*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****
On Sep 20, 2014, at 8:12 AM, James Pawley <[log in to unmask]> wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi David,
>
> Could you tell us a little more? How thick is the "surface label" (molecular or a layer of plastic?)?
fluorescent protein bound to the surface of a latex bead so molecularly thin on the scale of an optical microscope.
> Are we correct in assuming that the WF focus plane coincides with the centre of the bead?
yes
> What is the RI of the beads and the embedding medium?
live imaging of beads inside phagosomes inside cells with oil immersion and cells in media using a glass bottom dish
> Using NA 1.3-1.4 implies "all oil or glass to the focus plane". Anything else involves all sorts of refraction, reflection games near the bead surface.
related to this issue, it is not clear to me the extent to which latex beads are transparent and that would be one explanation if excitation or emission is restricted by the inability of light from the focal planes above the middle getting through the lower part of the bead to get back to the objective.
>
> As a not-too-close analogy, it has recently been recognized that, if you really fill the BFP of such an objective with excitation light when viewing a watery specimen, you get enhanced excitation right near the glass/water interface from the TIRF cause by the high-NA being (mostly) reflected at this interface.
>
> More prosaically, light created in a high RI plastic bead my have trouble getting out because of total or partial reflections. For instance, more of it than we would expect may leak out near where the bead touches the glass as this is a break in the RI barrier.
>
> If the bead is in oil and has about the same RI as oil, then I guess that I would expect that when the focus plane passes through the centre of the bead, you would record a doughnut in both confocal and WF. Let's assume that the fluorescent layer is very thin. In WF, the whole surface will be excited about evenly (not true for a plastic bead in water) but the light emitted from the upper and lower surface will be 1.5 µm out of focus at the image plane and at NA1.3 that is a long way. The light will make it to the CCD but it will be blurred over a larger area and so look dimmer. Although not as much dimmer as the confocal image where you have two PSFs to make it dimmer.
>
> Best,
>
> Jim Pawley
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> An after thought: in the confocal microscope, given a pinhole size of 0.8 - 1.0 AU, the shell thickness should appear thinest when centrally focused on. This is in fact a good test of one's optical system.
>>
>> Dan
>>
>> On Sep 19, 2014, at 2:43 PM, Knecht, David <[log in to unmask]> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your posting.
>>> *****
>>>
>>> We have been imaging fluorescent beads in wide field and confocal microscopes. The beads are 3 µm in diameter and surface labeled. In the confocal, they look like doughnuts. My expectation was that in wide field (60-100x 1.3-1.4 NA), they would look more uniformly labeled. In reality, they look like doughnuts. Can someone explain why?
>>> Dr. David Knecht
>>> Professor of Molecular and Cell Biology
>>> Core Microscopy Facility Director
>>> University of Connecticut
>>> Storrs, CT 06269
>>> 860-486-2200
>
>
> --
> ****************************************
> James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC, Canada, V0N3A0,
> Phone 604-885-0840, email <[log in to unmask]>
> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146
Dr. David Knecht
Professor of Molecular and Cell Biology
Core Microscopy Facility Director
University of Connecticut
Storrs, CT 06269
860-486-2200
Dr. David Knecht
Professor of Molecular and Cell Biology
Core Microscopy Facility Director
University of Connecticut
Storrs, CT 06269
860-486-2200
|
