How "deep" can you see in a brain slice?

[Jim Mansfield]

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Hi Jeremy,

Perhaps I am misunderstanding something, but I'm not sure the narrowness or
broadness of the excitation filter matters in the way you seem to think it
does.

For non-autofluorescent samples, photon collection and detection is
definitely the limiting factor for sensitivity. When there is background
autofluorescence, however, things are in a different regime and different
factors some into play.

But since the emission spectrum of a fluor is independent of the wavelength
of absorption, what matters is not what wavelength is being used for
excitation per se, but how many photons are being absorbed. So clearly the
absorption spectrum matters - a higher percentage of photons will be
absorbed at the peak absorption wavelength of the fluor than at other
wavelengths - but exactly which wavelength it is does not matter except in
an efficiency sense.

There is no harm in having a broad excitation range, at least in terms of
excitation efficiency. Every absorbed photon is a good photon, and since
most widefield light sources are white (or whiteish ;-) letting through
extra photons to the blue of the max absorption is helpful and increases
sensitivity ... although, having said that, one needs to consider the impact
of those on photobleaching and the tradeoffs vis-à-vis sensitivity.

For fluors with a small Stokes shift, there is also a tradeoff between
absorption efficiency and emission efficiency for any given
excitation/emission filter set since the excitation and emission wavelengths
cannot overlap. Ideally one would want to hit the max absorption peak and
image at the max emission peak but that can be difficult in practice, even
with the new filter sets that go from blocking to 99% transmission in only a
few nanometers.

I am not aware of any general rule for which way is best to lean in
practice: more towards hitting the absorption peak and shifting a bit to the
red on the emission; or more towards max emission throughput and shifting
the excitation a bit to the blue. Perhaps someone else can comment on that.

I hope that is helpful!

Jim

James R. Mansfield | Director, Tissue Analysis Applications
Caliper – a PerkinElmer Company | Life Sciences & Technology
68 Elm Street
Hopkinton, MA  01748 USA
Phone:  + 1 774 278 2802
Mobile:  [phone]
Email: [hidden email]
www.perkinelmer.com
www.caliperls.com

Need to send me large files? Use my YouSendIt DropBox:
https://dropbox.yousendit.com/Mansfield-Dropbox




-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Jeremy Adler
Sent: Thursday, February 09, 2012 6:01 AM
To: [hidden email]
Subject: efficient detection of fluorescence - widefield

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In fluorescence emitted photons are often the limiting factor so in  
widefield fluorescence filter blocks need to be optimised.

A possible problem is that for a molecule with a Stokes shift of say  
20nm the detector will only detect the shortest emitted photons it  
includes a range that starts at less than the Stokes shift, say  
excitation + 15nm. But looking at the width of excitation filters used  
in widefield fluoresence the excitation range,  perhaps 460/40, then  
many of the emitted photons will not even reach a detector with an  
emission filter set to exclude excitation light, perhaps a 490nm Long  
pass.
This argues that if photon detection efficiency is the primary concern  
then excitation ranges need to be narrow. In the real world the power  
output of widefield light sources is limited and an excitation range  
is needed to effective produce fluorescence is useful amounts and the  
emission spectrum of most fluorophores has a very long tail - but  
should consideration be given to the the excitation range with respect  
to the Stokes shift ?






Jeremy Adler
IGP
Rudbeckslaboratoriet
Daghammersköljdsväg 20
751 85 Uppsala
Sweden

0046 (0)18 471 4607

[Jeremy Adler-4]

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Hi Jim,

maybe I made my point badly.

It is not quite accurate to say that the emission spectrum is  
independant of the wavelength of absorbtion - emission involves a red  
shift and a molecule of alexa555 excited at 530nm can emit over a  
540+nm range (starting at the lower limit of the emission spectrum)  
while an excitation at 555nm would have a range starting at perhaps  
565+nm - missing the low end of emission spectrum.

For many fluorophores there is an overlap between the excitation and  
emission spectra. For Alexa555 it is attractive to excite between  
540-560nm and collect at 570+nm. The Stokes shift is around 15nm. With  
this combination of filters   fluorophores excited at the bottom end  
of excitation range (540nm) will emit a portion of their total  
emission in the 550-570nm range, assuming a minimum red shift of 10nm,  
and therefore these photons will not be detected. By contrast with a  
small excitation range 554-556nm almost all the emitted photons will  
be detected - which is more efficient. My question really comes down  
to wondering if the broad excitation ranges used in widefield filter  
blocks are really useful or there some attendant loss of detection  
efficiency

It also follows that excitation of alexa555 at 520nm, away from the  
absorption peak at 555nm, would allow for the dectection of all the  
emitted photons with at 540LP filter. Is there any downside to  
excitation away from the absorption peak ? - clearly more energy is  
being delivered for given emission and any related autofluorescence  
will be increased.



Quoting Jim Mansfield <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Jeremy,
>
> Perhaps I am misunderstanding something, but I'm not sure the narrowness or
> broadness of the excitation filter matters in the way you seem to think it
> does.
>
> For non-autofluorescent samples, photon collection and detection is
> definitely the limiting factor for sensitivity. When there is background
> autofluorescence, however, things are in a different regime and different
> factors some into play.
>
> But since the emission spectrum of a fluor is independent of the wavelength
> of absorption, what matters is not what wavelength is being used for
> excitation per se, but how many photons are being absorbed. So clearly the
> absorption spectrum matters - a higher percentage of photons will be
> absorbed at the peak absorption wavelength of the fluor than at other
> wavelengths - but exactly which wavelength it is does not matter except in
> an efficiency sense.
>
> There is no harm in having a broad excitation range, at least in terms of
> excitation efficiency. Every absorbed photon is a good photon, and since
> most widefield light sources are white (or whiteish ;-) letting through
> extra photons to the blue of the max absorption is helpful and increases
> sensitivity ... although, having said that, one needs to consider the impact
> of those on photobleaching and the tradeoffs vis-à-vis sensitivity.
>
> For fluors with a small Stokes shift, there is also a tradeoff between
> absorption efficiency and emission efficiency for any given
> excitation/emission filter set since the excitation and emission wavelengths
> cannot overlap. Ideally one would want to hit the max absorption peak and
> image at the max emission peak but that can be difficult in practice, even
> with the new filter sets that go from blocking to 99% transmission in only a
> few nanometers.
>
> I am not aware of any general rule for which way is best to lean in
> practice: more towards hitting the absorption peak and shifting a bit to the
> red on the emission; or more towards max emission throughput and shifting
> the excitation a bit to the blue. Perhaps someone else can comment on that.
>
> I hope that is helpful!
>
> Jim
>
> James R. Mansfield | Director, Tissue Analysis Applications
> Caliper – a PerkinElmer Company | Life Sciences & Technology
> 68 Elm Street
> Hopkinton, MA  01748 USA
> Phone:  + 1 774 278 2802
> Mobile:  [phone]
> Email: [hidden email]
> www.perkinelmer.com
> www.caliperls.com
>
> Need to send me large files? Use my YouSendIt DropBox:
> https://dropbox.yousendit.com/Mansfield-Dropbox
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On
> Behalf Of Jeremy Adler
> Sent: Thursday, February 09, 2012 6:01 AM
> To: [hidden email]
> Subject: efficient detection of fluorescence - widefield
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> In fluorescence emitted photons are often the limiting factor so in
> widefield fluorescence filter blocks need to be optimised.
>
> A possible problem is that for a molecule with a Stokes shift of say
> 20nm the detector will only detect the shortest emitted photons it
> includes a range that starts at less than the Stokes shift, say
> excitation + 15nm. But looking at the width of excitation filters used
> in widefield fluoresence the excitation range,  perhaps 460/40, then
> many of the emitted photons will not even reach a detector with an
> emission filter set to exclude excitation light, perhaps a 490nm Long
> pass.
> This argues that if photon detection efficiency is the primary concern
> then excitation ranges need to be narrow. In the real world the power
> output of widefield light sources is limited and an excitation range
> is needed to effective produce fluorescence is useful amounts and the
> emission spectrum of most fluorophores has a very long tail - but
> should consideration be given to the the excitation range with respect
> to the Stokes shift ?
>
>
>
>
>
>
> Jeremy Adler
> IGP
> Rudbeckslaboratoriet
> Daghammersköljdsväg 20
> 751 85 Uppsala
> Sweden
>
> 0046 (0)18 471 4607
>

Jeremy Adler
IGP
Rudbeckslaboratoriet
Daghammersköljdsväg 20
751 85 Uppsala
Sweden

0046 (0)18 471 4607

[mmodel]

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*****

I am not sure this is correct. No matter what frequency has been absorbed, the electron will slide to the lowest vibrational level of the excited band before it drops further down emitting a photon, and the emission spectrum will still be independent of the absorbed wavelength.  

Mike

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jeremy Adler
Sent: Thursday, February 09, 2012 9:58 AM
To: [hidden email]
Subject: Re: efficient detection of fluorescence - widefield

*****
To join, leave or search the confocal microscopy listserv, go to:
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Hi Jim,

maybe I made my point badly.

It is not quite accurate to say that the emission spectrum is  
independant of the wavelength of absorbtion - emission involves a red  
shift and a molecule of alexa555 excited at 530nm can emit over a  
540+nm range (starting at the lower limit of the emission spectrum)  
while an excitation at 555nm would have a range starting at perhaps  
565+nm - missing the low end of emission spectrum.

[Mike Ignatius]

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****


Hi,


Slightly late addition to this, but the depth penetration is of course also dependent on the reagent used.  


Upconverting (anti-stokes) reagents are excited at 980nm, with either 2P or any 980nm laser light and emit around 800nm while others emit in the visible.


Non-toxic, don't blink or bleach either.  They have very long lifetimes, so time gating is easily done.  
Their 2P brightness exceeds Qdot® fluors (see "Nanoparticles for highly efficient multiphoton fluorescence bioimaging." Laura Martinez Maestro, et al, Optics Express Vol 18, Nov 2010.)
So called luminescent upconverting phosphor nanocrystals (or Sunstones® for short) they are making headway in vivo imaging, cell tracking and the near elimination of backgrounds due to their upconversion makes them appealing to IHC apps as well.

Sigma site has much more info under Sunstones® @ http://www.sigmaaldrich.com/technical-documents/articles/biology/upconverting-ucp-nanocrystals.html 

I would be interested to hear comments from anyone that has tried them.


Mike Ignatius, Ph.D.






-----Original Message-----
From: Barbara Foster <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Sun, Feb 5, 2012 10:46 pm
Subject: Re: How "deep" can you see in a brain slice?


*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Hi,

I was shocked when I visited Neuroscience this past Fall and saw what
they were doing with special clearing agents and special objectives
with multiphoton.  The new limit is.... (ready?) at least 4 mm (no,
that is not microns) and I think that there may be some new
combinations which go as far as 8mm. This will be one of the topics
for the Editor's Page for the April issue of American Lab.  I will be
writing that article this next week and can get you the current stats then.

Good hunting!
Barbara Foster, President and Sr. Consultant

Microscopy/Microscopy Education
P: [phone]
W: www.MicroscopyEducation.com

We are now scheduling courses through June 2012

At 12:08 PM 2/5/2012, you wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>
>I'm no medical guy but I work for Japanese microscope company that
>will not be named.
>Brain isn't very translucent so I wouldn't hold out much hope. I
>would say 100um tops, maybe even closer to 50um. After this your
>image will start to degrade quite a bit. Any deeper than that and
>you are going to want a 2P-Confocal. I've imaged colloidal
>suspensions that are ~100um thick, and they are very close to being
>index matched. At the maximum depth the image wasn't that pretty.
>
>Maybe some more Neuro people will have a better answer for you.
>
>Good luck
>
>H
>
>Hugh Newman
>
>Graduate Researcher
>
>Dept. Physics and Physical Oceanography
>
>Memorial University
>
>St. Johns, Newfoundland, Canada
>
>
>
> > Date: Sun, 5 Feb 2012 13:53:33 -0600
> > From: [hidden email]
> > Subject: How "deep" can you see in a brain slice?
> > To: [hidden email]
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Dear all,
> > I am trying to view fluorescently labeled glioma cells invading
> into a 400um
> > thick brain slice on an Olypus FV300. Has anyone experience with this and
> > how "deep" it is reasonable to expect to see in the slice using a confocal
> > microscope? How can you maximize this depth? (selection of objectives,
> > processing of the slice....)
> > Thanks for any suggestions, Petr.
> >
> > Petr Busek, MD, PhD
> > Charles University in Prague
> > First Faculty of Medicine
> > Laboratory of Cancer Cell Biology
> > Institute of Biochemistry and Experimental Oncology
> > U Nemocnice 5
> > 128 53 Prague 2
> > Czech Republic
> > www.lf1.cuni.cz/lbnb
> >  Fax [phone]
>

 

 

[Lemasters, John J.]

Hi Mike,

Where are you now? Please reply privately to [hidden email].

Best regards, John

--
John J. Lemasters, MD, PhD
Professor and GlaxoSmithKline Distinguished Endowed Chair
Director, Center for Cell Death, Injury & Regeneration
Departments of Pharmaceutical & Biomedical Sciences and Biochemistry & Molecular Biology
Medical University of South Carolina
DD504 Drug Discovery Building
70 President Street, MSC 140
Charleston, SC 29425

Office: 843-876-2360
Lab: 843-876-2354
Fax: [phone]
Email: [hidden email]
http://academicdepartments.musc.edu/ccdir


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mike Ignatius
Sent: Friday, February 10, 2012 2:54 PM
To: [hidden email]
Subject: Re: How "deep" can you see in a brain slice?

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


Hi,


Slightly late addition to this, but the depth penetration is of course also dependent on the reagent used.  


Upconverting (anti-stokes) reagents are excited at 980nm, with either 2P or any 980nm laser light and emit around 800nm while others emit in the visible.


Non-toxic, don't blink or bleach either.  They have very long lifetimes, so time gating is easily done.  
Their 2P brightness exceeds Qdot® fluors (see "Nanoparticles for highly efficient multiphoton fluorescence bioimaging." Laura Martinez Maestro, et al, Optics Express Vol 18, Nov 2010.) So called luminescent upconverting phosphor nanocrystals (or Sunstones® for short) they are making headway in vivo imaging, cell tracking and the near elimination of backgrounds due to their upconversion makes them appealing to IHC apps as well.

Sigma site has much more info under Sunstones® @ http://www.sigmaaldrich.com/technical-documents/articles/biology/upconverting-ucp-nanocrystals.html 

I would be interested to hear comments from anyone that has tried them.


Mike Ignatius, Ph.D.






-----Original Message-----
From: Barbara Foster <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Sun, Feb 5, 2012 10:46 pm
Subject: Re: How "deep" can you see in a brain slice?


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

Hi,

I was shocked when I visited Neuroscience this past Fall and saw what they were doing with special clearing agents and special objectives with multiphoton.  The new limit is.... (ready?) at least 4 mm (no, that is not microns) and I think that there may be some new combinations which go as far as 8mm. This will be one of the topics for the Editor's Page for the April issue of American Lab.  I will be writing that article this next week and can get you the current stats then.

Good hunting!
Barbara Foster, President and Sr. Consultant

Microscopy/Microscopy Education
P: [phone]
W: www.MicroscopyEducation.com

We are now scheduling courses through June 2012

At 12:08 PM 2/5/2012, you wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>
>I'm no medical guy but I work for Japanese microscope company that will
>not be named.
>Brain isn't very translucent so I wouldn't hold out much hope. I would
>say 100um tops, maybe even closer to 50um. After this your image will
>start to degrade quite a bit. Any deeper than that and you are going to
>want a 2P-Confocal. I've imaged colloidal suspensions that are ~100um
>thick, and they are very close to being index matched. At the maximum
>depth the image wasn't that pretty.
>
>Maybe some more Neuro people will have a better answer for you.
>
>Good luck
>
>H
>
>Hugh Newman
>
>Graduate Researcher
>
>Dept. Physics and Physical Oceanography
>
>Memorial University
>
>St. Johns, Newfoundland, Canada
>
>
>
> > Date: Sun, 5 Feb 2012 13:53:33 -0600
> > From: [hidden email]
> > Subject: How "deep" can you see in a brain slice?
> > To: [hidden email]
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Dear all,
> > I am trying to view fluorescently labeled glioma cells invading
> into a 400um
> > thick brain slice on an Olypus FV300. Has anyone experience with
> > this and how "deep" it is reasonable to expect to see in the slice
> > using a confocal microscope? How can you maximize this depth?
> > (selection of objectives, processing of the slice....) Thanks for
> > any suggestions, Petr.
> >
> > Petr Busek, MD, PhD
> > Charles University in Prague
> > First Faculty of Medicine
> > Laboratory of Cancer Cell Biology
> > Institute of Biochemistry and Experimental Oncology U Nemocnice 5
> > 128 53 Prague 2
> > Czech Republic
> > www.lf1.cuni.cz/lbnb
> >  Fax [phone]
>

 

 

[Koo, Lily (NIH/NIAID) [E]]

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To join, leave or search the confocal microscopy listserv, go to:
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*****

Dear list,

I am curious about these clearing reagents but have not had any hands-on experience with them - I assume the underlying mechanism of "optical clearing" is refractive index matching?  Does this process also alleviate or eliminate autofluorescence?

Thanks,

Lily

-----Original Message-----
From: Mark Cannell [mailto:[hidden email]]
Sent: Monday, February 06, 2012 10:49 AM
To: [hidden email]
Subject: Re: How "deep" can you see in a brain slice?

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

Following a PM, here are some more references that come up in the obvious Pub Med search:

"Clearing tetrahydrofuran"

Am J Clin Pathol. 1959 Apr;31(4):357-61.
Tetrahydrofuran (THF) for routine dehydration, clearing, and infiltration.
HAUST MD.

Arch Dermatol. 1960 Nov;82:798-803.
Use of tetrahydrofuran for routine and rapid dehydration and clearing.
MALKINSON FD, POTTER B.

Still dismayed at the lack of references to prior work -or did they pluck this chemical out of thin air?

Regards Mark



On 6/02/2012, at 11:59 AM, Mark Cannell wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I must admit to being a bit surprised that the Nature methods paper did not cite the prior use of THF in clearing insects... This was in 1958!
>
> Tetrahydrofuran and its Use in Insect Histology
>
> T. N. Salthouse. The Canadian Entomologist, 1958, 90:555-557,
> 10.4039/Ent90555-9 Regards Mark
>
>
> On 5/02/2012, at 11:38 PM, George McNamara wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>>
>> Fixed and cleared: all the way:
>>
>> Three-dimensional imaging of the unsectioned adult spinal cord to
>> assess axon regeneration and glial responses after injury.
>> </pubmed/22198277> Ertürk A, Mauch CP, Hellal F, Förstner F, Keck T,
>> Becker K, Jährling N, Steffens H, Richter M, Hübener M, Kramer E,
>> Kirchhoff F, Dodt HU, Bradke F. Nat Med. 2011 Dec 25;18(1):166-71.
>> doi: 10.1038/nm.2600. PMID: 22198277
>>
>> Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. </pubmed/21878933> Hama H, Kurokawa H, Kawano H, Ando R, Shimogori T, Noda H, Fukami K, Sakaue-Sawano A, Miyawaki A. Nat Neurosci. 2011 Aug 30;14(11):1481-8. doi: 10.1038/nn.2928. PMID: 21878933.
>>
>> A colleague here at the U told me his lab had much better clearing and imaging with the Erturk et al method than with the versions of Hama et al's Scale that they tried (no, I do not know which many variants they tried or how extensively they tested each). This colleague told me that with the Erturk et al method they needed to image the same day (and the sooner the better). The Erturk et al method uses tetrahydrofuran (THF) to strip the lipids from the tissue, followed by immersion in benzyl alcohol:benzyl benzoate (BABB). BABB has a long history of use in optical clearing - see various papers by Bob Zucker, for examples:
>>
>> Whole insect and mammalian embryo imaging with confocal microscopy:
>> morphology and apoptosis. </pubmed/17051584>* *Zucker RM. Cytometry
>> A. 2006 Nov 1;69(11):1143-52. PMID: 17051584
>>
>> Confocal laser scanning microscopy of whole mouse ovaries: excellent
>> morphology, apoptosis detection, and spectroscopy.
>> </pubmed/16969804>* *Zucker RM, Jeffay SC. Cytometry A. 2006 Aug
>> 1;69(8):930-9. PMID: 16969804
>>
>> I will hypothesize here that 2,2'-thiodiethanol (TDE) might be a better ultimate destination after THF. For TDE see:
>>
>> 2,2'-thiodiethanol: a new water soluble mounting medium for high
>> resolution optical microscopy. </pubmed/17131355>* *Staudt T, Lang
>> MC, Medda R, Engelhardt J, Hell SW. Microsc Res Tech. 2007
>> Jan;70(1):1-9. PMID: 17131355
>>
>> See also Stan Vitha's post(s) here on transitioning specimens into TDE and imaging.
>>
>>
>> For fresh tissue - that is, hemisectioned mouse brain: sac the mouse, flush the RBCs, take out the brain, slice in half  (along a line that will bisect the glioma mass that you introduced by stereotaxic injection, being careful not to have cells up the needle track), bring to the confocal - a user of mine in L.A. on a Leica SP1 confocal, 10x objective lens (probably 0.4 NA), went 800 um. On a City of Hope LSM510/MP, I helped  image hemisectioned mouse brains previously implanted with GFP+ neural stem cells (Argon ion laser) plus DAPI (Coherent Chameleon laser, probably 750 nm excitation) several hundred micrometers deep. Again, one of the keys is to flush out the blood cells from the mouse vasculature - they scatter a lot more than mouse brain tissue. I have never been involved with brain slices - hopefully those protocols flush the blood cells after sac'ing the mouse.
>>
>> George
>>
>>
>>
>>
>> On 2/5/2012 2:53 PM, Petr Busek wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Dear all,
>>> I am trying to view fluorescently labeled glioma cells invading into
>>> a 400um thick brain slice on an Olypus FV300. Has anyone experience
>>> with this and how "deep" it is reasonable to expect to see in the
>>> slice using a confocal microscope? How can you maximize this depth?
>>> (selection of objectives, processing of the slice....) Thanks for
>>> any suggestions, Petr.
>>>
>>> Petr Busek, MD, PhD
>>> Charles University in Prague
>>> First Faculty of Medicine
>>> Laboratory of Cancer Cell Biology
>>> Institute of Biochemistry and Experimental Oncology U Nemocnice 5
>>> 128 53 Prague 2
>>> Czech Republic
>>> www.lf1.cuni.cz/lbnb
>>> Fax [phone]
>>>
>>>
>>
>>
>> --
>>
>>
>> George McNamara, PhD
>> Analytical Imaging Core Facility
>> University of Miami

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Dear Confocal Listserv,

PubSpectra dataset and more are now available at
http://works.bepress.com/gmcnamara/

I encourage those of you with old PMT based confocal microscopes to
check out the before and after replacement images in the .LIF file at

http://works.bepress.com/gmcnamara/8/

This is from our almost 5 year old Leica SP5 confocal microscope.


For those of you who are using - or should be using - the Leica SP5
confocal microscope for interference reflection contrast microscopy
(IRM), I encourage you to check out

http://works.bepress.com/gmcnamara/7/

and let me know by email if your instrument performs similarly.


The free Leica file viewer is available from
ftp://ftp.llt.de/softlib/LAS_AF_Lite/


Neutrophil fans and S. aureus fans may find my "The Chase" set of files
of interest.

Enjoy,

George

p.s. for those of you who downloaded Multi-Probe Microscopy from my
"selected works" site - thanks for downloading! Hopefully no one hit the
print button. I recently received the following usage statistics:

"McNamara 2011 MPMicro - Multi-Probe Microscopy (10/31/2011)"
174 full-text downloads since date of posting (2011-10-31)
http://works.bepress.com/gmcnamara/2

"Synergy STED multicolor 3D-EasySTED Excel file"
16 full-text downloads since date of posting (2011-11-04)
http://works.bepress.com/gmcnamara/3

"Tiki_Goddess (Pathscan)"
5 full-text downloads since date of posting (2011-10-29)
http://works.bepress.com/gmcnamara/1
Tiki is also available athttp://home.earthlink.net/~tiki_goddess/

 

George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility (AICF)
University of Miami, Miller School of Medicine

http://www.sylvester.org/AICF   (AICF home page)
PubSpectra data (XLSX file inside)
    http://www.sylvester.org/documents/PubSpectra.zip (download 2000+ spectra)
    http://works.bepress.com/gmcnamara/
PubSpectra / UA Graphing Site
     http://www.mcb.arizona.edu/ipc/fret/index.html   (Carl Boswell, now retired)
New UA Spectra Database Site
     http://www.spectra.arizona.edu/                  (Urs Utzinger)
UMiami Scholarly Repository "selected works"
     http://works.bepress.com/gmcnamara
Care to link?
     http://www.linkedin.com/in/georgemcnamara


Ready for imaging in 2012? Check out:


Miami 2012 Winter Symposium: Nanotechnology in Biomedicine
February 26-29, 2012, Miami, FL
Nature Publishing Group / University of Miami / Scripps Florida
http://www.nature.com/natureconferences/miami/mws2012/speakers.html

Association of Biomolecular Resource Facilities (ABRF)
International Symposium
March 17-20, 2012, Orlando, FL
http://conf.abrf.org/index.cfm

Biomedical Optics 2012 (OSA BIOMED) - Optical Society of America
April 29-May 2, 2012, Miami, FL
http://www.osa.org/meetings/topical_meetings/BIOMED/default.aspx



"Old soldiers never die, they just fade away." - Douglas Macarthur.
"Old antibodies die, please throw them away." - GM.

“Well of course you can’t understand your data, you have too many controls” - Anna M. Wu, quoted in Andreas Markus Loening, Ph.D. dissertation, UCLA, 2006.
"If you do all the controls, you'll never publish." - GM.
"If you don't do the controls, you shouldn't publish." ... alternative: "If you don't do the controls, don't waste everyone's time in lab meeting." - GM.