water immersion objectives for deep nonlinear imaging

[Nick Durr]

Dear List,
We've been using the older olympus XLUMPFL 20x 0.95 NA objective, and have
been getting decent images at large imaging depths. But for two reasons we're
looking into purchasing a new objective. First, when we do point spread
functions measurements for two-photon fluorescence at 780nm excitation with
this lens, we always get measurements that are significantly larger than the
size predicted from the diffraction limit (~30% bigger laterally and ~50% bigger
axially). We're careful about index matching, overfilling the back aperture, etc.,
and can get close to diffraction limited PSFs with other lenses, so I believe this
is a limitation of the lens, probably at NIR wavelengths. Second, we'd like to
get a lens that can be used with a coverslip (preferably one with a correction
collar).

It seems there are at least two objective lenses I can find that would be good
for our upgrade (we also want to have a working distance ~> 1mm): the
olympus 25x 1.05 NA W, and the nikon CFI Apo LWD 25x 1.1 NA W.

Their specs are very similar, so I'm wondering if anyone has had any experience
characterizing them, or can suggest other options that I haven't found. They
both say they are "corrected" out to 950-1000nm, but based on our experience
with the older olympus, I'm doubtful they perform at the diffraction limit in this
range. The Leica HCX 20x 1.0 also seems nice, but doesn't have a correction
collar. Has anyone done PSF measurements on any of these objectives? Or
compared them in any way?
Best Wishes,
-Nick

[mmodel]

Dear Nick -

Objectives vary even within the same brand/type. I think that most people who wish to get a perfect objective test them before buying.

Mike

________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Nick Durr [[hidden email]]
Sent: Thursday, June 10, 2010 9:17 PM
To: [hidden email]
Subject: water immersion objectives for deep nonlinear imaging

Dear List,
We've been using the older olympus XLUMPFL 20x 0.95 NA objective, and have
been getting decent images at large imaging depths. But for two reasons we're
looking into purchasing a new objective. First, when we do point spread
functions measurements for two-photon fluorescence at 780nm excitation with
this lens, we always get measurements that are significantly larger than the
size predicted from the diffraction limit (~30% bigger laterally and ~50% bigger
axially). We're careful about index matching, overfilling the back aperture, etc.,
and can get close to diffraction limited PSFs with other lenses, so I believe this
is a limitation of the lens, probably at NIR wavelengths. Second, we'd like to
get a lens that can be used with a coverslip (preferably one with a correction
collar).

It seems there are at least two objective lenses I can find that would be good
for our upgrade (we also want to have a working distance ~> 1mm): the
olympus 25x 1.05 NA W, and the nikon CFI Apo LWD 25x 1.1 NA W.

Their specs are very similar, so I'm wondering if anyone has had any experience
characterizing them, or can suggest other options that I haven't found. They
both say they are "corrected" out to 950-1000nm, but based on our experience
with the older olympus, I'm doubtful they perform at the diffraction limit in this
range. The Leica HCX 20x 1.0 also seems nice, but doesn't have a correction
collar. Has anyone done PSF measurements on any of these objectives? Or
compared them in any way?
Best Wishes,
-Nick

[Julio Vazquez]

Hi Nick, 

Nikon and Leica lenses will probably not fit on your Olympus microscope. Zeiss lenses (old W 0.8 mount, not the new M27 mount) will fit on Olympus, and we have sometimes used them interchangeably, although I have not checked PSFs to see how well they work in this context. Anyway, Zeiss has two relatively new lenses that look promising (though expensive!):

LD LCI PlanApo 25x/0,8 DIC Imm Corr (UV) VIS-IR (0.57 mm WD)

and

LD C-Apochromat 40x/1.1 W Corr UV-VIS-IR (0.62 mm WD)

Seems that those might have been designed specifically for 2P work. Haven't used them, so can't comment on their imaging properties.

one lens we found pretty decent for 2P work is the Zeiss  40x/0.8 dipping lens; 3 mm WD and quite affordable.  It actually works fine on inverted stand through a coverslip, although a bit tricky to use...

--

Julio Vazquez

Fred Hutchinson Cancer Research Center

Seattle, WA 

http://www.fhcrc.org/ 

On Jun 10, 2010, at 6:17 PM, Nick Durr wrote:

Dear List,

We've been using the older olympus XLUMPFL 20x 0.95 NA objective, and have 

been getting decent images at large imaging depths. But for two reasons we're 

looking into purchasing a new objective. First, when we do point spread 

functions measurements for two-photon fluorescence at 780nm excitation with 

this lens, we always get measurements that are significantly larger than the 

size predicted from the diffraction limit (~30% bigger laterally and ~50% bigger 

axially). We're careful about index matching, overfilling the back aperture, etc., 

and can get close to diffraction limited PSFs with other lenses, so I believe this 

is a limitation of the lens, probably at NIR wavelengths. Second, we'd like to 

get a lens that can be used with a coverslip (preferably one with a correction 

collar).

It seems there are at least two objective lenses I can find that would be good 

for our upgrade (we also want to have a working distance ~> 1mm): the 

olympus 25x 1.05 NA W, and the nikon CFI Apo LWD 25x 1.1 NA W.

Their specs are very similar, so I'm wondering if anyone has had any experience 

characterizing them, or can suggest other options that I haven't found. They 

both say they are "corrected" out to 950-1000nm, but based on our experience 

with the older olympus, I'm doubtful they perform at the diffraction limit in this 

range. The Leica HCX 20x 1.0 also seems nice, but doesn't have a correction 

collar. Has anyone done PSF measurements on any of these objectives? Or 

compared them in any way?

Best Wishes,

-Nick

[Nick Durr]

Hello Julio,
Thanks for the suggestions. We don't have an Olympus microscope, just the Olympus lens. All the parts for our microscope are put together individually and
are interchangeable, so we've used Nikon, Zeiss, Olympus lenses. The 40x Zeiss
does look nice, but we are hoping to get more than a 600um working distance
(20x is also better for deeper imaging). Best,
-Nick

On Thu, 10 Jun 2010 19:59:47 -0700, Julio Vazquez <[hidden email]>
wrote:

>Hi Nick,
>
>Nikon and Leica lenses will probably not fit on your Olympus
>microscope. Zeiss lenses (old W 0.8 mount, not the new M27 mount)
>will fit on Olympus, and we have sometimes used them interchangeably,
>although I have not checked PSFs to see how well they work in this
>context. Anyway, Zeiss has two relatively new lenses that look
>promising (though expensive!):
>
>LD LCI PlanApo 25x/0,8 DIC Imm Corr (UV) VIS-IR (0.57 mm WD)
>
>and
>
>LD C-Apochromat 40x/1.1 W Corr UV-VIS-IR (0.62 mm WD)
>
>Seems that those might have been designed specifically for 2P work.
>Haven't used them, so can't comment on their imaging properties.
>
>
>one lens we found pretty decent for 2P work is the Zeiss  40x/0.8
>dipping lens; 3 mm WD and quite affordable.  It actually works fine
>on inverted stand through a coverslip, although a bit tricky to use...
>
>
>--
>Julio Vazquez
>Fred Hutchinson Cancer Research Center
>Seattle, WA
>
>http://www.fhcrc.org/
>
>
>
>
>On Jun 10, 2010, at 6:17 PM, Nick Durr wrote:
>
>> Dear List,
>> We've been using the older olympus XLUMPFL 20x 0.95 NA objective,
>> and have
>> been getting decent images at large imaging depths. But for two
>> reasons we're
>> looking into purchasing a new objective. First, when we do point
>> spread
>> functions measurements for two-photon fluorescence at 780nm
>> excitation with
>> this lens, we always get measurements that are significantly larger
>> than the
>> size predicted from the diffraction limit (~30% bigger laterally
>> and ~50% bigger
>> axially). We're careful about index matching, overfilling the back
>> aperture, etc.,
>> and can get close to diffraction limited PSFs with other lenses, so
>> I believe this
>> is a limitation of the lens, probably at NIR wavelengths. Second,
>> we'd like to
>> get a lens that can be used with a coverslip (preferably one with a
>> correction
>> collar).
>>
>> It seems there are at least two objective lenses I can find that
>> would be good
>> for our upgrade (we also want to have a working distance ~> 1mm): the
>> olympus 25x 1.05 NA W, and the nikon CFI Apo LWD 25x 1.1 NA W.
>>
>> Their specs are very similar, so I'm wondering if anyone has had
>> any experience
>> characterizing them, or can suggest other options that I haven't
>> found. They
>> both say they are "corrected" out to 950-1000nm, but based on our
>> experience
>> with the older olympus, I'm doubtful they perform at the
>> diffraction limit in this
>> range. The Leica HCX 20x 1.0 also seems nice, but doesn't have a
>> correction
>> collar. Has anyone done PSF measurements on any of these
>> objectives? Or
>> compared them in any way?
>> Best Wishes,
>> -Nick
>
>

[Andreas Bruckbauer]

Dear Nick,
my own PSF measurements with the Olympus 25x NA 1.05 Objective when filling the back aperture (1/e^2 diameter equals he back focal aperture) gave PSFs which are about 18% larger in x and 20% in z at 800 nm than the theoretical value, maybe this can be improved when overfilling. The theoretical value was calculated following: Hess, S. T. & Webb, W. W. (2002) Biophys J, 83, 2300-2317 .The objective has  a higher transmission for IR than the 20x 0.95 from Olympus. According to the manufacturer, the lens is corrected only for the IR, not for visible light. This is sufficient for two photon with non descanned detectors. When you want to do confocal with visible excitation the 20x 0.95 is the better choice.

best wishes

Andreas

-----Original Message-----
From: Nick Durr <[hidden email]>
To: [hidden email]
Sent: Fri, 11 Jun 2010 2:17
Subject: water immersion objectives for deep nonlinear imaging

Dear List,


We've been using the older olympus XLUMPFL 20x 0.95 NA objective, and have 


been getting decent images at large imaging depths. But for two reasons we're 


looking into purchasing a new objective. First, when we do point spread 


functions measurements for two-photon fluorescence at 780nm excitation with 


this lens, we always get measurements that are significantly larger than the 


size predicted from the diffraction limit (~30% bigger laterally and ~50% bigger 





axially). We're careful about index matching, overfilling the back aperture, 


etc., 


and can get close to diffraction limited PSFs with other lenses, so I believe 


this 


is a limitation of the lens, probably at NIR wavelengths. Second, we'd like to 


get a lens that can be used with a coverslip (preferably one with a correction 


collar).





It seems there are at least two objective lenses I can find that would be good 


for our upgrade (we also want to have a working distance ~> 1mm): the 


olympus 25x 1.05 NA W, and the nikon CFI Apo LWD 25x 1.1 NA W.





Their specs are very similar, so I'm wondering if anyone has had any experience 


characterizing them, or can suggest other options that I haven't found. They 


both say they are "corrected" out to 950-1000nm, but based on our experience 


with the older olympus, I'm doubtful they perform at the diffraction limit in 


this 


range. The Leica HCX 20x 1.0 also seems nice, but doesn't have a correction 


collar. Has anyone done PSF measurements on any of these objectives? Or 


compared them in any way?


Best Wishes,


-Nick