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Rapid Staining Technique that provides high- contrast fluorescence microscopy imaging of resin embedded samples, reminiscent of low magnification transmission electron microscopy


Fluorophore-Infiltrated Resin Microscopy (FIRM)


n Works with Epoxy and Acrylic type resins


n Simply mount your sections on glass slides, air dry, and overlay with 100 microliters FIRM for 30 seconds, wash and coverslip with water or an aqueous mountant. View in Rhodamine channel of a standard wide field Fluorescence Microscope.


n FIRM staining is unique in that the brightly fluorescent dye infiltrates the resin, revealing tissue structures in negative relief, with extremely high contrast and resolution..


n Perfect for previewing specimens prior to EM Analysis


n High Throughput, high contrast image phenotyping of model organisms such as drosophila


n Ideal for material and food science analytical microscopy


Bridging the gap between light and electron microscopy.


Fluorophore-Infiltrated Resin Microscopy (FIRM) is a novel staining method providing ultra-high contrast imaging of tissue subcellular structure using a standard widefield fluorescence microscope. FIRM images closely resemble those obtained by transmission electron microscopy (TEM) at low magnifications. The key concept is the infiltration of thin resin sections with a fluorophore. FIRM can be used either in epoxy or acrylate resins. When illuminated by epifluorescence, the brightly fluorescent resin reveals cell and tissue structures in negative relief, according to the light absorbing and scattering properties of such structures. The method provides much greater contrast of fine subcellular structures than obtained by typical semithin section brightfield stains, such as toluidine blue. FIRM images of multiple (>50) serial semithin sections of complex structures, such as kidney glomeruli, are easily rendered in 3-D by open source software programs.


GENERAL METHODS: During the testing of a large number of fluorescent dyes as candidate counterstains for thinsection immunofluorescence in LR white sections, one with special properties was encountered. Most dyes, such as sodium fluorescein, non-selectively stained tissue and provided little contrast (not shown). However, one such dye, when applied to either LR White or Epon sections, provided very high contrast images using standard fluorescence microscopy, with the resin appearing brightly fluorescent and tissue structures appearing primarily in negative relief (Figure 1).


FIGURE 1: Human anterior pituitary, FIRM, rhodamine channel. Note that the fluorophore illuminates the LR White resin, providing an image of tissue structure primarily in negative relief. Bar, 100 μm. All subsequent FIRM images are shown in monochrome.


FIGURE 3: Human anterior pituitary, FIRM image taken with 150X glycerin objective. Individual secretory granules are easily visualized, ranging from 150- 350nm in diameter. Scale bar, 5 μm.


AB AB


FIGURE 2: Human anterior pituitary, formalin-fixed autopsy specimen, comparison of H&E paraffin section (A) with FIRM (B). 20x dry objective used for both. Scale bar, 20 μm.


FIGURE 4: Correlative FIRM/fluorescent lectin labeling of human pituitary. A single LR White thin section was labeled first with Concanavalin AAlexa-488 (B) imaged, then stained for FIRM imaging (A). Densely granulated cells (arrows in A) are strongly labeled with Con-A (arrows in B). Dark structures resembling large lysosomes or residual bodies (arrowheads in A) are also strongly labeled by Con-A, consistent with reports in the literature.


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