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Long working distance oil immersion objective

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Lucas Cahill

Long working distance oil immersion objective

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Hi all,

Does anyone have recommendations for long working distance oil immersion objectives?

I am currently using a 25X Ziess objective with ~0.5mm working distance to image BABB cleared tissue but would like image deeper.

Best,

Lucas

Lucas Cahill

PhD Candidate | Medical Engineering & Medical Physics

Harvard-MIT Health Sciences & Technology

Armstrong, Brian

Re: Long working distance oil immersion objective

Hi Lucas, you could use something like: Zeiss W Plan-Apo 20x/1.0 with 2.3mm Working distance. This lens, and most lenses for intra-vital imaging applications, will be water immersion and not oil. I believe Leica made a BABB lens for a better match of RI.

Zeiss does make a lens for CLARITY, and they have a lens with 5.6mm of working distance, but it has a 32mm thread. In short, these lenses are designed with specific uses and microscopes in mind. These lenses are large and may not fit on your microscope unless you have an upright fixed stage MP system. I realize that there is an RI mismatch with water and BABB but water immersion lenses may still be your best choice.

Best of luck,

Brian Armstrong PhD

Associate Research Professor

Developmental and Stem Cell Biology

Diabetes and Metabolic Diseases

Director, Light Microscopy Core

Beckman Research Institute, City of Hope

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Lucas Cahill
Sent: Monday, December 12, 2016 3:45 PM
To: [hidden email]
Subject: Long working distance oil immersion objective

Hi all,

Does anyone have recommendations for long working distance oil immersion objectives?

I am currently using a 25X Ziess objective with ~0.5mm working distance to image BABB cleared tissue but would like image deeper.

Best,

Lucas

Lucas Cahill

PhD Candidate | Medical Engineering & Medical Physics

Harvard-MIT Health Sciences & Technology

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Douglas Richardson

Re: Long working distance oil immersion objective

Do not use a water dipping objective with BABB. The solvent will dissolve the glue that holds your front lens in place and destroy the objective over time. Also the spherical aberration from the RI mismatch will not allow you to image very deep.

The 25x multi immersion mention by Lucas is the best I've found from Zeiss. I asked about a longer WD low mag oil objective 1-2 years ago, but got the impression I was the only one asking. If more people think this would be useful, please pass it along to your reps!

We also routinely use their 10x/0.45NA air objective when we need a longer working distance in solvent cleared samples. The Z resolution suffers a bit, but can be somewhat restored by deconvolution. Usually if you're imaging something that big, you're not looking for Sub-cellular resolution anyway.

Also, you may want to consider switching to DBE (3dico, Idisco) it's a little safer for everyone else in the lab.

Doug

Sent from my iPhone

On Dec 12, 2016, at 7:22 PM, Armstrong, Brian <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Lucas, you could use something like: Zeiss W Plan-Apo 20x/1.0 with 2.3mm Working distance. This lens, and most lenses for intra-vital imaging applications, will be water immersion and not oil. I believe Leica made a BABB lens for a better match of RI.

Zeiss does make a lens for CLARITY, and they have a lens with 5.6mm of working distance, but it has a 32mm thread. In short, these lenses are designed with specific uses and microscopes in mind. These lenses are large and may not fit on your microscope unless you have an upright fixed stage MP system. I realize that there is an RI mismatch with water and BABB but water immersion lenses may still be your best choice.

Best of luck,

Brian Armstrong PhD

Associate Research Professor

Developmental and Stem Cell Biology

Diabetes and Metabolic Diseases

Director, Light Microscopy Core

Beckman Research Institute, City of Hope

From: Confocal Microscopy List [[hidden email]] On Behalf Of Lucas Cahill
Sent: Monday, December 12, 2016 3:45 PM
To: [hidden email]
Subject: Long working distance oil immersion objective

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi all,

Does anyone have recommendations for long working distance oil immersion objectives?

I am currently using a 25X Ziess objective with ~0.5mm working distance to image BABB cleared tissue but would like image deeper.

Best,

Lucas

--

Lucas Cahill

PhD Candidate | Medical Engineering & Medical Physics

Harvard-MIT Health Sciences & Technology

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Craig Brideau

Re: Long working distance oil immersion objective

It's interesting there hasn't been much development in a lens like this. Many of the clearing methods use harsh solvents that also need to be used as the mounting/immersion medium. Given that these solvents will dissolve a typical microscope objective, the sample is usually placed in an imaging chamber with a coverslip lid. It seems to me that a LWD oil lens with a collar to compensate for the solvent/medium and sample depth would be ideal for something like this.

Craig

On Mon, Dec 12, 2016 at 6:55 PM, Doug Richardson <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****
Do not use a water dipping objective with BABB. The solvent will dissolve the glue that holds your front lens in place and destroy the objective over time. Also the spherical aberration from the RI mismatch will not allow you to image very deep.

The 25x multi immersion mention by Lucas is the best I've found from Zeiss. I asked about a longer WD low mag oil objective 1-2 years ago, but got the impression I was the only one asking. If more people think this would be useful, please pass it along to your reps!

We also routinely use their 10x/0.45NA air objective when we need a longer working distance in solvent cleared samples. The Z resolution suffers a bit, but can be somewhat restored by deconvolution. Usually if you're imaging something that big, you're not looking for Sub-cellular resolution anyway.

Also, you may want to consider switching to DBE (3dico, Idisco) it's a little safer for everyone else in the lab.

Doug

Sent from my iPhone

On Dec 12, 2016, at 7:22 PM, Armstrong, Brian <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Lucas, you could use something like: Zeiss W Plan-Apo 20x/1.0 with 2.3mm Working distance. This lens, and most lenses for intra-vital imaging applications, will be water immersion and not oil. I believe Leica made a BABB lens for a better match of RI.

Zeiss does make a lens for CLARITY, and they have a lens with 5.6mm of working distance, but it has a 32mm thread. In short, these lenses are designed with specific uses and microscopes in mind. These lenses are large and may not fit on your microscope unless you have an upright fixed stage MP system. I realize that there is an RI mismatch with water and BABB but water immersion lenses may still be your best choice.

Best of luck,

Brian Armstrong PhD

Associate Research Professor

Developmental and Stem Cell Biology

Diabetes and Metabolic Diseases

Director, Light Microscopy Core

Beckman Research Institute, City of Hope

From: Confocal Microscopy List [[hidden email]] On Behalf Of Lucas Cahill
Sent: Monday, December 12, 2016 3:45 PM
To: [hidden email]
Subject: Long working distance oil immersion objective

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi all,

Does anyone have recommendations for long working distance oil immersion objectives?

I am currently using a 25X Ziess objective with ~0.5mm working distance to image BABB cleared tissue but would like image deeper.

Best,

Lucas

--

Lucas Cahill

PhD Candidate | Medical Engineering & Medical Physics

Harvard-MIT Health Sciences & Technology

---------------------------------------------------------------------
*SECURITY/CONFIDENTIALITY WARNING:
This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender. (fpc5p)
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James Pawley

Re: Long working distance oil immersion objective

In reply to this post by Lucas Cahill

Really can’t suggest using an NA 1, dipping water (RI = 1.33) lens with BABB (RI = 1.5681) because, at such high-NA, you would end up wth massive spherical aberration even a few micrometers into the BABB.

Zeiss used to make relatively long working distance, oil (RI = 1.518) objectives for 160mm tube length microscopes, but they seem to have vanished with the switch to infinity correction and as I remember they only had a limited WD..

The problem is that manufacturers see two situations in which one might want to have a long distance between the front of the objective and the focus plane: When looking through air (and the bottom of a culture dish) to a focus plane in the cells above the glass and 2) using a dipping lens to look through material close to the RI of water to a focus plane a mm or so inside a biological specimen that is immersed in water (or saline). In neither of these cases does one find a continuous RI of about 1.52 all the way from the front of the objective to the focus plane mm in front of the objective (as would (almost) be the case if you looked at your BABB specimen floating in oil). I neglect here the large number of long-WD “air” objectives as these are only corrected to focus either with nothing but air or only a coverslip (or dish) of specific thickness between the focused plane and the front of the objective.

You could use an air lens designed for looking through the bottom of a Petri Dish at the cells above and having a spherical aberration correction collar allowing you to correct for between 0 and 2 mm of the glass in the bottom of the dish. Although the RI of coverglass is not quite the same as that of oil or BABB, it is close and the collar can be adjusted continuously, so a fudge is possible.

For example, the Nikon CFI 40x .55NA has a correction collar that corrects for 0-2.0 mm of glass and allows a free WD of 1.7-2.7 mm (depending, I suppose on the setting of the correction collar). If you put a thin coverslip on your specimen and set the collar for 2 mm of glass you would get a fairly aberration-free image if you focused on a plane inside your specimen that was about 2 mm on the other side of the air-glass surface of the coverslip. The problem is that the aberrations would build up when the air gap changed as you focused above or below this plane. When shifting focus in the direction of the coverslip, you could compensate for this change by resetting the collar to less than 2 mm but this would be fussy and you would be likely to bump the specimen out of alignment.

Only one company that I know of allows you to optically adjust SA-correction away from the objective and that is the mSAC system of III

https://www.intelligent-imaging.com/accessories.php#msac

But this is quite expensive and I don’t know for sure how much SA it could correct in your situation as the system is normally used with aqueous specimens.

One last thought. Optical engineers can only correct for SA in a high-NA optical system if the positions of the object and image planes are fixed and known. As a result, even assuming that you have an oil objective and an oil-immersed specimen, the image will only be sharp as long as the intermediate image picked up by the camera projection lens (or the eyepiece) is that formed exactly 1cm below the edge of the ocular tube. Move the camera lens or the ocular backward or forward (hard to do without a hacksaw and not recommended!) and the SA correction will no longer be correct. So, if you were to construct your own microscope in which a long-WD 160mm-tubelength) 40x NA.8 aqueous dipping lens (or an infinity tube length objective and its matching tube lens) looked down into a dish containing your oily (or watery) specimen, it would produce a sharp image at its focus plane if either:

1) the object were immersed in water and located at the focus plane about equal to the WD in front of the objective AND the camera was at the “normal” location above the fixed-conjugate objective or the infinity objective plus tube lens (i.e, normal operation, the same optics as in the microscope)

2) if the object were completely immersed in some liquid having a higher RI than water (i.e., BABB) and the camera or ocular were placed much closer to the back of the objective. I don’t know how much closer but quite a lot.

Moving the new intermediate image plane closer to the objective will of course reduce the magnification by a proportional amount and it is possible that, because the RI is so far from that for which the lens is designed, the image at the non-ab erratic plane, might be only 10x larger than the object even though you are using a 40x objective (i.e. the camera will only be about 25% of the normal distance from the tube lens. This is why you can’t really do it "On the microscope"). But don’t despair, with a little effort, you can magnify this relatively aberration-free 10x intermediate image as much as you want by changing the camera optics or the eyepiece. Be sure to use a dipping water lens for this game, not an air lens because although 1.33 is a LONG way from 1.57, it is a lot closer than 1.0.

I know this sounds bleak, but as SA only becomes very severe at high NA, you may have to just settle for low-NA or get someone to build you a special long-WD oil lens.

Good luck and tell us if you get something that works,

Jim Pawley

James and Christine Pawley, 5446 Burley Place, Box 2348, Sechelt BC, Canada, V0N3A0 [hidden email], Phone 1-604-885-0840, cell 1-604-989-6146

On Dec 12, 2016, at 3:45 PM, Lucas Cahill <[hidden email]> wrote:

BABB

Rosemary.White

Re: Long working distance oil immersion objective

Warning - mention of specific companies, no commercial interest.

Hi all,

Re. Jim’s comment about adjusting the correction collar on the fly, Leica sell 40x and 63x objectives with motorised correction collars that might work if you needed to adjust this continuously while focusing through a big sample. http://www.leica-microsystems.com/products/confocal-microscopes/details/product/leica-motcorr/

I imagine the other companies do something similar. An expensive solution, but perhaps one that might be needed for many cleared samples in future.

cheers,

Rosemary

p.s. No, we don’t have one of these, though would be curious to test one.

Dr Rosemary White

CSIRO Black Mountain

GPO Box 1700

Canberra, ACT 2601

Australia

Adjunct Prof, EH Graham Centre, CSU & Research School of Biology, ANU

T 61 2 6246 5475

E [hidden email]

From: Confocal Microscopy List <[hidden email]> on behalf of JAMES B PAWLEY <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Date: Tuesday, 13 December 2016 2:57 pm
To: "[hidden email]" <[hidden email]>
Subject: Re: Long working distance oil immersion objective

Only one company that I know of allows you to optically adjust SA-correction away from the objective and that is the mSAC system of III

https://www.intelligent-imaging.com/accessories.php#msac

But this is quite expensive and I don’t know for sure how much SA it could correct in your situation as the system is normally used with aqueous specimens.

I know this sounds bleak, but as SA only becomes very severe at high NA, you may have to just settle for low-NA or get someone to build you a special long-WD oil lens.

Good luck and tell us if you get something that works,

Jim Pawley

James and Christine Pawley, 5446 Burley Place, Box 2348, Sechelt BC, Canada, V0N3A0 [hidden email], Phone 1-604-885-0840, cell 1-604-989-6146

On Dec 12, 2016, at 3:45 PM, Lucas Cahill <[hidden email]> wrote:

BABB

Armstrong, Brian

Re: Long working distance oil immersion objective

In reply to this post by Douglas Richardson

Hi Lucas, just a couple more thoughts. I would not suggest dipping your water lens into BABB, I assumed you were dipping your 25x water immersion lens into water. Not only would you risk your lens but because the lens was designed with the 1.33 RI of water, dipping the lens into a solution such as BABB, Focus Clear or RIMS etc.. would not be beneficial anyway. We do a fair bit of imaging of cleared tissues that are large. We have many lenses including the 25x you are using as well as the 20x/1.0W and many different water and oil immersion lenses. I would have recommended the lens that you are already using because it is a fair compromise for your needs. It is because you want to exceed this performance where it becomes difficult (or expensive). The reality is that after all the tests and trials we ended up using the 10x/0.45 air lens for 90% of the imaging we perform on cleared tissues in part because we are capturing large tiled areas with many z-sections. Moreover, the labs I know that have these beautiful expensive specialized lenses leave them in their lovely wooden boxes on the shelf and use the 10x/0.45 air lens for the majority of their imaging as well.

With that being said, the proof is in the pudding and you should try some different lenses yourself and use what performs the best for you on your sample and microscope set-up, and do not feel disappointed if you end up using a 10x air objective.

Cheers,

Brian Armstrong PhD

Associate Research Professor

Developmental and Stem Cell Biology

Diabetes and Metabolic Diseases

Director, Light Microscopy Core

Beckman Research Institute, City of Hope

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Doug Richardson
Sent: Monday, December 12, 2016 5:55 PM
To: [hidden email]
Subject: Re: Long working distance oil immersion objective

Also, you may want to consider switching to DBE (3dico, Idisco) it's a little safer for everyone else in the lab.

Doug

Sent from my iPhone

On Dec 12, 2016, at 7:22 PM, Armstrong, Brian <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Lucas, you could use something like: Zeiss W Plan-Apo 20x/1.0 with 2.3mm Working distance. This lens, and most lenses for intra-vital imaging applications, will be water immersion and not oil. I believe Leica made a BABB lens for a better match of RI.

Zeiss does make a lens for CLARITY, and they have a lens with 5.6mm of working distance, but it has a 32mm thread. In short, these lenses are designed with specific uses and microscopes in mind. These lenses are large and may not fit on your microscope unless you have an upright fixed stage MP system. I realize that there is an RI mismatch with water and BABB but water immersion lenses may still be your best choice.

Best of luck,

Brian Armstrong PhD

Associate Research Professor

Developmental and Stem Cell Biology

Diabetes and Metabolic Diseases

Director, Light Microscopy Core

Beckman Research Institute, City of Hope

From: Confocal Microscopy List [[hidden email]] On Behalf Of Lucas Cahill
Sent: Monday, December 12, 2016 3:45 PM
To: [hidden email]
Subject: Long working distance oil immersion objective

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi all,

Does anyone have recommendations for long working distance oil immersion objectives?

I am currently using a 25X Ziess objective with ~0.5mm working distance to image BABB cleared tissue but would like image deeper.

Best,

Lucas

--

Lucas Cahill

PhD Candidate | Medical Engineering & Medical Physics

Harvard-MIT Health Sciences & Technology

---------------------------------------------------------------------
*SECURITY/CONFIDENTIALITY WARNING:
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