Posted by
Vitaly Boyko on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Basic-live-cell-imaging-question-tp5687698p5709014.html
Effective NA 1.2 of a buffer sample is good news - better
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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...
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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:
>>>
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>>> *****
>>>
>>> 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
>>>
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