Positioning Solutions for Total Internal Reflection Fluorescence Microscopy (TIRFM)

In total internal reflection fluorescence microscopy (TIRFM), the laser beam hits the glass plate of a microscope slide, a microtiter plate or Petri dish at a flat angle in order to stimulate the fluorescence.

Nevertheless, the electromagnetic field of the laser beam does penetrate the sample volume on the other side of the glass plate and forms an evanescent field with a penetration depth between 100 to 200 nm. As a result of the reduction of the stimulated area in the Z direction, a better Z resolution is achieved than the standard wide field - or confocal fluorescence microscopy. Here, the Z resolution is typically 500 nm. That is the reason why TIRFM is more frequently used in fluorescence microscopy as a comparatively simple and inexpensive super resolution method.

Setting the Critical Angle of Incidence

The laser beam for TIRF fluorescence stimulation is coupled for this purpose by shifting a radiation cage with mirror and lenses vertically at the edge of the objective at a specific angle.

This can be done with two >> Q-545 Q-Motion® Precision Linear Stages. Thanks to their piezoelectric inertia principle, they provide high nanometer-accurate position resolution, are compact and on top of that, inexpensive. Control is done by the >> E-870 PIShift Driver Electronics.

  • Compact design
  • High positioning resolution in the nanometer range
  • Fast positioning

High-Precision Positioning of Samples

PILine® XY stages position the sample on the plane quickly and accurately to a tenth of a micrometer, controlled by >> C-867 PILine® Motion Controller.
Very low-profile high-precision PInano® XYZ piezo systems can be mounted on the PILine® stages, e.g., a >> P-545.3R8S. They achieve a positioning precision of one nanometer in all three dimensions (x, y, and z).

In order to produce in-depth insights and revealing images, it scans the entire sample in nanometer steps in the X and Y direction. Extremely fine motion allows 3-D measurements to be made in the Z direction and this realizes the associated shift in focus. In addition, the PInano® offers low-noise positioning with a very fast and reliable >> E-727 Controller.