Posted by
Barbara Foster on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Basic-live-cell-imaging-question-tp5687698p5709696.html
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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
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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:
>
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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
>> 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:
>>>
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>>>
>>> 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
>>>>
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