3D volume rendering and measurement

[Joan Sim]

Dear all,
 
I am completely new to image analysis from confocal microscopy and I would
like to seek advices about which softwares to use for 3D volume rendering and
calculation of the volume after obtaining Z-stacks of immunostained Drosophila
larvae brains. I briefly know that ImageJ (free) and Amira (expensive) are
some of the available softwares, but can ImageJ perform 3D volume rendering
and calculation?
 
I would greatly appreciate any advices given. Thank you!
 
Best regards,
Joan, PhD student
Singapore

[Tim O'Brien Sr.]

One program that you might want to try is ImageSurfer.  It is free, which is a plus, and can do 3D rendering as an isosurface or volume rendering, and several color datasets can be shown together.  It has a really convenient slice extractor tool that lets you measure relationships between the datasets.  (It cannot measure volumes, per se, yet).  It is available from our CISMM website at UNC.  We are an NIBIB resource, so are interested in providing tools that Biologists (and other imaging scientists) will actually use. Download the program from  http://cismm.cs.unc.edu/downloads/.  There are also a number of other free tools for analyzing data from microscopy.  The software is constantly being updated vis a vis feedback from users.  It is good to have a good graphics card and a fair amount of ram memory when using it. 

Thanks,
Tim



firstname Joan Sim wrote:

Dear all, 
 
I am completely new to image analysis from confocal microscopy and I would 
like to seek advices about which softwares to use for 3D volume rendering and 
calculation of the volume after obtaining Z-stacks of immunostained Drosophila 
larvae brains. I briefly know that ImageJ (free) and Amira (expensive) are 
some of the available softwares, but can ImageJ perform 3D volume rendering 
and calculation? 
 
I would greatly appreciate any advices given. Thank you! 
 
Best regards,
Joan, PhD student
Singapore 

  

[Beavers, Nick]

Hi Joan,

 

If you are interested in Commerical Software for 3D rendering and measurement of confocal immunostained z-stacks you can also check out Image-Pro Plus 3D from Media Cybernetics.

 

http://www.mediacy.com/index.aspx?page=IP_3D_analysis

 

You can separately view, threshold, count, and measure channels of fluorescence and then overlay them, change transparency, calculate intensities, and create movies of your renderings to share later.

 

I work and train with the product every day and I’ve never seen anything quite as robust on the market. There are many great free packages available to you as others have mentioned, but if you’re interested in something with a Tech Support line to call and online and in-person training classes, I would definitely take a look at Image-Pro Plus 3D.

 

I hope this is helpful.

 

Regards,

 

 

Nick Beavers

Media Cybernetics, Inc.

phone: [phone]

web: www.mediacy.com

email: [hidden email]

 

 

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Tim O'Brien
Sent: Wednesday, September 16, 2009 5:17 PM
To: [hidden email]
Subject: Re: 3D volume rendering and measurement

 

One program that you might want to try is ImageSurfer.  It is free, which is a plus, and can do 3D rendering as an isosurface or volume rendering, and several color datasets can be shown together.  It has a really convenient slice extractor tool that lets you measure relationships between the datasets.  (It cannot measure volumes, per se, yet).  It is available from our CISMM website at UNC.  We are an NIBIB resource, so are interested in providing tools that Biologists (and other imaging scientists) will actually use. Download the program from  http://cismm.cs.unc.edu/downloads/.  There are also a number of other free tools for analyzing data from microscopy.  The software is constantly being updated vis a vis feedback from users.  It is good to have a good graphics card and a fair amount of ram memory when using it. 

Thanks,
Tim



firstname Joan Sim wrote:

Dear all, 

I am completely new to image analysis from confocal microscopy and I would 

like to seek advices about which softwares to use for 3D volume rendering and 

calculation of the volume after obtaining Z-stacks of immunostained Drosophila 

larvae brains. I briefly know that ImageJ (free) and Amira (expensive) are 

some of the available softwares, but can ImageJ perform 3D volume rendering 

and calculation? 

I would greatly appreciate any advices given. Thank you! 

Best regards,

Joan, PhD student

Singapore 

 

 

 

 

 

 

---

CONFIDENTIALITY NOTICE: THIS E-MAIL TRANSMISSION AND ITS ATTACHMENTS CONTAIN CONFIDENTIAL & PROPRIETARY INFORMATION OF PRINCETON INSTRUMENTS, ACTON RESEARCH, MEDIA CYBERNETICS, AND THEIR AFFILIATES AND IS INTENDED FOR THE EXCLUSIVE AND CONFIDENTIAL USE OF THE INTENDED RECIPIENT.  ANY USE, DISSEMINATION, PRINTING OR COPYING OF THIS TRANSMISSION AND ITS ATTACHMENT(S) IS STRICTLY PROHIBITED.  IF YOU ARE NOT THE INTENDED RECIPIENT, PLEASE DO NOT READ, PRINT, COPY, DISTRIBUTE OR TAKE ACTION IN RELIANCE UPON THIS MESSAGE.  IF YOU HAVE RECEIVED THIS MESSAGE IN ERROR, PLEASE NOTIFY THE SENDER IMMEDIATELY BY TELEPHONE OR RETURN E-MAIL AND PROMPTLY DELETE ALL COPIES OF THE ORGINAL TRANSMISSION AND ITS ATTACHMENTS FROM YOUR COMPUTER SYSTEM.

---

 

 

 

 

 

 

 

 

 

[Gitta Hamel]

Hello Joan,

As a commercial solution for volume rendering and 3D analysis I'd like to draw your attention to the Huygens software for analysis, rendering and deconvolution in 2D up to 5D.

The analysis and rendering are closely integrated to give you all possibilities to measure for example 3D volumes, and to quantify and co-localize your data.

Huygens has by default modules to deconvolve your images. Image stacks (also multi-channel or time series) will benefit greatly from deconvolution and give much more resolution and remove blur while restoring all your signal. This will significantly improve your data and subsequent analysis.  

http://www.svi.nl/support/wiki/ObjectAnalyzer

Movies can be made and very soon object tracking will be available.

Huygens runs on Windows, Mac and Linux and any standard graphical card will do.
Test-licenses are freely available on http://www.svi.nl/download

Our WIKI gives you 24 hour support while we offer training both via the web and in small groups with max 6 persons, we analyze tough data with you and can be called from 8-18 hours for telephone support while email support is covered from 8 to 22 hours during weekdays.

With best regards,

Gitta Hamel

**************************************
Gitta Hamel
Managing Director Scientific Volume Imaging bv
[hidden email]
tel: ++ 31 35 6 42 16 26
***************************************

Beavers, Nick wrote:

Hi Joan,

 

If you are interested in Commerical Software for 3D rendering and measurement of confocal immunostained z-stacks you can also check out Image-Pro Plus 3D from Media Cybernetics.

 

http://www.mediacy.com/index.aspx?page=IP_3D_analysis

 

You can separately view, threshold, count, and measure channels of fluorescence and then overlay them, change transparency, calculate intensities, and create movies of your renderings to share later.

 

I work and train with the product every day and I’ve never seen anything quite as robust on the market. There are many great free packages available to you as others have mentioned, but if you’re interested in something with a Tech Support line to call and online and in-person training classes, I would definitely take a look at Image-Pro Plus 3D.

 

I hope this is helpful.

 

Regards,

 

 

Nick Beavers

Media Cybernetics, Inc.

phone: [phone]

web: www.mediacy.com

email: [hidden email]

 

 

 

From: Confocal Microscopy List [[hidden email]] On Behalf Of Tim O'Brien
Sent: Wednesday, September 16, 2009 5:17 PM
To: [hidden email]
Subject: Re: 3D volume rendering and measurement

 

One program that you might want to try is ImageSurfer.  It is free, which is a plus, and can do 3D rendering as an isosurface or volume rendering, and several color datasets can be shown together.  It has a really convenient slice extractor tool that lets you measure relationships between the datasets.  (It cannot measure volumes, per se, yet).  It is available from our CISMM website at UNC.  We are an NIBIB resource, so are interested in providing tools that Biologists (and other imaging scientists) will actually use. Download the program from  http://cismm.cs.unc.edu/downloads/.  There are also a number of other free tools for analyzing data from microscopy.  The software is constantly being updated vis a vis feedback from users.  It is good to have a good graphics card and a fair amount of ram memory when using it. 

Thanks,
Tim



firstname Joan Sim wrote:

Dear all, 

I am completely new to image analysis from confocal microscopy and I would 

like to seek advices about which softwares to use for 3D volume rendering and 

calculation of the volume after obtaining Z-stacks of immunostained Drosophila 

larvae brains. I briefly know that ImageJ (free) and Amira (expensive) are 

some of the available softwares, but can ImageJ perform 3D volume rendering 

and calculation? 

I would greatly appreciate any advices given. Thank you! 

Best regards,

Joan, PhD student

Singapore 

 

 

 

 

 

 

---

CONFIDENTIALITY NOTICE: THIS E-MAIL TRANSMISSION AND ITS ATTACHMENTS CONTAIN CONFIDENTIAL & PROPRIETARY INFORMATION OF PRINCETON INSTRUMENTS, ACTON RESEARCH, MEDIA CYBERNETICS, AND THEIR AFFILIATES AND IS INTENDED FOR THE EXCLUSIVE AND CONFIDENTIAL USE OF THE INTENDED RECIPIENT.  ANY USE, DISSEMINATION, PRINTING OR COPYING OF THIS TRANSMISSION AND ITS ATTACHMENT(S) IS STRICTLY PROHIBITED.  IF YOU ARE NOT THE INTENDED RECIPIENT, PLEASE DO NOT READ, PRINT, COPY, DISTRIBUTE OR TAKE ACTION IN RELIANCE UPON THIS MESSAGE.  IF YOU HAVE RECEIVED THIS MESSAGE IN ERROR, PLEASE NOTIFY THE SENDER IMMEDIATELY BY TELEPHONE OR RETURN E-MAIL AND PROMPTLY DELETE ALL COPIES OF THE ORGINAL TRANSMISSION AND ITS ATTACHMENTS FROM YOUR COMPUTER SYSTEM.

---

 

 

 

 

 

 

 

 

 

[Renato A. Mortara]

Hello,

 

I wonder if I could get some feedback on how two doubts;

 

1) How well does the solid-state 405 laser performs with DAPI-labeled samples (Cells, tissues, etc.) with not-so-UV-transparent objectives ?

 

I currently run an old BioRad 1024UV and with the Coherent 622 Inova laser (351/363 lines) even with  not-so-UV-transparent objectives such as a 63x PlanApo and a 100X 1.4 PlanApo (both from Zeiss), by using DAPI at a relatively high concentration (20-50 uM) we get very nice signal.

 

2) Has anyone tried the 150x 1.45 NA Olympus objective (presumably designed for TIRF) with conventional/confocal fluorescence ? Is the small WD a problem ?

 

 

Any input will be greatly appreciated,

 

Best regards,

Renato

 

Renato A. Mortara

Parasitology Division

UNIFESP - Escola Paulista de Medicina

Rua Botucatu, 862, 6th floor

São Paulo, SP

04023-062

Brazil

Phone: [phone]

Fax:     [phone]

email: [hidden email]

home page: www.ecb.epm.br/~ramortara

[Colin Rickman-2]

Hi,

I can't help for the first point but the 150x Olympus objective is a
fantastic objective. I have used it for TIRFM, 2D, 3D and 4D widefield
fluorescence microscopy, single molecule imaging and PALM. If you can
afford it and can live with the quite long lead time I can definitely
recommend it.

Regards

Colin

--
Dr Colin Rickman
Centre for Integrative Physiology
School of Biomedical Sciences
University of Edinburgh
Hugh Robson Building
George Square
Edinburgh
EH8 9XD

Tel: [phone]
Fax: [phone]



Renato Mortara wrote:

> Hello,
>  
> I wonder if I could get some feedback on how two doubts;
>  
> 1) How well does the solid-state 405 laser performs with DAPI-labeled
> samples (Cells, tissues, etc.) with not-so-UV-transparent objectives ?
>  
> I currently run an old BioRad 1024UV and with the Coherent 622 Inova
> laser (351/363 lines) even with  not-so-UV-transparent objectives such
> as a 63x PlanApo and a 100X 1.4 PlanApo (both from Zeiss), by using
> DAPI at a relatively high concentration (20-50 uM) we get very nice
> signal.
>  
> 2) Has anyone tried the 150x 1.45 NA Olympus objective (presumably
> designed for TIRF) with conventional/confocal fluorescence ? Is the
> small WD a problem ?
>  
>  
> Any input will be greatly appreciated,
>  
> Best regards,
>
> Renato
>  
> Renato A. Mortara
> Parasitology Division
> UNIFESP - Escola Paulista de Medicina
> Rua Botucatu, 862, 6th floor
> São Paulo, SP
> 04023-062
> Brazil
> Phone: [phone]
> Fax:     [phone]
> email: [hidden email] <mailto:[hidden email]>
> home page: www.ecb.epm.br/~ramortara

The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.

[Goodhouse, Joseph G.]

405 works perfectly fine with either Dapi or Hoechst

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato Mortara
Sent: Tuesday, September 22, 2009 9:39 AM
To: [hidden email]
Subject: DAPI on a 405 laser and 150x 1.45 objective

 

Hello,

 

I wonder if I could get some feedback on how two doubts;

 

1) How well does the solid-state 405 laser performs with DAPI-labeled samples (Cells, tissues, etc.) with not-so-UV-transparent objectives ?

 

I currently run an old BioRad 1024UV and with the Coherent 622 Inova laser (351/363 lines) even with  not-so-UV-transparent objectives such as a 63x PlanApo and a 100X 1.4 PlanApo (both from Zeiss), by using DAPI at a relatively high concentration (20-50 uM) we get very nice signal.

 

2) Has anyone tried the 150x 1.45 NA Olympus objective (presumably designed for TIRF) with conventional/confocal fluorescence ? Is the small WD a problem ?

 

 

Any input will be greatly appreciated,

 

Best regards,

Renato

 

Renato A. Mortara

Parasitology Division

UNIFESP - Escola Paulista de Medicina

Rua Botucatu, 862, 6th floor

São Paulo, SP

04023-062

Brazil

Phone: [phone]

Fax:     [phone]

email: [hidden email]

home page: www.ecb.epm.br/~ramortara

[Ghafar Sarvestani]

Hello Renato,

We have a Coherent solid-state 405 laser in our Nikon confocal microscope and we use it regularly for capturing images from DAPI-labelled samples. It performs very well with Nikon 20x and 60x Plan Apo Water objectives. Of course it depends to the intensity level of Dapi-labelled samples and the level of 405 laser power you use.

Ghafar

***********************************

Ghafar T Sarvestani, PhD

Detmold Imaging Centre

Hanson Institute/IMVS

 

T 08 8222 3603 M 040 25177123

F 08 8222 3162

www.imvs.sa.gov.au

 

IMVS

PO Box 14 Rundle Mall, Adelaide, SA, 5000

 

Quality Pathology supporting Training and Research

 

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato Mortara
Sent: Tuesday, 22 September 2009 11:09 PM
To: [hidden email]
Subject: DAPI on a 405 laser and 150x 1.45 objective

Hello,

 

I wonder if I could get some feedback on how two doubts;

 

1) How well does the solid-state 405 laser performs with DAPI-labeled samples (Cells, tissues, etc.) with not-so-UV-transparent objectives ?

 

I currently run an old BioRad 1024UV and with the Coherent 622 Inova laser (351/363 lines) even with  not-so-UV-transparent objectives such as a 63x PlanApo and a 100X 1.4 PlanApo (both from Zeiss), by using DAPI at a relatively high concentration (20-50 uM) we get very nice signal.

 

2) Has anyone tried the 150x 1.45 NA Olympus objective (presumably designed for TIRF) with conventional/confocal fluorescence ? Is the small WD a problem ?

 

 

Any input will be greatly appreciated,

 

Best regards,

Renato

 

Renato A. Mortara

Parasitology Division

UNIFESP - Escola Paulista de Medicina

Rua Botucatu, 862, 6th floor

São Paulo, SP

04023-062

Brazil

Phone: [phone]

Fax:     [phone]

email: [hidden email]

home page: www.ecb.epm.br/~ramortara

[Stephen Cody-2]

G'day Renato,

 

I'm not sure I would describe 405nm as UV, I tend to call it visible violet. 405nm should be transmitted very well by most objectives. Transmission curves for Olympus objectives can be found at http://microscope.olympus.com/uis2/ . I could not find any objective listed there with poor transmission at 405nm.

 

When doing Ca2+ imaging with Fura-2 a combination of 340nm and 380nm is often used for excitation and certainly in order to transmitt 405nm efficiently a UV objective should be used. But if no UV transmitting objectives are available it ispossible to use 360nm and 380nm combination instead. You should not have problems with Visible Violet transmission at 405nm with any objective.

 

Having said that some objectives are chormatically corrected at 405nm, which can improve imaging especially when using confocal. 

Cheers

Stephen Cody

 

2009/9/22 Renato Mortara <[hidden email]>

Hello,

 

I wonder if I could get some feedback on how two doubts;

 

1) How well does the solid-state 405 laser performs with DAPI-labeled samples (Cells, tissues, etc.) with not-so-UV-transparent objectives ?

 

I currently run an old BioRad 1024UV and with the Coherent 622 Inova laser (351/363 lines) even with  not-so-UV-transparent objectives such as a 63x PlanApo and a 100X 1.4 PlanApo (both from Zeiss), by using DAPI at a relatively high concentration (20-50 uM) we get very nice signal.

 

2) Has anyone tried the 150x 1.45 NA Olympus objective (presumably designed for TIRF) with conventional/confocal fluorescence ? Is the small WD a problem ?

 

 

Any input will be greatly appreciated,

 

Best regards,

Renato

 

Renato A. Mortara

Parasitology Division

UNIFESP - Escola Paulista de Medicina

Rua Botucatu, 862, 6th floor

São Paulo, SP

04023-062

Brazil

Phone: [phone]

Fax:     [phone]

email: [hidden email]

home page: www.ecb.epm.br/~ramortara

--
Stephen H. Cody

[Craig Brideau]

Stephen: I agree, technically 405 is 'violet' not UV.  At best you
might call it 'near UV' in the same way a Ti:Saph laser is 'near IR'.
Most objectives that can handle 'blue' can usually handle 'violet', in
terms of raw power transmission through the objective.  Your mileage
may vary for chromatic correction, however.

Craig


On Tue, Sep 22, 2009 at 10:41 PM, Stephen Cody <[hidden email]> wrote:

> G'day Renato,
>
> I'm not sure I would describe 405nm as UV, I tend to call it visible violet.
> 405nm should be transmitted very well by most objectives. Transmission
> curves for Olympus objectives can be found at
> http://microscope.olympus.com/uis2/ . I could not find any objective listed
> there with poor transmission at 405nm.
>
> When doing Ca2+ imaging with Fura-2 a combination of 340nm and 380nm is
> often used for excitation and certainly in order to transmitt 405nm
> efficiently a UV objective should be used. But if no UV transmitting
> objectives are available it ispossible to use 360nm and 380nm combination
> instead. You should not have problems with Visible Violet transmission at
> 405nm with any objective.
>
> Having said that some objectives are chormatically corrected at 405nm, which
> can improve imaging especially when using confocal.
> Cheers
> Stephen Cody
>
> 2009/9/22 Renato Mortara <[hidden email]>
>>
>> Hello,
>>
>> I wonder if I could get some feedback on how two doubts;
>>
>> 1) How well does the solid-state 405 laser performs with DAPI-labeled
>> samples (Cells, tissues, etc.) with not-so-UV-transparent objectives ?
>>
>> I currently run an old BioRad 1024UV and with the Coherent 622 Inova laser
>> (351/363 lines) even with  not-so-UV-transparent objectives such as a 63x
>> PlanApo and a 100X 1.4 PlanApo (both from Zeiss), by using DAPI at a
>> relatively high concentration (20-50 uM) we get very nice signal.
>>
>> 2) Has anyone tried the 150x 1.45 NA Olympus objective (presumably
>> designed for TIRF) with conventional/confocal fluorescence ? Is the small WD
>> a problem ?
>>
>>
>> Any input will be greatly appreciated,
>>
>> Best regards,
>> Renato
>>
>> Renato A. Mortara
>> Parasitology Division
>> UNIFESP - Escola Paulista de Medicina
>> Rua Botucatu, 862, 6th floor
>> São Paulo, SP
>> 04023-062
>> Brazil
>> Phone: [phone]
>> Fax:     [phone]
>> email: [hidden email]
>> home page: www.ecb.epm.br/~ramortara
>
>
> --
> Stephen H. Cody
>

[Stephen Cody-2]

Sorry List my previous email was a little confusing. It should have read:

 

When doing Ca2+ imaging with Fura-2 a combination of 340nm and 380nm is often used for excitation and certainly in order to transmitt "340nm" efficiently a UV objective should be used. But if no UV transmitting objectives are available it is possible to use 360nm and 380nm combination instead. You should not have problems with Visible Violet transmission at 405nm with any objective.

2009/9/23 Stephen Cody <[hidden email]>

G'day Renato,

 

I'm not sure I would describe 405nm as UV, I tend to call it visible violet. 405nm should be transmitted very well by most objectives. Transmission curves for Olympus objectives can be found at http://microscope.olympus.com/uis2/ . I could not find any objective listed there with poor transmission at 405nm.

 

When doing Ca2+ imaging with Fura-2 a combination of 340nm and 380nm is often used for excitation and certainly in order to transmitt 405nm efficiently a UV objective should be used. But if no UV transmitting objectives are available it ispossible to use 360nm and 380nm combination instead. You should not have problems with Visible Violet transmission at 405nm with any objective.

 

Having said that some objectives are chormatically corrected at 405nm, which can improve imaging especially when using confocal. 

Cheers

Stephen Cody

 

2009/9/22 Renato Mortara <[hidden email]>

Hello,

 

I wonder if I could get some feedback on how two doubts;

 

1) How well does the solid-state 405 laser performs with DAPI-labeled samples (Cells, tissues, etc.) with not-so-UV-transparent objectives ?

 

I currently run an old BioRad 1024UV and with the Coherent 622 Inova laser (351/363 lines) even with  not-so-UV-transparent objectives such as a 63x PlanApo and a 100X 1.4 PlanApo (both from Zeiss), by using DAPI at a relatively high concentration (20-50 uM) we get very nice signal.

 

2) Has anyone tried the 150x 1.45 NA Olympus objective (presumably designed for TIRF) with conventional/confocal fluorescence ? Is the small WD a problem ?

 

 

Any input will be greatly appreciated,

 

Best regards,

Renato

 

Renato A. Mortara

Parasitology Division

UNIFESP - Escola Paulista de Medicina

Rua Botucatu, 862, 6th floor

São Paulo, SP

04023-062

Brazil

Phone: [phone]

Fax:     [phone]

email: [hidden email]

home page: www.ecb.epm.br/~ramortara

--
Stephen H. Cody

--
Stephen H. Cody

[Rietdorf, Jens]

Dear Renato,
 
even though 405 is clearly suboptimal for DAPI excitation I have never had any problems with any of the lenses in our facility. There is an alternative to DAPI though that even improves the overall efficiency by at least 10fold in our hands, I repost a comment from Jason A. Kilgore on Hoechst 34580 below. Overstaining with Dapi typically generates background caused by binding to other nucleic acid species, extensive illumination with 405 and below can increase autofluorescence, both of which will reduce S/N remarckably.
 
Concerning your question about the 150xTIRF, that is an objective tuned for single molecule work, I see no reason to use it for conventional or confocal, except maybe for the widefield in combination with a camera with very large pixels, in which case still I would rather try an optovar or other additional magnifying lens before I spend this amount of money.
 
Best wishes, jens
 
------
 
Hoechst 34580 would be a better choice than DAPI (or the other Hoechst
dyes), as it excites at 392nm (DAPI excites around 350) and is readily
excited at 405nm.

Hoechst 34580 is available from Molecular Probes (#H21486).

Cheers,

Jason

*Commercial association acknowledged*


Jason A. Kilgore

________________________________

From: Confocal Microscopy List on behalf of Renato Mortara
Sent: Tue 9/22/2009 15:39
To: [hidden email]
Subject: DAPI on a 405 laser and 150x 1.45 objective


Hello,
 
I wonder if I could get some feedback on how two doubts;
 
1) How well does the solid-state 405 laser performs with DAPI-labeled samples (Cells, tissues, etc.) with not-so-UV-transparent objectives ?
 
I currently run an old BioRad 1024UV and with the Coherent 622 Inova laser (351/363 lines) even with  not-so-UV-transparent objectives such as a 63x PlanApo and a 100X 1.4 PlanApo (both from Zeiss), by using DAPI at a relatively high concentration (20-50 uM) we get very nice signal.
 
2) Has anyone tried the 150x 1.45 NA Olympus objective (presumably designed for TIRF) with conventional/confocal fluorescence ? Is the small WD a problem ?
 
 
Any input will be greatly appreciated,
 
Best regards,

Renato
 
Renato A. Mortara
Parasitology Division
UNIFESP - Escola Paulista de Medicina
Rua Botucatu, 862, 6th floor
São Paulo, SP
04023-062
Brazil
Phone: [phone]
Fax:     [phone]
email: [hidden email]
home page: www.ecb.epm.br/~ramortara