor 568. Depending on the
instrument (excitation source, emission wavelength selection, detector
sensitivity), and detection antibody, one may be better than the other.
Cy3 is still very popular, and similar spectra to Alexa Fluor 555.
Advantages include (i) green (Alexa Fluor 488, EGFP, mNeonGreen,
mClover, UnaG) often has overlap with autofluorescence, (ii) NIR channel
(Alexa Fluor 647, Cy5, etc ... see NIRvana Sciences for new NIR
fluorophores) is typically not visible to the eye, so hard to search.
there are filter sets that enable some users to see Cy5 by eye - see

Direct eye visualization of *Cy5* fluorescence for immunocytochemistry
and in situ hybridization. <http://www.ncbi.nlm.nih.gov/pubmed/10681398>*
Ferri* GL, Isola J, Berger P, Giro G.

J Histochem Cytochem. 2000 Mar;48(3):437-44.
PMID: 10681398

Quadruple immunofluorescence: a direct visualization method.
<http://www.ncbi.nlm.nih.gov/pubmed/9016305>

*Ferri* GL, Gaudio RM, Castello IF, Berger P, Giro G.

J Histochem Cytochem. 1997 Feb;45(2):155-8.

PMID:
    9016305


I am also still a big fan of tyramide signal amplification - available
from both Invitrogen and PerkinElmer - available with several
fluorophores from each of these vendors. Generally to decrease primary
antibody from conventional immunostaining, and may need to decrease the
HRP-2ndary Ab as well.

***

The Central Dogma of DNA makes RNA makes protein implies that detecting
the mRNA should also help build confidence that the immunostaining is
correct (thee are exceptions, such as short lived RNA, transfer of RNA
and/or protein from cell to cell, etc). For mRNA detection, I recommend
Stellaris RNA FISH - see http://stellarisfish.smugmug.com/
It is compatible with immunofluorescence (I would do TSA first, if
possible). Some immunofluorescence + RNA FISH galleries are at
http://stellarisfish.smugmug.com/Immunofluorescence-RNA-FISH
See also Arjun Raj's 2013 papers on iceFISH (intron detection of nascent
transcripts ... currently at 20plex), SNP FISH (talk about the need for
stringent controls!) and Turbo FISH (15 minutes from specimen on slide
to ready for image ... though 4x more reagent).

Turbo FISH: A Method for Rapid Single Molecule RNA FISH.
<http://www.ncbi.nlm.nih.gov/pubmed/24066168>

Shaffer SM, Wu MT, *Levesque* MJ, *Raj* A.

PLoS One. 2013 Sep 16;8(9):e75120. doi: 10.1371/journal.pone.0075120.

PMID:
    24066168


Visualizing SNVs to quantify allele-specific expression in single cells.
<http://www.ncbi.nlm.nih.gov/pubmed/23913259>

*Levesque* MJ, Ginart P, Wei Y, *Raj* A.

Nat Methods. 2013 Sep;10(9):865-7. doi: 10.1038/nmeth.2589. Epub 2013 Aug 4.

PMID:
    23913259


Single-chromosome transcriptional profiling reveals chromosomal gene
expression regulation. <http://www.ncbi.nlm.nih.gov/pubmed/23416756>

*Levesque* MJ, *Raj* A.

Nat Methods. 2013 Mar;10(3):246-8. doi: 10.1038/nmeth.2372. Epub 2013
Feb 17. Erratum in: Nat Methods. 2013 May;10(5):445.

PMID:
    23416756


See also Long Cai's multiplex detection with this type of probes (in
yeast so far):

Turning single cells into microarrays by super-resolution barcoding.
<http://www.ncbi.nlm.nih.gov/pubmed/23178478>

*Cai L*.

Brief Funct Genomics. 2013 Mar;12(2):75-80. doi: 10.1093/bfgp/els054.
Epub 2012 Nov 22. Review.

PMID:
    23178478


Single-cell systems biology by super-resolution imaging and
combinatorial labeling. <http://www.ncbi.nlm.nih.gov/pubmed/22660740>

Lubeck E, Cai L.

Nat Methods. 2012 Jun 3;9(7):743-8. doi: 10.1038/nmeth.2069.

PMID:
    22660740


Disclosure: Our lab/dept is hosting a wet lab workshop from Biosearch
Tech (Stellaris FISH) next week.

Somewhat different probe design ("molecular beacons") enables live cell
detection:

Mechanism of mRNA transport in the nucleus.
<http://www.ncbi.nlm.nih.gov/pubmed/16284251>

Vargas DY, Raj A, Marras SA, Kramer FR, Tyagi S.

Proc Natl Acad Sci U S A. 2005 Nov 22;102(47):17008-13. Epub 2005 Nov 11.

PMID:
    16284251


This paper is cute in that they designed their molecular beacons by
looking at the rules for siRNA/miRNA and breaking all of them:

Live-cell, temporal gene expression analysis of osteogenic
differentiation in adipose-derived stem cells.
<http://www.ncbi.nlm.nih.gov/pubmed/22840182>

Desai HV, Voruganti IS, Jayasuriya C, Chen Q, Darling EM.

Tissue Eng Part A. 2013 Jan;19(1-2):40-8. doi:
10.1089/ten.TEA.2012.0127. Epub 2012 Sep 5.

PMID:
    22840182



An alternative to the Biosearch oligo probes approach is to use branched
DNA - see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338343/ 
(acdbio.com) and Panomics/Affymetrix QuantiGene ViewRNA ISH
http://www.panomics.com/products/rna-in-situ-analysis/viewrna-ish-cell-assay/overview 
... two companies licensed the same approach. Branched DNA ISH has been
around for a long time - Dr. Farhad Moatamed, West los Angeles VA,
showed me this circa 1995 when I was at UIC (MetaMorph customer).

Enjoy,

George


On 12/3/2013 9:47 PM, Feinstein, Timothy wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> For the many of you who have more experience in histology, do you prefer any particular label for immunostaining a challenging protein?  I understand that some proteins cannot be reliably imaged in their native state using confocal.  Nonetheless it seems like there must be better and worse label choices for those close calls.
>
> Years ago I might have warned people away from red and far red labels due to Low PMT semsitivity, but GaAsP detectors in those channels seem sensitive enough to moot that.  Red or far red seem preferable since most tissues have relatively low autofluorescence in that range.  Maybe labels in the 594-647 range are now better, or was that always true?
>
> Any input appreciated.
>
> Cheers,
>
>
> TF
>
> Timothy Feinstein, Ph.D. | Confocal Manager, Van Andel Institute
> 333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503<x-apple-data-detectors://0/0>
> Phone: [phone]<tel:[phone]>  | Email: [hidden email]<mailto:[hidden email]>
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/26/

[Martin Wessendorf-2]

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Dear Dr. Feinstein--

On 12/3/2013 9:47 PM, Feinstein, Timothy wrote:

> For the many of you who have more experience in histology, do you prefer any particular label for immunostaining a challenging protein?  I understand that some proteins cannot be reliably imaged in their native state using confocal.  Nonetheless it seems like there must be better and worse label choices for those close calls.

I vote for Cy3.  Get it from Jackson ImmunoResearch (--I'm a happy user
but not commercially associated) and make certain not to run the
antibody through a freeze-thaw cycle----reconstitute it in 50:50
water-glycerol and store it at -20.

Good luck!

Martin Wessendorf
--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Jacqueline Ross]

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Hi Tim,

My vote goes for Alexa 594 especially if you plan to combine it with a green fluorophore as it has less bleedthrough issues and is very stable. If you have autofluorescence problems, then I would go for Alexa 488 as it's easier to discriminate the autofluorescence by eye (green/yellow) if you have a LP barrier filter in your microscope. You can then make sure that you avoid capturing the autofluorescence when using the confocal (use a narrow filter or select narrow emission band if spectral).

Cheers,

Jacqui

Jacqueline Ross
Biomedical Imaging Microscopist
Biomedical Imaging Research Unit 
School of Medical Sciences 
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142, NEW ZEALAND

Tel: [phone]
Fax: [phone]

http://www.fmhs.auckland.ac.nz/sms/biru/

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Feinstein, Timothy
Sent: Wednesday, 4 December 2013 4:47 p.m.
To: [hidden email]
Subject: Best label for low-abundance staining

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*****

For the many of you who have more experience in histology, do you prefer any particular label for immunostaining a challenging protein?  I understand that some proteins cannot be reliably imaged in their native state using confocal.  Nonetheless it seems like there must be better and worse label choices for those close calls.

Years ago I might have warned people away from red and far red labels due to Low PMT semsitivity, but GaAsP detectors in those channels seem sensitive enough to moot that.  Red or far red seem preferable since most tissues have relatively low autofluorescence in that range.  Maybe labels in the 594-647 range are now better, or was that always true?

Any input appreciated.

Cheers,


TF

Timothy Feinstein, Ph.D. | Confocal Manager, Van Andel Institute
333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503<x-apple-data-detectors://0/0>
Phone: [phone]<tel:[phone]> | Email: [hidden email]<mailto:[hidden email]>

[Glen MacDonald-2]

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My vote would also be for Alexa594, but don’t even think about combining it with a far red label such as Cy5.  It will excite with the 633-650 wavelength.  If using far red, then stick with Alexa 568.  You should also use a bandpass filter for the green channel if labeling with A594.
You may also consider display as grayscale or pseudo color to green in color merge displays.    Which would argue for Alexa488.  I’ve heard the DyLights are equally bright, but no experience with them.  

Glen MacDonald
        Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
        Cellular Morphology Core
Center on Human Development and Disability
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]







On Dec 4, 2013, at 3:10 PM, Jacqui Ross <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Tim,
>
> My vote goes for Alexa 594 especially if you plan to combine it with a green fluorophore as it has less bleedthrough issues and is very stable. If you have autofluorescence problems, then I would go for Alexa 488 as it's easier to discriminate the autofluorescence by eye (green/yellow) if you have a LP barrier filter in your microscope. You can then make sure that you avoid capturing the autofluorescence when using the confocal (use a narrow filter or select narrow emission band if spectral).
>
> Cheers,
>
> Jacqui
>
> Jacqueline Ross
> Biomedical Imaging Microscopist
> Biomedical Imaging Research Unit
> School of Medical Sciences
> Faculty of Medical & Health Sciences
> The University of Auckland
> Private Bag 92019
> Auckland 1142, NEW ZEALAND
>
> Tel: [phone]
> Fax: [phone]
>
> http://www.fmhs.auckland.ac.nz/sms/biru/
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Feinstein, Timothy
> Sent: Wednesday, 4 December 2013 4:47 p.m.
> To: [hidden email]
> Subject: Best label for low-abundance staining
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> For the many of you who have more experience in histology, do you prefer any particular label for immunostaining a challenging protein?  I understand that some proteins cannot be reliably imaged in their native state using confocal.  Nonetheless it seems like there must be better and worse label choices for those close calls.
>
> Years ago I might have warned people away from red and far red labels due to Low PMT semsitivity, but GaAsP detectors in those channels seem sensitive enough to moot that.  Red or far red seem preferable since most tissues have relatively low autofluorescence in that range.  Maybe labels in the 594-647 range are now better, or was that always true?
>
> Any input appreciated.
>
> Cheers,
>
>
> TF
>
> Timothy Feinstein, Ph.D. | Confocal Manager, Van Andel Institute
> 333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503<x-apple-data-detectors://0/0>
> Phone: [phone]<tel:[phone]> | Email: [hidden email]<mailto:[hidden email]>

[Feinstein, Timothy]

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Many thanks to everyone who replied on and off list.  Your replies were
very helpful.  

Cheers,


TF

Timothy Feinstein, Ph.D. | Confocal Manager
333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503
Phone: [phone] | Email: [hidden email]







On 12/4/13, 7:04 PM, "Glen MacDonald" <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://scanmail.trustwave.com/?c=129&d=38Of0mX5NOS-IBaxqS6mT5ZNDzZSaptHAfr
>uVTA0Zg&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalmic
>roscopy
>*****
>
>My vote would also be for Alexa594, but don¹t even think about combining
>it with a far red label such as Cy5.  It will excite with the 633-650
>wavelength.  If using far red, then stick with Alexa 568.  You should
>also use a bandpass filter for the green channel if labeling with A594.
>You may also consider display as grayscale or pseudo color to green in
>color merge displays.    Which would argue for Alexa488.  I¹ve heard the
>DyLights are equally bright, but no experience with them.
>
>Glen MacDonald
> Core for Communication Research
>Virginia Merrill Bloedel Hearing Research Center
> Cellular Morphology Core
>Center on Human Development and Disability
>Box 357923
>University of Washington
>Seattle, WA 98195-7923  USA
>[phone]
>[hidden email]
>
>
>
>
>
>
>
>On Dec 4, 2013, at 3:10 PM, Jacqui Ross <[hidden email]>
>wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>>
>>http://scanmail.trustwave.com/?c=129&d=4MOf0gutlkQPetnA-2plDJR7JhiSxD7jcs
>>IrK5-OKg&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalm
>>icroscopy
>> *****
>>
>> Hi Tim,
>>
>> My vote goes for Alexa 594 especially if you plan to combine it with a
>>green fluorophore as it has less bleedthrough issues and is very stable.
>>If you have autofluorescence problems, then I would go for Alexa 488 as
>>it's easier to discriminate the autofluorescence by eye (green/yellow)
>>if you have a LP barrier filter in your microscope. You can then make
>>sure that you avoid capturing the autofluorescence when using the
>>confocal (use a narrow filter or select narrow emission band if
>>spectral).
>>
>> Cheers,
>>
>> Jacqui
>>
>> Jacqueline Ross
>> Biomedical Imaging Microscopist
>> Biomedical Imaging Research Unit
>> School of Medical Sciences
>> Faculty of Medical & Health Sciences
>> The University of Auckland
>> Private Bag 92019
>> Auckland 1142, NEW ZEALAND
>>
>> Tel: [phone]
>> Fax: [phone]
>>
>>
>>http://scanmail.trustwave.com/?c=129&d=4MOf0gutlkQPetnA-2plDJR7JhiSxD7jcp
>>QofZaPeA&u=http%3a%2f%2fwww%2efmhs%2eauckland%2eac%2enz%2fsms%2fbiru%2f
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>>[mailto:[hidden email]] On Behalf Of Feinstein, Timothy
>> Sent: Wednesday, 4 December 2013 4:47 p.m.
>> To: [hidden email]
>> Subject: Best label for low-abundance staining
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>>
>>http://scanmail.trustwave.com/?c=129&d=4MOf0gutlkQPetnA-2plDJR7JhiSxD7jcs
>>IrK5-OKg&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalm
>>icroscopy
>> *****
>>
>> For the many of you who have more experience in histology, do you
>>prefer any particular label for immunostaining a challenging protein?  I
>>understand that some proteins cannot be reliably imaged in their native
>>state using confocal.  Nonetheless it seems like there must be better
>>and worse label choices for those close calls.
>>
>> Years ago I might have warned people away from red and far red labels
>>due to Low PMT semsitivity, but GaAsP detectors in those channels seem
>>sensitive enough to moot that.  Red or far red seem preferable since
>>most tissues have relatively low autofluorescence in that range.  Maybe
>>labels in the 594-647 range are now better, or was that always true?
>>
>> Any input appreciated.
>>
>> Cheers,
>>
>>
>> TF
>>
>> Timothy Feinstein, Ph.D. | Confocal Manager, Van Andel Institute
>> 333 Bostwick Ave., N.E., Grand Rapids, Michigan
>>49503<x-apple-data-detectors://0/0>
>> Phone: [phone]<tel:[phone]> | Email:
>>[hidden email]<mailto:[hidden email]>