At 04:21 PM 3/22/01 -0500, Seijo, Edward R. wrote:
>Does anyone have any suggestions on how to measure the the thickness of
>bacteria layer adhering to a small segment of fiber optic cable. We have a
>Zeiss LSM 510 at our institution, but it is primarily used for biological
>applications and not materials.
>
>I would appreciate any ideas.
>
>Thanks.
Dear Edward,
The confocal can be used for sectioning and, therefore, for measuring,
assuming that there is some way to image the bacteria. Don't let the fact
that this instrument was purchased for bio applications limit your
thinking: the issues are the same for any type of matter, whether bio or
materials.
First, the imaging issue. You might want to test the bacteria for
autofluorescence. If the LSM has a series of dichroics, try each one to
see if you get an image. You might also try it in reflective mode. In
this case, if you get a strong artifact from the laser, try mounting a
quarter waveplate in the path. (I'd give you a more complete explanation
but I am working with an injured shoulder and will keep to the most
specific issues. Please send me another message if you need
details). Also, I have known instances where DAPI or DiI were used for
imaging biofilms... they might be helpful here, too. Molecular Probes, I
am sure, is also a good source for info... just let them know what kind of
bacteria you are working with.
Second: the depth measurement.
Depth measurements are derived from Snell's law and need to take into
account the refractive index of the mountant and the immersing
material. Below is a section from a chapter I am finishing up, which has
all the calculations. Instead of using the fine focus on the microscope,
you can use the Z drive on the LSM for much greater precision.
Hope this is helpful.
Best regards,
Barbara Foster
Microscopy/Microscopy Education
125 Paridon Street, Suite 102
Springfield, MA 01118
PH: 413-746-6931 FX: 413-746-9311 Web: www.MME-Microscopy.com/education
"Why didn't they teach us that sooner?" ... probably because no one
thought to call MME about customized, on-site courses. Offered in all
areas of microscopy, sample prep,and image analysis, they make an immediate
impact on your productivity.
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@
From ACS Handbook on Polymers/Chapter on Light microscopy (in press)
Depth
Depth measurements can be performed on any microscope fitted with a
graduated fine focus mechanism. For reference and to avoid backlash,
measure from the bottom to the top. (Reminder: as you focus AWAY, you are
moving from lower in the sample toward the top of the sample). Record the
initial reading on the fine focus, move to the top and record the second
reading. The apparent depth is the difference between these two:
Z’ = |Z1 - Z2|
where
Z’ = apparent depth
Z1, Z2 = initial and second readings
Equation 18. Apparent depth
Since Snell’s Law is very much in effect, you must compensate for
differences in refractive index:
Z = Z’ (n/n’)
where
Z = true depth
Z’ = apparent depth
n = ri, embedding or mounting medium
n’ = ri, immersion medium between sample and objective
Equation 19. Actual depth
Experiment: Use a marker to put dots on the top and bottom of a coverslip,
then measure its thickness. Two reminders: (a) most #1 ½ coverslips range
between 0.13-0.17mm in thickness and (b) the refractive index of glass is
typically about 1.5212
|
