Constructing a Filter Cube 01/01/09 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 Group Meeting - Name

Gathering Fluorescence Objective Detector Sample Fluorescence is the absorption and re-radiation of light by organic and inorganic specimens. 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 cube graphics; emission/excitation data for Kevin’s samples. Filter cube: F11, DC 2, F13 Filter cubes allow one to quickly and cheaply assess samples with fluorescence with easily replicated results. Light pathway through a filter cube

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). 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

Fluorescence Imaging Fluorescent imaging requires that one determines excitation/emission spectra for the fluorphore (eg: Cy3 dye). Determine which filter transmits at the excitement wavelength. This is the appropriate excitation filter Determine excitation/emission spectra for your dye. 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.

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.

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.

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. Other Activities Results of investigation of auto-fluorescence of ONR film QNM of ONR film DMA SOP’s Nanomechanical standards for Bruker AFM