Constructing a Filter Cube
For using the Olympus IX70 to image fluorescent emissions
Virginia E. Vance, Kevin Pollack, Jeffery E. Raymond & Karen L. Wooley
Texas A&M University
Departments of Chemistry and Chemical Engineering
Group Meeting, 2013/04/12
Gathering Fluorescence
Sample
Fluorescence is the absorption and reradiation of light by organic and inorganic
specimens.
Objective
By determining the wavelengths of
light that cause a sample to emit
light, one can excite at only those
wavelengths, and exclude
extraneous emissions.
Filter cubes allow one to
quickly and cheaply
assess samples with
fluorescence with easily
replicated results.
Detector
Light pathway through a filter cube
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Collecting Fluorescence
A filter cube consists of three filters
housed in a filter cube or box: the
excitation filter, the emission filter, and
the dichroic (DC).
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•
•
Excitation filter:
transmits only selected
wavelengths that excite
the fluorophore.
Emission filter:
transmits light emitted
from excited specimen,
sample, or dye
Dichroic: separates the
emission and excitation
wavelengths
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Fluorescence Imaging
Fluorescent imaging requires that
one determines
excitation/emission spectra for
the fluorphore (eg: Cy3 dye).
Excitation and emission
spectra of unconjugated Cy3
dye in phosphate buffered
saline.
Excitation maximum (nm)
Emission maximum (nm)
548
562
Determine which filter transmits at the excitement
wavelength. This is the appropriate excitation filter
Determine which filter transmits the emission
wavelength. This is the emission filter.
Determine which dichroic reflects the excitation
wavelength and transmits the emissive wavelength.
Constructing a Filter Cube
Using selected filter,
gently place the
excitation filter into
the cube, with the
arrow on the side of
the filter pointing in
the same direction
the light beam will
travel.
Some filters have a
indistinguishable
black arrow; look
carefully.
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Constructing a Filter Cube
Gently screw
the retainer
over the filter
using clean
fingers.
When the retainer is secure,
place the key in the
indentations of the retainer
and tighten gently.
Repeat procedure for the emission filter.
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Constructing a Filter Cube
Loosen screws, but do not remove. Slide DC into
place under the clips, and tighten screws.
Assemble cube and screw
together. Label filters used or
label cube.
Installing Filter Cubes
Remove the filter cube
turret from the
Olympus IX70.
Loosen screw,
and slide filter
cube in place.
Be sure that the
excitation filter
is properly
aligned.
Replace
magnetic lid
and reinstall
under
eyepiece.
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Future Work
• Filtercubes: Get a list of chromophores that
the group is using and generate filter sets/
filter cubes for those projects.
• Complete filter/dichroic characterization.
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•
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Other Activities
Results of investigation of auto-fluorescence
of ONR film
QNM of ONR film
DMA SOP’s
Nanomechanical standards for Bruker AFM
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