2X objective

[Caroline Bass]

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Hey Guys,

 

I suppose this isn't the proper forum for this question since I'm not using a confocal (although the company claims the scope is "confocal-like"), but I thought someone would be able to direct me.  I'm not very good with fluorescence really, so sorry if this question is naive.  I've been using an Olympus IX71 inverted fluorescent microscope.  The illumination source is an X-cite series 120.  There are three filters on the scope, a 470/525 for GFP, 560/630 for texas red/rfp, and some sort of multi filter that can visualize dapi and several other dyes. 

 

So here's the question, I'm visualizing GFP in rat brain, and I have a fabulous signal, but the lowest magnification objective is 10X.  Can someone suggest an objective that would allow me to take a larger view of the signal?  Something in the 2X to 4X range would be good.

 

I tried a 1.6X and 2X objective I pilfered off of another scope and it didn't work at all.  I couldn't focus on the tissue.  My current objectives are:

 

CPlan N

10X/0.25 PhC

 

LCPlanFl

20X/0.40 Ph1

 

LCPlanFL

40X/0.60 Ph2

 

LCPlan FL

60X/0.70 Ph2

 

There are two open slots in the turret.  Any suggestions would be helpful in terms of viewing this image.  I've tried mosaics, but they are very time consuming.

 

Thanks,

 

Caroline

 

[Craig Brideau]

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

What were the two objectives you tried that didn't work?

Craig


On 10/28/07, Caroline Bass <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hey Guys,
>
> I suppose this isn't the proper forum for this question since I'm not using
> a confocal (although the company claims the scope is "confocal-like"), but I
> thought someone would be able to direct me.  I'm not very good with
> fluorescence really, so sorry if this question is naive.  I've been using an
> Olympus IX71 inverted fluorescent microscope.  The illumination source is an
> X-cite series 120.  There are three filters on the scope, a 470/525 for GFP,
> 560/630 for texas red/rfp, and some sort of multi filter that can visualize
> dapi and several other dyes.
>
> So here's the question, I'm visualizing GFP in rat brain, and I have a
> fabulous signal, but the lowest magnification objective is 10X.  Can someone
> suggest an objective that would allow me to take a larger view of the
> signal?  Something in the 2X to 4X range would be good.
>
> I tried a 1.6X and 2X objective I pilfered off of another scope and it
> didn't work at all.  I couldn't focus on the tissue.  My current objectives
> are:
>
> CPlan N
> 10X/0.25 PhC
>
> LCPlanFl
> 20X/0.40 Ph1
>
> LCPlanFL
> 40X/0.60 Ph2
>
> LCPlan FL
> 60X/0.70 Ph2
>
> There are two open slots in the turret.  Any suggestions would be helpful in
> terms of viewing this image.  I've tried mosaics, but they are very time
> consuming.
>
> Thanks,
>
> Caroline
>

[Alexei Markevitch]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hello everybody,

 

Has you had experience integrating a Coherent Cube laser with Leica confocal microscope? I know a person who would like to do it (not subscribed here), and he would like to pick up brains from those who have done it already.

 

Thanks,

Alexei

---

Вы уже с Yahoo!? Испытайте обновленную и улучшенную. Yahoo! Почту!

[Craig Brideau]

Heh.  We're probably going to be replacing an Argon-Ion with
Spectra-Physics' cube-equvalent in a month or so.  I'll let you know
how it goes...

Craig

On 10/28/07, Alexei Markevitch <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> Hello everybody,
>
> Has you had experience integrating a Coherent Cube laser with Leica confocal
> microscope? I know a person who would like to do it (not subscribed here),
> and he would like to pick up brains from those who have done it already.
>
> Thanks,
> Alexei
>
>
>  ________________________________
>
> Вы уже с Yahoo!? Испытайте обновленную и улучшенную. Yahoo! Почту!
>
>

[K.K.Veeraraghavan]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

What was the Camera you were using to capture these images?

Probably you could try using a reducing C Mount lens to your CCD Camera to
get a wider field of view.

K.K.Veeraraghavan

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Craig Brideau
Sent: 29 October 2007 06:02
To: [hidden email]
Subject: Re: 2X objective

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

What were the two objectives you tried that didn't work?

Craig


On 10/28/07, Caroline Bass <[hidden email]> wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hey Guys,
>
> I suppose this isn't the proper forum for this question since I'm not
using
> a confocal (although the company claims the scope is "confocal-like"), but
I
> thought someone would be able to direct me.  I'm not very good with
> fluorescence really, so sorry if this question is naive.  I've been using
an
> Olympus IX71 inverted fluorescent microscope.  The illumination source is
an
> X-cite series 120.  There are three filters on the scope, a 470/525 for
GFP,
> 560/630 for texas red/rfp, and some sort of multi filter that can
visualize
> dapi and several other dyes.
>
> So here's the question, I'm visualizing GFP in rat brain, and I have a
> fabulous signal, but the lowest magnification objective is 10X.  Can
someone
> suggest an objective that would allow me to take a larger view of the
> signal?  Something in the 2X to 4X range would be good.
>
> I tried a 1.6X and 2X objective I pilfered off of another scope and it
> didn't work at all.  I couldn't focus on the tissue.  My current
objectives

> are:
>
> CPlan N
> 10X/0.25 PhC
>
> LCPlanFl
> 20X/0.40 Ph1
>
> LCPlanFL
> 40X/0.60 Ph2
>
> LCPlan FL
> 60X/0.70 Ph2
>
> There are two open slots in the turret.  Any suggestions would be helpful

in
> terms of viewing this image.  I've tried mosaics, but they are very time
> consuming.
>
> Thanks,
>
> Caroline
>

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[Martin Wessendorf]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Caroline Bass wrote:

> So here's the question, I'm visualizing GFP in rat brain, and I have a
> fabulous signal, but the lowest magnification objective is 10X.  Can
> someone suggest an objective that would allow me to take a larger view
> of the signal?  Something in the 2X to 4X range would be good.

Dear Carolyn--

We have an Olympus BX50 upright and have both a 2x/0.05 NA Plan, and a
4x/0.16 NA UPlan on it.  With strong labeling, the 4x is a nice little
objective and we use it a lot for run-of-the-mill scanning of
fluorescently labeling tissue and for low-mag imaging of a variety of
fluorophores.  The 2x is much less useful.  This is probably due to its
very low NA.  As you probably know, the brightness of a fluorescent
imaging system will be directly proportional to NA raised to the 4th
power and inversely proportional to magnification squared.  In this
case, that works out to (.16/.05=3.2)^4 = 104.8, divided by 2^2 = 4, or
26.2.  Thus the 4x objective should give a fluorescence image over 25
times brighter than the 2x objective.  In my experience, that's probably
about right.  The 2x just doesn't work well for most fluorescent
specimens.

I'd get the 4x.

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: martinw[at]med.umn.edu