Confocal Microscopy List

Basic live cell imaging question...

Classic

List

Threaded

35 messages Options

Dolphin, Colin

Basic live cell imaging question...

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

We would like to do live cell imaging - mammalian cell lines - but only have direct access to an upright Olympus BX61. We don't really need complicated perfusion chambers, etc just something simple. We're real neophytes so all suggestions gratefully received.

Colin

Mika Hukkanen

Re: Basic live cell imaging question...

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

Hi Colin, we do it almost on a daily basis and with various settings; simple
and relatively inexpensive - just get a long distance dip type objective
from Olympus. We have got 2 sets from Leica; 1 set for mammalian cells and 1
for infectious samples (viruses, bacteria etc) - all for live cell studies.
Numerical aperture is not high, but sufficient enough for single cell
studies. High NA dip objectives are very, very bulky and expensive, but
these ones with ceramic head (meant for physiological imaging) are within
very manageable budget. Olympus manufactures really excellent objectives,
just contact the local rep - or us for further info on the use of these 'dip
type' objectives etc.

Mika

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Dolphin, Colin
Sent: 29. lokakuuta 2010 22:01
To: [hidden email]
Subject: Basic live cell imaging question...

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

We would like to do live cell imaging - mammalian cell lines - but only have
direct access to an upright Olympus BX61. We don't really need complicated
perfusion chambers, etc just something simple. We're real neophytes so all
suggestions gratefully received.

Colin

leoncio vergara

Re: Basic live cell imaging question...

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

... for infectious samples, how do you handle the biosafety issues and decontamination?

________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Mika Hukkanen [[hidden email]]
Sent: Friday, October 29, 2010 3:07 PM
To: [hidden email]
Subject: Re: Basic live cell imaging question...

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

Hi Colin, we do it almost on a daily basis and with various settings; simple
and relatively inexpensive - just get a long distance dip type objective
from Olympus. We have got 2 sets from Leica; 1 set for mammalian cells and 1
for infectious samples (viruses, bacteria etc) - all for live cell studies.
Numerical aperture is not high, but sufficient enough for single cell
studies. High NA dip objectives are very, very bulky and expensive, but
these ones with ceramic head (meant for physiological imaging) are within
very manageable budget. Olympus manufactures really excellent objectives,
just contact the local rep - or us for further info on the use of these 'dip
type' objectives etc.

Mika

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Dolphin, Colin
Sent: 29. lokakuuta 2010 22:01
To: [hidden email]
Subject: Basic live cell imaging question...

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

We would like to do live cell imaging - mammalian cell lines - but only have
direct access to an upright Olympus BX61. We don't really need complicated
perfusion chambers, etc just something simple. We're real neophytes so all
suggestions gratefully received.

Colin

mmodel

Re: Basic live cell imaging question...

In reply to this post by Mika Hukkanen

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

If you don't need fluorescence you can observe live cells in Petri dishes using phase of Hoffman contrast.

Mike

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mika Hukkanen
Sent: Friday, October 29, 2010 4:08 PM
To: [hidden email]
Subject: Re: Basic live cell imaging question...

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

Hi Colin, we do it almost on a daily basis and with various settings; simple
and relatively inexpensive - just get a long distance dip type objective
from Olympus. We have got 2 sets from Leica; 1 set for mammalian cells and 1
for infectious samples (viruses, bacteria etc) - all for live cell studies.
Numerical aperture is not high, but sufficient enough for single cell
studies. High NA dip objectives are very, very bulky and expensive, but
these ones with ceramic head (meant for physiological imaging) are within
very manageable budget. Olympus manufactures really excellent objectives,
just contact the local rep - or us for further info on the use of these 'dip
type' objectives etc.

Mika

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Dolphin, Colin
Sent: 29. lokakuuta 2010 22:01
To: [hidden email]
Subject: Basic live cell imaging question...

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

We would like to do live cell imaging - mammalian cell lines - but only have
direct access to an upright Olympus BX61. We don't really need complicated
perfusion chambers, etc just something simple. We're real neophytes so all
suggestions gratefully received.

Colin

Dan Focht

Re: Basic live cell imaging question...commercial response

In reply to this post by Dolphin, Colin

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

Colin

Please take a detailed look at the Bioptechs website for a variety of live-cell imaging environmental control options.
www.bioptechs.com

Bioptechs develops and manufactures time-lapse microscopy chambering systems for all types of specimens.
It helps us a lot if you fill out our user profile questionnaire available from our home page.
This enables us to address all the important compatibility factors in selecting an environmental system.

You will find that we do not just sell something that heats and say good luck, we make sure that we provide you the best and most cost effective solution for your needs.

If you call us during 9:00 to 5:00 eastern time the phone will always be answered by a live human being.
You will also find we don't hide anything on our web site and you don't have to sign in or subscribe.
We make it as easy as we can for you.

Dan

On Oct 29, 2010, at 3:00 PM, Dolphin, Colin wrote:

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

We would like to do live cell imaging - mammalian cell lines - but only have direct access to an upright Olympus BX61. We don't really need complicated perfusion chambers, etc just something simple. We're real neophytes so all suggestions gratefully received.

Colin

Dan Focht
Bioptechs, Inc.
3560 Beck Rd.
Butler, PA 16002
www.bioptechs.com
P: (724)282-7145
F: (724)282-0745
[hidden email]

Mika Hukkanen

Re: Basic live cell imaging question...

In reply to this post by leoncio vergara

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

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Vergara, Leoncio A.
Sent: 29. lokakuuta 2010 23:12
To: [hidden email]
Subject: Re: Basic live cell imaging question...

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

... for infectious samples, how do you handle the biosafety issues and
decontamination?

________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf
Of Mika Hukkanen [[hidden email]]
Sent: Friday, October 29, 2010 3:07 PM
To: [hidden email]
Subject: Re: Basic live cell imaging question...

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

Hi Colin, we do it almost on a daily basis and with various settings; simple
and relatively inexpensive - just get a long distance dip type objective
from Olympus. We have got 2 sets from Leica; 1 set for mammalian cells and 1
for infectious samples (viruses, bacteria etc) - all for live cell studies.
Numerical aperture is not high, but sufficient enough for single cell
studies. High NA dip objectives are very, very bulky and expensive, but
these ones with ceramic head (meant for physiological imaging) are within
very manageable budget. Olympus manufactures really excellent objectives,
just contact the local rep - or us for further info on the use of these 'dip
type' objectives etc.

Mika

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Dolphin, Colin
Sent: 29. lokakuuta 2010 22:01
To: [hidden email]
Subject: Basic live cell imaging question...

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

We would like to do live cell imaging - mammalian cell lines - but only have
direct access to an upright Olympus BX61. We don't really need complicated
perfusion chambers, etc just something simple. We're real neophytes so all
suggestions gratefully received.

Colin

Gert van Cappellen-2

Re: Basic live cell imaging question...

In reply to this post by Dolphin, Colin

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

Culture your cells on a round coverslip. Take an object glas glue a
square piece of non-toxic rubber with a round hole on it. Fill this with
CO2 satured medium somewaht more as the volume of the hole. Put your
coverslip on it, with the cells to the medium off course. Press it
gently down and the glass will seal itself to the rubber ring. Now your
cells will survive for a couple of hours, so you can do the first
imaging. For real experiments you have to find a way to heat the object
glass to 37C.

Good luck, Gert

Op 29-10-2010 21:00, Dolphin, Colin schreef:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> We would like to do live cell imaging - mammalian cell lines - but only have direct access to an upright Olympus BX61. We don't really need complicated perfusion chambers, etc just something simple. We're real neophytes so all suggestions gratefully received.
>
> Colin
>

Axel Kurt Preuss

Re: Basic live cell imaging question...

You need a water immersion object or have to build one

Cheers

Axel
—————
Axel K Preuss, PhD,
Central Imaging, IMCB, A*Star, 61 Biopolis Dr, 6-19B, Singapore 138673, sent from 9271.5622

On Nov 4, 2010, at 4:06 AM, Gert van Cappellen <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Culture your cells on a round coverslip. Take an object glas glue a
> square piece of non-toxic rubber with a round hole on it. Fill this with
> CO2 satured medium somewaht more as the volume of the hole. Put your
> coverslip on it, with the cells to the medium off course. Press it
> gently down and the glass will seal itself to the rubber ring. Now your
> cells will survive for a couple of hours, so you can do the first
> imaging. For real experiments you have to find a way to heat the object
> glass to 37C.
>
> Good luck, Gert
>
> Op 29-10-2010 21:00, Dolphin, Colin schreef:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> We would like to do live cell imaging - mammalian cell lines - but only have direct access to an upright Olympus BX61. We don't really need complicated perfusion chambers, etc just something simple. We're real neophytes so all suggestions gratefully received.
>>
>> Colin
>>

Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

Axel Kurt Preuss

Re: Basic live cell imaging question...

In reply to this post by Gert van Cappellen-2

I just see previous comment . Yep, that works fine

Thanks
Cheers

Axel
—————
Axel K Preuss, PhD,
Central Imaging, IMCB, A*Star, 61 Biopolis Dr, 6-19B, Singapore 138673, sent from 9271.5622

On Nov 4, 2010, at 4:06 AM, Gert van Cappellen <[hidden email]> wrote:

Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

JOEL B. SHEFFIELD

Re: Basic live cell imaging question...

In reply to this post by Axel Kurt Preuss

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

I think Gert's response makes a lot sense. For a newbie, with limited goals
(initially), it is not necessary to have an elaborate setup. In the (very)
old days, there was something called a "hanging drop culture" in which
tissue fragments were placed in a drop of medium on a slide, the slide was
inverted and examined with a conventional microscope. Gert is suggesting a
modern version of the same, assuming that you would visualize the cells
through the cover slip to which they are attached, Ujnfortunately, the
original writer did not specify the kind of optics they intended to use. If
the cells are, indeed, attached to the cover slip, it is possible to use
properly adjusted phase or Hoffman optics in transmissionto increase the
contrast. If fluorescence is needed, then you have to be concerned with
light toxicity, etc., but the optics should work fine, as long as you don't
want to see nuclear speckles, or details of mitochondria.

Joel

On Wed, Nov 3, 2010 at 9:03 PM, Axel Kurt Preuss <
[hidden email]> wrote:

> You need a water immersion object or have to build one
>
>
> Cheers
>
> Axel
> —————
> Axel K Preuss, PhD,
> Central Imaging, IMCB, A*Star, 61 Biopolis Dr, 6-19B, Singapore 138673,
> sent from 9271.5622
>
>
> On Nov 4, 2010, at 4:06 AM, Gert van Cappellen <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Culture your cells on a round coverslip. Take an object glas glue a
> > square piece of non-toxic rubber with a round hole on it. Fill this with
> > CO2 satured medium somewaht more as the volume of the hole. Put your
> > coverslip on it, with the cells to the medium off course. Press it
> > gently down and the glass will seal itself to the rubber ring. Now your
> > cells will survive for a couple of hours, so you can do the first
> > imaging. For real experiments you have to find a way to heat the object
> > glass to 37C.
> >
> > Good luck, Gert
> >
> > Op 29-10-2010 21:00, Dolphin, Colin schreef:
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> We would like to do live cell imaging - mammalian cell lines - but only
> have direct access to an upright Olympus BX61. We don't really need
> complicated perfusion chambers, etc just something simple. We're real
> neophytes so all suggestions gratefully received.
> >>
> >> Colin
> >>
>
> Note: This message may contain confidential information. If this Email/Fax
> has been sent to you by mistake, please notify the sender and delete it
> immediately. Thank you.
>

--

Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL: http://astro.temple.edu/~jbs

Axel Kurt Preuss

Re: Basic live cell imaging question...

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

Joel, I totally agree.

All what Gert needs is to avoid cell jitter or media perturbations. That s best achieved by having an immersion objective or by placing a coverslid on the cells with some space left for them to "breathe". In other words, not get squished or dry up. Or, to grow them on a coverslip and flip that in one way or the other upside down

There are three ways to achieve that
1) and 2) are leaving cells on their substrate and cover them with a coverslip
1) by placing spacers on the slid and place that on the cell culture
2) by placing the lid on the objective and glue it (reversibly with some non damaging glue or wax if it has to be) on the coverslip and make sure the edges of the slid are cemented in a way that they don't let media in
3)
The third way is the upside down approach . In this way, cells are grown on a coverslip and flipped upside down and best placed on some mold with enough medium volume. If the molded carrier is glass he may not need Hoffman. I think that s what Gert meant.

You also can buy a cheap small perfusion chamber which you can place upside down, (cells grown on coverslip are mounted into chamber, chamber gets flipped upside down, cells face downwards and their coverslip upwards towards objective, the whole chamber gets perfused and you can put it upside down and with some luck you don't get air bubbles killing your cells).

Colin needed to tell us whether he wants to stimulate the cells or not, and whether the BX has a water objective.

Thanks, Cheers
Best Regards
Axel cell +65 9271.5622
------------------
"We focus on your objectives!" -Axel K Preuss PhD, Central Imaging @IMCB, 6-19B, Singapore 138673

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of JOEL B. SHEFFIELD
Sent: Thursday, November 04, 2010 9:40 AM
To: [hidden email]
Subject: Re: Basic live cell imaging question...

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

I think Gert's response makes a lot sense. For a newbie, with limited goals
(initially), it is not necessary to have an elaborate setup. In the (very)
old days, there was something called a "hanging drop culture" in which
tissue fragments were placed in a drop of medium on a slide, the slide was
inverted and examined with a conventional microscope. Gert is suggesting a
modern version of the same, assuming that you would visualize the cells
through the cover slip to which they are attached, Ujnfortunately, the
original writer did not specify the kind of optics they intended to use. If
the cells are, indeed, attached to the cover slip, it is possible to use
properly adjusted phase or Hoffman optics in transmissionto increase the
contrast. If fluorescence is needed, then you have to be concerned with
light toxicity, etc., but the optics should work fine, as long as you don't
want to see nuclear speckles, or details of mitochondria.

Joel

On Wed, Nov 3, 2010 at 9:03 PM, Axel Kurt Preuss <
[hidden email]> wrote:

> You need a water immersion object or have to build one
>
>
> Cheers
>
> Axel
> -----
> Axel K Preuss, PhD,
> Central Imaging, IMCB, A*Star, 61 Biopolis Dr, 6-19B, Singapore 138673,
> sent from 9271.5622
>
>
> On Nov 4, 2010, at 4:06 AM, Gert van Cappellen <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Culture your cells on a round coverslip. Take an object glas glue a
> > square piece of non-toxic rubber with a round hole on it. Fill this with
> > CO2 satured medium somewaht more as the volume of the hole. Put your
> > coverslip on it, with the cells to the medium off course. Press it
> > gently down and the glass will seal itself to the rubber ring. Now your
> > cells will survive for a couple of hours, so you can do the first
> > imaging. For real experiments you have to find a way to heat the object
> > glass to 37C.
> >
> > Good luck, Gert
> >
> > Op 29-10-2010 21:00, Dolphin, Colin schreef:
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> We would like to do live cell imaging - mammalian cell lines - but only
> have direct access to an upright Olympus BX61. We don't really need
> complicated perfusion chambers, etc just something simple. We're real
> neophytes so all suggestions gratefully received.
> >>
> >> Colin
> >>
>
> Note: This message may contain confidential information. If this Email/Fax
> has been sent to you by mistake, please notify the sender and delete it
> immediately. Thank you.
>

--

Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL: http://astro.temple.edu/~jbs

Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

Gert van Cappellen-2

Re: Basic live cell imaging question...

In reply to this post by Axel Kurt Preuss

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

I'm quite sure the cells will also survive an oil immersion lens and
normally this gives enough information for single cells. However a water
immersion lens is better but certainly not necessary.

Best regards,
Gert

Op 4-11-2010 2:03, Axel Kurt Preuss schreef:

> You need a water immersion object or have to build one
>
>
> Cheers
>
> Axel
> —————
> Axel K Preuss, PhD,
> Central Imaging, IMCB, A*Star, 61 Biopolis Dr, 6-19B, Singapore 138673, sent from 9271.5622
>
>
> On Nov 4, 2010, at 4:06 AM, Gert van Cappellen<[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Culture your cells on a round coverslip. Take an object glas glue a
>> square piece of non-toxic rubber with a round hole on it. Fill this with
>> CO2 satured medium somewaht more as the volume of the hole. Put your
>> coverslip on it, with the cells to the medium off course. Press it
>> gently down and the glass will seal itself to the rubber ring. Now your
>> cells will survive for a couple of hours, so you can do the first
>> imaging. For real experiments you have to find a way to heat the object
>> glass to 37C.
>>
>> Good luck, Gert
>>
>> Op 29-10-2010 21:00, Dolphin, Colin schreef:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> We would like to do live cell imaging - mammalian cell lines - but only have direct access to an upright Olympus BX61. We don't really need complicated perfusion chambers, etc just something simple. We're real neophytes so all suggestions gratefully received.
>>>
>>> Colin
>>>
> Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

Straatman, Kees (Dr.)

Re: Basic live cell imaging question...

I always wonder if a water immersion lens is better than an oil immersion lens for live cell imaging. Both have the wrong RI for cells. Water too low, oil too high. However, the NA of an oil lens is higher than from a water lens, so the oil objective should be more light efficient and your resolution should be a little better. Or am I wrong....?

kees

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gert van Cappellen
Sent: 04 November 2010 20:38
To: [hidden email]
Subject: Re: Basic live cell imaging question...

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

I'm quite sure the cells will also survive an oil immersion lens and
normally this gives enough information for single cells. However a water
immersion lens is better but certainly not necessary.

Best regards,
Gert

Op 4-11-2010 2:03, Axel Kurt Preuss schreef:

lechristophe

Re: Basic live cell imaging question...

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

There are now silicon oil objectives from Olympus at 30X NA 1.05 and 60X NA
1.3, that are somewhere between water and oil:
http://www.olympusamerica.com/oai_HeadlinesDetails.asp?pressNo=806

Christophe

On Fri, Nov 5, 2010 at 09:51, Straatman, Kees R. (Dr.) <[hidden email]
> wrote:

> I always wonder if a water immersion lens is better than an oil immersion
> lens for live cell imaging. Both have the wrong RI for cells. Water too low,
> oil too high. However, the NA of an oil lens is higher than from a water
> lens, so the oil objective should be more light efficient and your
> resolution should be a little better. Or am I wrong....?
>
> kees
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Gert van Cappellen
> Sent: 04 November 2010 20:38
> To: [hidden email]
> Subject: Re: Basic live cell imaging question...
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I'm quite sure the cells will also survive an oil immersion lens and
> normally this gives enough information for single cells. However a water
> immersion lens is better but certainly not necessary.
>
> Best regards,
> Gert
>
> Op 4-11-2010 2:03, Axel Kurt Preuss schreef:
> > You need a water immersion object or have to build one
> >
> >
> > Cheers
> >
> > Axel
> > —————
> > Axel K Preuss, PhD,
> > Central Imaging, IMCB, A*Star, 61 Biopolis Dr, 6-19B, Singapore 138673,
> sent from 9271.5622
> >
> >
> > On Nov 4, 2010, at 4:06 AM, Gert van Cappellen<
> [hidden email]> wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Culture your cells on a round coverslip. Take an object glas glue a
> >> square piece of non-toxic rubber with a round hole on it. Fill this with
> >> CO2 satured medium somewaht more as the volume of the hole. Put your
> >> coverslip on it, with the cells to the medium off course. Press it
> >> gently down and the glass will seal itself to the rubber ring. Now your
> >> cells will survive for a couple of hours, so you can do the first
> >> imaging. For real experiments you have to find a way to heat the object
> >> glass to 37C.
> >>
> >> Good luck, Gert
> >>
> >> Op 29-10-2010 21:00, Dolphin, Colin schreef:
> >>> *****
> >>> To join, leave or search the confocal microscopy listserv, go to:
> >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >>> *****
> >>>
> >>> We would like to do live cell imaging - mammalian cell lines - but only
> have direct access to an upright Olympus BX61. We don't really need
> complicated perfusion chambers, etc just something simple. We're real
> neophytes so all suggestions gratefully received.
> >>>
> >>> Colin
> >>>
> > Note: This message may contain confidential information. If this
> Email/Fax has been sent to you by mistake, please notify the sender and
> delete it immediately. Thank you.
>

Guy Cox-2

Re: Basic live cell imaging question...

In reply to this post by Straatman, Kees (Dr.)

High NA water lenses have correction collars to adjust for the refractive index. So long as you know how to adjust it – and it’s simple, but it is essential – it will give you a far better result than an oil lens. The supposed higher NA of an oil lens is imaginary – if the sample is in ‘medium’ the effective NA is RI of ‘medium’ x sin alpha. The figure written on the lens implies that the sample is in a mountant of RI 1.515. If the sample is in water the real NA of your NA 1.4 lens is 1.2. But the extreme uncorrected spherical aberration involved means that you won’t get anything like the resolution you’d expect from a lens of NA 1.2.

The whole concept of “Numerical Aperture” is an unfortunate accident of history. If we stuck to the actual parameter – RI x sin alpha – all these misconceptions would not arise.

Guy

Optical Imaging Techniques in Cell Biology

by Guy Cox CRC Press / Taylor & Francis

http://www.guycox.com/optical.htm <http://www.guycox.com/optical.htm>

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Australian Centre for Microscopy & Microanalysis,

Madsen Building F09, University of Sydney, NSW 2006

Phone +61 2 9351 3176 Fax +61 2 9351 7682

Mobile 0413 281 861

______________________________________________

http://www.guycox.net <http://www.guycox.net>

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Straatman, Kees R. (Dr.)
Sent: Friday, 5 November 2010 7:51 PM
To: [hidden email]
Subject: Re: Basic live cell imaging question...

I always wonder if a water immersion lens is better than an oil immersion lens for live cell imaging. Both have the wrong RI for cells. Water too low, oil too high. However, the NA of an oil lens is higher than from a water lens, so the oil objective should be more light efficient and your resolution should be a little better. Or am I wrong....?

kees

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gert van Cappellen
Sent: 04 November 2010 20:38
To: [hidden email]
Subject: Re: Basic live cell imaging question...

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

I'm quite sure the cells will also survive an oil immersion lens and
normally this gives enough information for single cells. However a water
immersion lens is better but certainly not necessary.

Best regards,
Gert

Op 4-11-2010 2:03, Axel Kurt Preuss schreef:

________________________________

No virus found in this message.
Checked by AVG - www.avg.com
Version: 10.0.1153 / Virus Database: 424/3238 - Release Date: 11/04/10

________________________________

No virus found in this message.
Checked by AVG - www.avg.com
Version: 10.0.1153 / Virus Database: 424/3238 - Release Date: 11/04/10

Vitaly Boyko

Re: Basic live cell imaging question...

Effective NA 1.2 of a buffer sample is good news - better
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Guy,

Effective NA 1.2 of a buffer sample is good news - better sampling
(reduced/negligeable undersampling) when 100x NA 1.4 is used either with the
16um/pixel EMCCD or 2x2 binned 6.4um/pixel CCD. Is there a (simple) solution to
spherical aberrations which are pronounced especially in the image corners?

Vitaly
301-515-7833

________________________________
From: Guy Cox <[hidden email]>
To: [hidden email]
Sent: Fri, November 5, 2010 7:22:05 AM
Subject: Re: Basic live cell imaging question...

High NA water lenses have correction collars to adjust for the refractive
index. So long as you know how to adjust it – and it’s simple, but it is
essential – it will give you a far better result than an oil lens. The supposed
higher NA of an oil lens is imaginary – if the sample is in ‘medium’ the
effective NA is RI of ‘medium’ x sin alpha. The figure written on the lens
implies that the sample is in a mountant of RI 1.515. If the sample is in water
the real NA of your NA 1.4 lens is 1.2. But the extreme uncorrected spherical
aberration involved means that you won’t get anything like the resolution you’d
expect from a lens of NA 1.2.

The whole concept of “Numerical Aperture” is an unfortunate accident of
history. If we stuck to the actual parameter – RI x sin alpha – all these
misconceptions would not arise.

Guy

Optical Imaging Techniques in Cell Biology

by Guy Cox CRC Press / Taylor & Francis

http://www.guycox.com/optical.htm <http://www.guycox.com/optical.htm>

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Australian Centre for Microscopy & Microanalysis,

Madsen Building F09, University of Sydney, NSW 2006

Phone +61 2 9351 3176 Fax +61 2 9351 7682

Mobile 0413 281 861

______________________________________________

http://www.guycox.net <http://www.guycox.net>

From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Straatman, Kees R. (Dr.)
Sent: Friday, 5 November 2010 7:51 PM
To: [hidden email]
Subject: Re: Basic live cell imaging question...

I always wonder if a water immersion lens is better than an oil immersion lens
for live cell imaging. Both have the wrong RI for cells. Water too low, oil too
high. However, the NA of an oil lens is higher than from a water lens, so the
oil objective should be more light efficient and your resolution should be a
little better. Or am I wrong....?

kees

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Gert van Cappellen
Sent: 04 November 2010 20:38
To: [hidden email]
Subject: Re: Basic live cell imaging question...

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

I'm quite sure the cells will also survive an oil immersion lens and
normally this gives enough information for single cells. However a water
immersion lens is better but certainly not necessary.

Best regards,
Gert

Op 4-11-2010 2:03, Axel Kurt Preuss schreef:

> You need a water immersion object or have to build one
>
>
> Cheers
>
> Axel
> —————
> Axel K Preuss, PhD,
> Central Imaging, IMCB, A*Star, 61 Biopolis Dr, 6-19B, Singapore 138673, sent
>from 9271.5622
>
>
> On Nov 4, 2010, at 4:06 AM, Gert van Cappellen<[hidden email]>
>wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Culture your cells on a round coverslip. Take an object glas glue a
>> square piece of non-toxic rubber with a round hole on it. Fill this with
>> CO2 satured medium somewaht more as the volume of the hole. Put your
>> coverslip on it, with the cells to the medium off course. Press it
>> gently down and the glass will seal itself to the rubber ring. Now your
>> cells will survive for a couple of hours, so you can do the first
>> imaging. For real experiments you have to find a way to heat the object
>> glass to 37C.
>>
>> Good luck, Gert
>>
>> Op 29-10-2010 21:00, Dolphin, Colin schreef:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> We would like to do live cell imaging - mammalian cell lines - but only have
>>>direct access to an upright Olympus BX61. We don't really need complicated
>>>perfusion chambers, etc just something simple. We're real neophytes so all
>>>suggestions gratefully received.
>>>
>>> Colin
>>>
> Note: This message may contain confidential information. If this Email/Fax has
>been sent to you by mistake, please notify the sender and delete it immediately.
>Thank you.

James Pawley

Re: Basic live cell imaging question...

In reply to this post by Straatman, Kees (Dr.)

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

>I always wonder if a water immersion lens is
>better than an oil immersion lens for live cell
>imaging. Both have the wrong RI for cells. Water
>too low, oil too high. However, the NA of an oil
>lens is higher than from a water lens, so the
>oil objective should be more light efficient and
>your resolution should be a little better. Or am
>I wrong....?
>
>kees

Hi Kees,

Yes, I am afraid that you are wrong. The higher
NA rays, that an NA 1.4 lens might accept if it
were used with an oiled specimen, are totally
reflected at the water-glass interface and do not
get into the objective. Oil lenses work well for
TIRF because the "specimen" is just the thin
layer near the interface in which fluorescence is
excited by the evanescent wave. But the cone of
light that actually forms the image (i.e., not
counting the part that is probably used for the
high-angle excitation) will still be about NA
1.25.

The other "thin aqueous biological specimens"
that were productively viewed with an oil
objective were the various fiber systems
(isolated microtubules, actin filaments etc.
lying on a slide, in media) that were visualized
using video-enhanced contrast DIC by Shinya Inoue
and Robert Allen back in the early 1980s.
Although the exact quantum-mechanical explanation
of the interactions near this specimen is beyond
me, it seems likely that these structures were so
close to the glass that they were essentially
part of it in terms of maximum angle at which
scattered rays could enter the objective. In any
case, the raw resolution of the recorded images
was about that of a normal NA 1.4 objective with
the green light that they used. The ability to
visualize much smaller structures was more a
"detection of their presence on a clear
background" than a matter of resolving them.

The rationale for using oil in those days was
that there really weren't any good, high-NA water
objectives available. (Here is a good place to
reemphasize Guy's point about the absolute
necessity of carefully adjusting the coverslip
correction collar. At NA 1.2, aim for an accuracy
of <+/- 2µm of water-replaced-by glass. This is
less important with oil objectives because oil
and coverslip have about the same RI.)

As the RI of water is about 1.33, one might
assume that one could use an objective with an NA
of up to 1.33. The reason this doesn't help much
is that, although the rays between 1.2 and 1.33
may not be totally reflected at the water-glass
interface, they are still highly reflected. (Just
remember how bright the image of the sun is when
it reflects off a pane of glass at glancing
incidence. Although some sunlight is still being
refracted through the glass into the building, it
will be dim because most of the light was
reflected.)

And finally, as mentioned by others, there is the
matter of spherical aberration (apparently my
favorite topic!). How is this important? If you
are looking at large fluorescent objects
(say >1µm), then it isn't quite so important.
Assuming it is not lost to reflection at the
glass-water interface, a larger NA lens will
accept more light and if it is not absorbed or
reflected while passing through the optics, this
light will end up in the image somewhere close to
its proper location, making the blob appear
brighter than it would be otherwise. However, if
you are hoping that the higher NA of the oil lens
will yield better resolution on a water
specimen, forget it. As Rimas Juskaitis makes
clear in Chapter 11 of The Handbook, even under
optimal conditions (i.e., the right oil, temp
etc) the best 1.4 objectives then available were
only free from phase error up to NA 1.35 (i.e.,
the rays between 1.35 and 1.4 were passing the
objective but not being focused properly and
hence not contributing the a reduction in the
imaged size of a point object.) I don't know how
the newer 1.45, 1.49 and 1.65 objectives would
perform under his very stringent test conditions
but I would like to point out that he only got
the old ones to work at 1.35 by controlling the
oil temperature to +/- 1deg C.

Once SA is present, the image of a point object
gets bigger in a complicated way (it is hard to
define the PSF of an aberrated image with a
single number.). This means not only that the
resolution is reduced, but that that the
brightest part of this image will be dimmer than
it would have been without the aberration. i.e.
The high-NA oil image of a point object will be
dimmer than the aberration-free image from a
water lens with slightly lower "faceplate" NA.
The best plan is to always include small (<.3µm)
fluorescent beads in your preparations and before
you start imaging "for real," focus up and down
on the beads "by eye" to make sure that the
slightly-out-of-focus image seen above focus,
looks very much like that seen the same amount
below focus. If this is true, then SA should not
be a problem.

What I am trying to say is that resolution isn't
always proportional to the number on the side of
the objective. Everything else has to be
"perfect" and it seldom is. As you point out,
cells are neither water or oil. It is worse than
that. Their internal RI is extremely variable,
which is why DIC and phase show strong contrast
of subcellular details.

But that is another story for another hour...

Regards,

Jim Pawley

*********************************************************************************
Prof. James B. Pawley, Ph. 608-263-3147
Room 223, Zoology Research
Building, FAX
608-265-5315
1117 Johnson Ave., Madison, WI, 53706
[hidden email]
3D Microscopy of Living Cells Course, June 11-23, 2011, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/
Applications still being accepted
"If it ain't diffraction, it must be statistics." Anon.

>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[hidden email]] On
>Behalf Of Gert van Cappellen
>Sent: 04 November 2010 20:38
>To: [hidden email]
>Subject: Re: Basic live cell imaging question...
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
> I'm quite sure the cells will also survive an oil immersion lens and
>normally this gives enough information for single cells. However a water
>immersion lens is better but certainly not necessary.
>
>Best regards,
>Gert
>
>Op 4-11-2010 2:03, Axel Kurt Preuss schreef:
>> You need a water immersion object or have to build one
>>
>>
>> Cheers
>>
>> Axel
>> -----
>> Axel K Preuss, PhD,
>> Central Imaging, IMCB, A*Star, 61 Biopolis Dr,
>>6-19B, Singapore 138673, sent from 9271.5622
>>
>>
>> On Nov 4, 2010, at 4:06 AM, Gert van
>>Cappellen<[hidden email]> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Culture your cells on a round coverslip. Take an object glas glue a
>>> square piece of non-toxic rubber with a round hole on it. Fill this with
>>> CO2 satured medium somewaht more as the volume of the hole. Put your
>>> coverslip on it, with the cells to the medium off course. Press it
>>> gently down and the glass will seal itself to the rubber ring. Now your
>>> cells will survive for a couple of hours, so you can do the first
>>> imaging. For real experiments you have to find a way to heat the object
>>> glass to 37C.
>>>
>>> Good luck, Gert
>>>
>>> Op 29-10-2010 21:00, Dolphin, Colin schreef:
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> We would like to do live cell imaging -
>>>>mammalian cell lines - but only have direct
>>>>access to an upright Olympus BX61. We don't
>>>>really need complicated perfusion chambers,
>>>>etc just something simple. We're real
>>>>neophytes so all suggestions gratefully
>>>>received.
>>>>
>>>> Colin
>>>>
>> Note: This message may contain confidential
>>information. If this Email/Fax has been sent to
>>you by mistake, please notify the sender and
>>delete it immediately. Thank you.

--
James and Christine Pawley, 21 N. Prospect Ave.
Madison, WI, 53726 Phone: 608-238-3953

Barbara Foster

Re: Basic live cell imaging question...

In reply to this post by Straatman, Kees (Dr.)

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

HI, Kees

This is a little difficult to discuss without benefit of diagrams, but let's give it a try.
Several things to keep in mind:
a. If the RI of an object exactly matches the RI of the surroundings there will be NO contrast and therefore you will not be able to see the object.
b. The cells are mounted in, essentially water.
c. If you think of a cell as a point, light emerging from that point will travel in all directions. The more you capture, the better, not only because the signal is stronger, but that light carries information about orientation, spacing, and edges (See diffraction theory)
d. As the imaging information leaves the cells, it is that information which is subject to the RI of the surrounding medium. As that light leaves the cell it will approach a variety of interfaces at the water/coverslip and the coverslip/air or oil.
e. Here are the RI's we need:
Water (in which the cells are mounted): 1.33
Glass coverslip: ~ 1.51
Air: 1.00
Oil: 1.5121
According to Snell's Law, light will refract (bend) as it crosses an interface at an angle. The amount of bend is directly related to the ratio of the RI's on either side of the interface. Further, if light passes from higher refractive index into lower refractive index, it will bend AWAY from the normal (in the simplest case, essentially, the optical axis of the microscope). If moving from lower into higher, it will bend TOWARD the optical axis. So, if light moves from water (1.33) into a glass coverslip (1.51), it will move TOWARD the optical axis. This is good because it allows the objective to capture more of that information-carrying light.
f. However, when the light emerges from the coverslip, one of three things happens.
<If it moves into air (from RI 1.51 to RI = 1.00), it is strongly refracted, often out of the collecting angle of the objective, resulting in a loss of both resolution and edge information.
<If it moves into water (water immersion objective... RI=1.51 to RI = 1.33), less information is lost
<If it moves into oil (oil immersion objective... RI=1.51 to RI=1.51), the maximum amount of information is retained.

So... the long answer to your question is: oil immersion is really the best for observing fine detail in living cells. Dipping objectives are also helpful because they significantly reduce the number of interfaces. One caveat: you must carefully attend to their cleanliness. In actual fact, to keep them healthy, live cells are mounted in saline solutions. If you don't clean off the objective after every use with a cloth dampened (not sopping wet) with distilled water, the salt will dry on the objective, corroding both the casing and marring the glass. I've visited some very prestigious labs where, unfortunately, that simple step was not practiced. The results were very sad, indeed.

Hope this is helpful.

Good hunting,
Barbara Foster, President and Sr. Consultant
Microscopy/Microscopy Education
W: www.MicroscopyEducation.com

Working in confocal or fluorescence? Take part in our latest survey. Visit www.MicroscopyEducation.com for details. Survey has been extended to November 5th.

At 09:00 PM 11/4/2010, you wrote:

>I always wonder if a water immersion lens is better than an oil immersion lens for live cell imaging. Both have the wrong RI for cells. Water too low, oil too high. However, the NA of an oil lens is higher than from a water lens, so the oil objective should be more light efficient and your resolution should be a little better. Or am I wrong....?
>
>kees
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gert van Cappellen
>Sent: 04 November 2010 20:38
>To: [hidden email]
>Subject: Re: Basic live cell imaging question...
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
> I'm quite sure the cells will also survive an oil immersion lens and
>normally this gives enough information for single cells. However a water
>immersion lens is better but certainly not necessary.
>
>Best regards,
>Gert
>
>Op 4-11-2010 2:03, Axel Kurt Preuss schreef:
>> You need a water immersion object or have to build one
>>
>>
>> Cheers
>>
>> Axel
>>
>> Axel
>> Axel K Preuss, PhD,
>> Central Imaging, IMCB, A*Star, 61 Biopolis Dr, 6-19B, Singapore 138673, sent from 9271.5622
>>
>>
>> On Nov 4, 2010, at 4:06 AM, Gert van Cappellen<[hidden email]> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Culture your cells on a round coverslip. Take an object glas glue a
>>> square piece of non-toxic rubber with a round hole on it. Fill this with
>>> CO2 satured medium somewaht more as the volume of the hole. Put your
>>> coverslip on it, with the cells to the medium off course. Press it
>>> gently down and the glass will seal itself to the rubber ring. Now your
>>> cells will survive for a couple of hours, so you can do the first
>>> imaging. For real experiments you have to find a way to heat the object
>>> glass to 37C.
>>>
>>> Good luck, Gert
>>>
>>> Op 29-10-2010 21:00, Dolphin, Colin schreef:
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> We would like to do live cell imaging - mammalian cell lines - but only have direct access to an upright Olympus BX61. We don't really need complicated perfusion chambers, etc just something simple. We're real neophytes so all suggestions gratefully received.
>>>>
>>>> Colin
>>>>
>> Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.

John Oreopoulos

Re: Basic live cell imaging question...

In reply to this post by James Pawley

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

Hi Jim,

I'm wondering if you can clear up a few things related to this topic. You said in your last email that the higher NA rays in a water-glass situation do not get into the objective, even in the case of TIRF. There is a very nice paper by Jorg Enderlein's group that uses a very simple technique to measure the actual NA of an objective which involves viewing the distribution of light rays in the back focal plane of an objective with the use of a Bertrand lens:

http://www.opticsinfobase.org/oe/abstract.cfm?uri=oe-13-23-9409

And another similar paper by Mattheyses and Axelrod:

http://spiedl.aip.org/getabs/servlet/GetabsServlet?prog=normal&id=JBOPFO000010000005054007000001&idtype=cvips&gifs=yes&ref=no

Both of these papers clearly show that a large percentage of the light actually does propagate into the objective lens beyond the critical angle. However, you did also allude to "thin aqueous biological" in vitro specimens, the ones that are typical of single-molecule experiments where the material under investigation has been separated from the cell, and both of these papers made their measurements under similar conditions, so maybe this is only a special case.

My question is, does this situation only occur for fluorescent objects that are very near the glass-water interface? That is to say, if I were to view a fluorescent cellular sample the same way (with a Bertrand lens, widefield illumination, focused slightly into the cell), would I see that most of the light propagates into the oil immersion objective BELOW the critical angle (and hence the effective lower NA)?

As for a "quantum mechanical explanation" of why the other situation works, it seems that Hellen and Axelrod came up with an explanation some time ago that does not involve quantum mechanics at all, and it can be explained using classical electrodynamics... but I've had trouble following this paper because it gets rather complicated with the math:

Hellen, E.H. and D. Axelrod, Fluorescence emission at dielectric and metal-film interfaces. Journal of the Optical Society of America B-Optical Physics, 1987. 4(3): p. 337-350.

Axelrod, D., Emission of fluorescence at an interface, in Methods in cell biology, T. Langsing and Y. Wang, Editors. 1989, Academic Press: San Diego. p. 399-416.

There is a very simple diagram in the second book chapter that basically shows very oblique light rays emitted by a fluorescent molecule near an interface (the near-field) can turn into propagating evanescent waves that get coupled back into the higher index medium. This website talks a bit more about this (see Figure 6):

http://micro.magnet.fsu.edu/primer/techniques/fluorescence/tirf/tirfintro.html

John Oreopoulos

On 2010-11-05, at 11:33 AM, James Pawley wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>> I always wonder if a water immersion lens is better than an oil immersion lens for live cell imaging. Both have the wrong RI for cells. Water too low, oil too high. However, the NA of an oil lens is higher than from a water lens, so the oil objective should be more light efficient and your resolution should be a little better. Or am I wrong....?
>>
>> kees
>
>
> Hi Kees,
>
> Yes, I am afraid that you are wrong. The higher NA rays, that an NA 1.4 lens might accept if it were used with an oiled specimen, are totally reflected at the water-glass interface and do not get into the objective. Oil lenses work well for TIRF because the "specimen" is just the thin layer near the interface in which fluorescence is excited by the evanescent wave. But the cone of light that actually forms the image (i.e., not counting the part that is probably used for the high-angle excitation) will still be about NA 1.25.
>
> The other "thin aqueous biological specimens" that were productively viewed with an oil objective were the various fiber systems (isolated microtubules, actin filaments etc. lying on a slide, in media) that were visualized using video-enhanced contrast DIC by Shinya Inoue and Robert Allen back in the early 1980s. Although the exact quantum-mechanical explanation of the interactions near this specimen is beyond me, it seems likely that these structures were so close to the glass that they were essentially part of it in terms of maximum angle at which scattered rays could enter the objective. In any case, the raw resolution of the recorded images was about that of a normal NA 1.4 objective with the green light that they used. The ability to visualize much smaller structures was more a "detection of their presence on a clear background" than a matter of resolving them.
>
> The rationale for using oil in those days was that there really weren't any good, high-NA water objectives available. (Here is a good place to reemphasize Guy's point about the absolute necessity of carefully adjusting the coverslip correction collar. At NA 1.2, aim for an accuracy of <+/- 2µm of water-replaced-by glass. This is less important with oil objectives because oil and coverslip have about the same RI.)
>
> As the RI of water is about 1.33, one might assume that one could use an objective with an NA of up to 1.33. The reason this doesn't help much is that, although the rays between 1.2 and 1.33 may not be totally reflected at the water-glass interface, they are still highly reflected. (Just remember how bright the image of the sun is when it reflects off a pane of glass at glancing incidence. Although some sunlight is still being refracted through the glass into the building, it will be dim because most of the light was reflected.)
>
> And finally, as mentioned by others, there is the matter of spherical aberration (apparently my favorite topic!). How is this important? If you are looking at large fluorescent objects (say >1µm), then it isn't quite so important. Assuming it is not lost to reflection at the glass-water interface, a larger NA lens will accept more light and if it is not absorbed or reflected while passing through the optics, this light will end up in the image somewhere close to its proper location, making the blob appear brighter than it would be otherwise. However, if you are hoping that the higher NA of the oil lens will yield better resolution on a water specimen, forget it. As Rimas Juskaitis makes clear in Chapter 11 of The Handbook, even under optimal conditions (i.e., the right oil, temp etc) the best 1.4 objectives then available were only free from phase error up to NA 1.35 (i.e., the rays between 1.35 and 1.4 were passing the objective but not being focused properly and hence not contributing the a reduction in the imaged size of a point object.) I don't know how the newer 1.45, 1.49 and 1.65 objectives would perform under his very stringent test conditions but I would like to point out that he only got the old ones to work at 1.35 by controlling the oil temperature to +/- 1deg C.
>
> Once SA is present, the image of a point object gets bigger in a complicated way (it is hard to define the PSF of an aberrated image with a single number.). This means not only that the resolution is reduced, but that that the brightest part of this image will be dimmer than it would have been without the aberration. i.e. The high-NA oil image of a point object will be dimmer than the aberration-free image from a water lens with slightly lower "faceplate" NA. The best plan is to always include small (<.3µm) fluorescent beads in your preparations and before you start imaging "for real," focus up and down on the beads "by eye" to make sure that the slightly-out-of-focus image seen above focus, looks very much like that seen the same amount below focus. If this is true, then SA should not be a problem.
>
> What I am trying to say is that resolution isn't always proportional to the number on the side of the objective. Everything else has to be "perfect" and it seldom is. As you point out, cells are neither water or oil. It is worse than that. Their internal RI is extremely variable, which is why DIC and phase show strong contrast of subcellular details.
>
> But that is another story for another hour...
>
> Regards,
>
> Jim Pawley
>
> *********************************************************************************
> Prof. James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-265-5315
> 1117 Johnson Ave., Madison, WI, 53706 [hidden email]
> 3D Microscopy of Living Cells Course, June 11-23, 2011, UBC, Vancouver Canada
> Info: http://www.3dcourse.ubc.ca/ Applications still being accepted
> "If it ain't diffraction, it must be statistics." Anon.
>
>
>
>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gert van Cappellen
>> Sent: 04 November 2010 20:38
>> To: [hidden email]
>> Subject: Re: Basic live cell imaging question...
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> I'm quite sure the cells will also survive an oil immersion lens and
>> normally this gives enough information for single cells. However a water
>> immersion lens is better but certainly not necessary.
>>
>> Best regards,
>> Gert
>>
>> Op 4-11-2010 2:03, Axel Kurt Preuss schreef:
>>> You need a water immersion object or have to build one
>>>
>>>
>>> Cheers
>>>
>>> Axel
>>> -----
>>> Axel K Preuss, PhD,
>>> Central Imaging, IMCB, A*Star, 61 Biopolis Dr, 6-19B, Singapore 138673, sent from 9271.5622
>>>
>>>
>>> On Nov 4, 2010, at 4:06 AM, Gert van Cappellen<[hidden email]> wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Culture your cells on a round coverslip. Take an object glas glue a
>>>> square piece of non-toxic rubber with a round hole on it. Fill this with
>>>> CO2 satured medium somewaht more as the volume of the hole. Put your
>>>> coverslip on it, with the cells to the medium off course. Press it
>>>> gently down and the glass will seal itself to the rubber ring. Now your
>>>> cells will survive for a couple of hours, so you can do the first
>>>> imaging. For real experiments you have to find a way to heat the object
>>>> glass to 37C.
>>>>
>>>> Good luck, Gert
>>>>
>>>> Op 29-10-2010 21:00, Dolphin, Colin schreef:
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> *****
>>>>>
>>>>> We would like to do live cell imaging - mammalian cell lines - but only have direct access to an upright Olympus BX61. We don't really need complicated perfusion chambers, etc just something simple. We're real neophytes so all suggestions gratefully received.
>>>>>
>>>>> Colin
>>>>>
>>> Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.
>
>
> --
> James and Christine Pawley, 21 N. Prospect Ave. Madison, WI, 53726 Phone: 608-238-3953

Andreas Bruckbauer

Re: Basic live cell imaging question...

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

Hi John,

I recently tested the Olympus 100x NA 1.65 objective which uses special coverslips and oil with refractive index 1.78 against our 100x NA 1.45 lens. For single molecule TIRF directly at the coverslip i get 2-3 times higher signal with the 1.65 NA lens. This was in good agreement to a calculation based on classical electrodynamics which gave 2.9x more signal in the case of the 1.65 lens. However when i calculted the case for focussing one micron into the cell, the 1.65 lens had a slight disadvantage, more reflection due to the higher difference of refractive indices. This calculation does not account for PSFs or aberations it just deals with plane waves and dipol excitation/emission. However it shows quite clearly that It is really the coupling of the near-field of the oscillating dipol to the propagating waves in the lens which gives rise to increased signal in the high NA case, this does not work if the molecule is more than 1/2 wavelength away from the interface glass/cell.

best wishes

Andreas

-----Original Message-----
From: John Oreopoulos <[hidden email]>
To: [hidden email]
Sent: Fri, 5 Nov 2010 18:31
Subject: Re: Basic live cell imaging question...

*****

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

http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy

*****

Hi Jim,

I'm wondering if you can clear up a few things related to this topic. You said

in your last email that the higher NA rays in a water-glass situation do not get

into the objective, even in the case of TIRF. There is a very nice paper by Jorg

Enderlein's group that uses a very simple technique to measure the actual NA of

an objective which involves viewing the distribution of light rays in the back

focal plane of an objective with the use of a Bertrand lens:

http://www.opticsinfobase.org/oe/abstract.cfm?uri=oe-13-23-9409

And another similar paper by Mattheyses and Axelrod:

http://spiedl.aip.org/getabs/servlet/GetabsServlet?prog=normal&id=JBOPFO000010000005054007000001&idtype=cvips&gifs=yes&ref=no

Both of these papers clearly show that a large percentage of the light actually

does propagate into the objective lens beyond the critical angle. However, you

did also allude to "thin aqueous biological" in vitro specimens, the ones that

are typical of single-molecule experiments where the material under

investigation has been separated from the cell, and both of these papers made

their measurements under similar conditions, so maybe this is only a special

case.

My question is, does this situation only occur for fluorescent objects that are

very near the glass-water interface? That is to say, if I were to view a

fluorescent cellular sample the same way (with a Bertrand lens, widefield

illumination, focused slightly into the cell), would I see that most of the

light propagates into the oil immersion objective BELOW the critical angle (and

hence the effective lower NA)?

As for a "quantum mechanical explanation" of why the other situation works, it

seems that Hellen and Axelrod came up with an explanation some time ago that

does not involve quantum mechanics at all, and it can be explained using

classical electrodynamics... but I've had trouble following this paper because

it gets rather complicated with the math:

Hellen, E.H. and D. Axelrod, Fluorescence emission at dielectric and metal-film

interfaces. Journal of the Optical Society of America B-Optical Physics, 1987.

4(3): p. 337-350.

Axelrod, D., Emission of fluorescence at an interface, in Methods in cell

biology, T. Langsing and Y. Wang, Editors. 1989, Academic Press: San Diego. p.

399-416.

There is a very simple diagram in the second book chapter that basically shows

very oblique light rays emitted by a fluorescent molecule near an interface (the

near-field) can turn into propagating evanescent waves that get coupled back

into the higher index medium. This website talks a bit more about this (see

Figure 6):

http://micro.magnet.fsu.edu/primer/techniques/fluorescence/tirf/tirfintro.html

John Oreopoulos

On 2010-11-05, at 11:33 AM, James Pawley wrote:

> *****

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

> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy

> *****

>

>> I always wonder if a water immersion lens is better than an oil immersion

lens for live cell imaging. Both have the wrong RI for cells. Water too low, oil

too high. However, the NA of an oil lens is higher than from a water lens, so

the oil objective should be more light efficient and your resolution should be a

little better. Or am I wrong....?

>>

>> kees

>

>

> Hi Kees,

>

> Yes, I am afraid that you are wrong. The higher NA rays, that an NA 1.4 lens

might accept if it were used with an oiled specimen, are totally reflected at

the water-glass interface and do not get into the objective. Oil lenses work

well for TIRF because the "specimen" is just the thin layer near the interface

in which fluorescence is excited by the evanescent wave. But the cone of light

that actually forms the image (i.e., not counting the part that is probably used

for the high-angle excitation) will still be about NA 1.25.

>

> The other "thin aqueous biological specimens" that were productively viewed

with an oil objective were the various fiber systems (isolated microtubules,

actin filaments etc. lying on a slide, in media) that were visualized using

video-enhanced contrast DIC by Shinya Inoue and Robert Allen back in the early

1980s. Although the exact quantum-mechanical explanation of the interactions

near this specimen is beyond me, it seems likely that these structures were so

close to the glass that they were essentially part of it in terms of maximum

angle at which scattered rays could enter the objective. In any case, the raw

resolution of the recorded images was about that of a normal NA 1.4 objective

with the green light that they used. The ability to visualize much smaller

structures was more a "detection of their presence on a clear background" than a

matter of resolving them.

>

> The rationale for using oil in those days was that there really weren't any

good, high-NA water objectives available. (Here is a good place to reemphasize

Guy's point about the absolute necessity of carefully adjusting the coverslip

correction collar. At NA 1.2, aim for an accuracy of <+/- 2µm of

water-replaced-by glass. This is less important with oil objectives because oil

and coverslip have about the same RI.)

>

> As the RI of water is about 1.33, one might assume that one could use an

objective with an NA of up to 1.33. The reason this doesn't help much is that,

although the rays between 1.2 and 1.33 may not be totally reflected at the

water-glass interface, they are still highly reflected. (Just remember how

bright the image of the sun is when it reflects off a pane of glass at glancing

incidence. Although some sunlight is still being refracted through the glass

into the building, it will be dim because most of the light was reflected.)

>

> And finally, as mentioned by others, there is the matter of spherical

aberration (apparently my favorite topic!). How is this important? If you are

looking at large fluorescent objects (say >1µm), then it isn't quite so

important. Assuming it is not lost to reflection at the glass-water interface, a

larger NA lens will accept more light and if it is not absorbed or reflected

while passing through the optics, this light will end up in the image somewhere

close to its proper location, making the blob appear brighter than it would be

otherwise. However, if you are hoping that the higher NA of the oil lens will

yield better resolution on a water specimen, forget it. As Rimas Juskaitis

makes clear in Chapter 11 of The Handbook, even under optimal conditions (i.e.,

the right oil, temp etc) the best 1.4 objectives then available were only free

from phase error up to NA 1.35 (i.e., the rays between 1.35 and 1.4 were passing

the objective but not being focused properly and hence not contributing the a

reduction in the imaged size of a point object.) I don't know how the newer

1.45, 1.49 and 1.65 objectives would perform under his very stringent test

conditions but I would like to point out that he only got the old ones to work

at 1.35 by controlling the oil temperature to +/- 1deg C.

>

> Once SA is present, the image of a point object gets bigger in a complicated

way (it is hard to define the PSF of an aberrated image with a single number.).

This means not only that the resolution is reduced, but that that the brightest

part of this image will be dimmer than it would have been without the

aberration. i.e. The high-NA oil image of a point object will be dimmer than the

aberration-free image from a water lens with slightly lower "faceplate" NA. The

best plan is to always include small (<.3µm) fluorescent beads in your

preparations and before you start imaging "for real," focus up and down on the

beads "by eye" to make sure that the slightly-out-of-focus image seen above

focus, looks very much like that seen the same amount below focus. If this is

true, then SA should not be a problem.

>

> What I am trying to say is that resolution isn't always proportional to the

number on the side of the objective. Everything else has to be "perfect" and it

seldom is. As you point out, cells are neither water or oil. It is worse than

that. Their internal RI is extremely variable, which is why DIC and phase show

strong contrast of subcellular details.

>

> But that is another story for another hour...

>

> Regards,

>

> Jim Pawley

>

> *********************************************************************************

> Prof. James B. Pawley, Ph. 608-263-3147 Room

223, Zoology Research Building, FAX 608-265-5315

> 1117 Johnson Ave., Madison, WI, 53706 [hidden email]

> 3D Microscopy of Living Cells Course, June 11-23, 2011, UBC, Vancouver Canada

> Info: http://www.3dcourse.ubc.ca/ Applications still being accepted

> "If it ain't diffraction, it must be statistics." Anon.

>

>

>

>

>> -----Original Message-----

>> From: Confocal Microscopy List [mailto:[hidden email]] On

Behalf Of Gert van Cappellen

>> Sent: 04 November 2010 20:38

>> To: [hidden email]

>> Subject: Re: Basic live cell imaging question...

>>

>> *****

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

>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy

>> *****

>>

>> I'm quite sure the cells will also survive an oil immersion lens and

>> normally this gives enough information for single cells. However a water

>> immersion lens is better but certainly not necessary.

>>

>> Best regards,

>> Gert

>>

>> Op 4-11-2010 2:03, Axel Kurt Preuss schreef:

>>> You need a water immersion object or have to build one

>>>

>>>

>>> Cheers

>>>

>>> Axel

>>> -----

>>> Axel K Preuss, PhD,

>>> Central Imaging, IMCB, A*Star, 61 Biopolis Dr, 6-19B, Singapore 138673,

sent from 9271.5622

>>>

>>>

>>> On Nov 4, 2010, at 4:06 AM, Gert van Cappellen<[hidden email]>

wrote:

>>>

>>>> *****

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

>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy

>>>> *****

>>>>

>>>> Culture your cells on a round coverslip. Take an object glas glue a

>>>> square piece of non-toxic rubber with a round hole on it. Fill this with

>>>> CO2 satured medium somewaht more as the volume of the hole. Put your

>>>> coverslip on it, with the cells to the medium off course. Press it

>>>> gently down and the glass will seal itself to the rubber ring. Now your

>>>> cells will survive for a couple of hours, so you can do the first

>>>> imaging. For real experiments you have to find a way to heat the object

>>>> glass to 37C.

>>>>

>>>> Good luck, Gert

>>>>

>>>> Op 29-10-2010 21:00, Dolphin, Colin schreef:

>>>>> *****

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

>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy

>>>>> *****

>>>>>

>>>>> We would like to do live cell imaging - mammalian cell lines - but only

have direct access to an upright Olympus BX61. We don't really need complicated

perfusion chambers, etc just something simple. We're real neophytes so all

suggestions gratefully received.

>>>>>

>>>>> Colin

>>>>>

>>> Note: This message may contain confidential information. If this Email/Fax

has been sent to you by mistake, please notify the sender and delete it

immediately. Thank you.

>

>

> --

> James and Christine Pawley, 21 N. Prospect Ave. Madison, WI, 53726 Phone:

608-238-3953