Designed as an accessory for the inverted IX-series of microscopes, the Olympus TIRFM illuminator acts to couple laser emission to the microscope via an external port. This tutorial explores alignment of the input fiber connector with the microscope optical path in order to optimize the incident light angle through high numerical aperture objectives.

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The tutorial initializes with green laser illumination at 550 nanometers passing through the fiber coupling and through several lenses a dichroic filter and a beamsplitter before entering the objective. Use the Wavelength slider to adjust the input laser median wavelength between a range of 400 and 700 nanometers. The Laser Alignment slider is employed to control a micrometer that adjusts the position of the fiber illuminator, which translates the laser beam upward and downward in the microscope optical pathway. Initially, the laser beam is positioned to travel through the center of the optical path and exit the objective without reflection or refraction. As the slider is moved (either to the left or right), the laser first experiences refraction at the boundary between the microscope slide and the medium of lower refractive index. Further movement of the slider will untimately cause the beam to undergo total internal reflection pass back through the microscope optical train.