Susana,

As Johannes points out in another response, there are no good UV fibers with
small cores.  Various labs have been trying to develop UV fibers for various
purposes, including Near Field Optical Microscopy (NSOM).  You should try to
focus your probe through your primary microscope objective or a secondary
objective.

Even a dry objective of 0.9NA can provide spot sizes of less than 1 um
diameter in the visible.  You might layout a system like an optical trap.
The laser typically overfills the back aperture of the objective by about
20%.  Use a beam expander to expand the beam to this size and defocus it
slightly, if  required, to make the focus of the laser match that of the
camera or eyepieces.  You may actually want to form a trap for other work
and to help align your probe (use ~ 3 um diameter fluorescent latex beads
for targets.  If you form a good trap, you might buy some 0.04 um beads and
observe them form an airy disk pattern.

The simplest thing is to leave the probe/trap in one fixed location and just
mark that location on your video.  This is not very difficult to do on a
research microscope.  Typically, you insert a dichroic beamsplitter in the
fluorescence path to fold the "trap" laser into the microscope.  If
required, movable mirrors may be set in the laser beam path to change the
location  of the focussed spot at the specimen plane.

Since optical traps have been used to trap cells, you may need to keep a
reduced power, e.g.  ~~ 10 mW at the objective can form a trap.  You
probably want a much lower power density for your work.

Since tunable lasers are expensive and/or difficult to maintain for most
people, you may want to start with a couple of different "convenient" laser
wavelengths.  Helium Neon lasers are easy to use and you can get wavelengths
at the usual 633 nm and also 543 nm (green), 594 nm (yellow) and 612 nm
(orange).  Some of these may be available at your school and some are
available used.  MWK (1-800-356-7714) has some of the green and orange ones
available now for about $350 and $400 respectively.  MWK and other used
equipment vendors also have other lasers of potential interest.


-----------------------
At 09:58 PM 2/24/97 -0500, you wrote:
>Dear members,
>
>        I am trying to deliver light (UV and visible) to a limited area of a
>cell prep in order to study rapid responses to this stimulus (eg.
>intracellular calcium levels). The set-up I am imagining is having a laser
>source (the ideal for us is a tunable one, in order to be able to examine
>responses to different wavelengths) and a fiber optic to carry the light to
>the vicinity of the cell (I guess a micromanipulator would be necessary to
>target the cell). Does anybody know which is the smallest spot of
>illumination possible nowadays? Will I be able to stimulate a single cell, a
>group of cells, an area on the surface of a cell? Also, is there an
>alternative to this (to avoid the obvious expense)? (I am sure all these are
>also questions you will like, Johannes...are you there?)
>
>        Another non-related question: I have just used DiOC18 to stain
>membranes. Since it has a long alkyl chain, I was expecting it to stain well
>the plasma membrane and may be, to some extent, intracellular membrane
>systems like endoplasmic reticulum. However, the latter was amazingly well
>stained. I am not disappointed by this at all, because it turned out to be a
>beautiful prep, but it was not what I had expected. Any explanations for
>this? Could anyone tell me his favorite way to use the dye? (it could be a
>preparation problem).
>
>        Thanks a lot,
>
>                                                        Susana
>
>

Neal Nicklaus
Senior Scientist
SEQ Limited

Voice:  609-452-6033 Ext. 13
Fax:    609-452-5955
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