Microscopy has proved immeasurably beneficial to researchers in the biological sciences. However, findings obtained with a confocal microscope are only as reliable as the performance of the microscope itself. Regular testing therefore is advised.Scientists can determine the overall performance of a microscope by measuring the axial response using a thin fluorescent layer. With a well-aligned microscope, this should yield a symmetric intensity profile with a width that corresponds to the theoretical resolution. Previously, researchers used chemically synthesized fluorescence layers to conduct such tests.In the January issue of the Journal of Microscopy, Michael A. Model and James L. Blank at Kent State University in Ohio reported that concentrated fluorescent dyes with a large extinction coefficient also can be used. Because light penetrates only a very thin layer in such liquids, the dyes can serve as two-dimensional fluorescent sources for confocal microscopes.Researchers have reported a novel and relatively simple means to test the alignment of a microscope, by measuring the axial response using a thin layer of a concentrated dye. Shown here are optical sections through a concentrated fluorescein sodium salt solution acquired with a well-aligned microscope (A), a microscope with a misaligned pinhole (B) and a microscope with a misaligned optical fiber (C). These images were taken with an Olympus Fluoview 300 and planapo 100×, 1.35-NA objective lens. Reprinted with permission of the Journal of Microscopy.They demonstrated this with concentrated solutions made from fluorescein sodium salt, acid fuchsin calcium salt or acid blue 9. For each, they placed several microliters of solution between a cover glass and a slide and acquired measurements with an Olympus laser scanning inverted confocal microscope outfitted with a 60×, 1.4-NA lens. They used a 488-nm line from an argon laser, a 543-nm line from a HeNe-G laser, and a 633-nm line from a HeNe-R laser, all from Melles Griot (now CVI Melles Griot), to probe the solutions.They collected three image stacks of the solutions: one on a well-aligned microscope, one with a misaligned pinhole and one with a misaligned optical fiber. The images showed that the misalignments led to a loss of axial resolution, a shift of the maximum intensity toward the edges of the field and growing heterogeneity across the X-Y projection.Thus the researchers used concentrated dyes to test a microscope’s alignment. They concluded that the method is a practical alternative to techniques involving chemical conjugation of dyes to glass, because it offers ease of preparation and homogeneity across the field, as well as photostability.