CaliCube: A Quantitative Tool for Epi Illumination

Microscope components are not perfect. Discover how.

More than 50% of the microscopes tested have epi illumination issues that compromise the performance of a microscope. Without an image of the epi illumination as seen from within your microscope, these problems are almost impossible to detect. 

The Real and Ideal

The specifications and tolerances associated with the optical components in an epi illuminated light path, are rarely optimal and often involve significant throughput and centration inefficientcies that go undetected in a microscope. Centration problems involve not only the optical systems within and external to a microscope, like a coupler or the internal relay optics, but also the fluorescent filter turret. Additionally the field size of the illumination is generic and often not well matched to the objective in use. CaliCube provides a diagnostic conjugate plane image of the epi light path, that reveals all of the hidden inefficiencies associated with the components that make up an epi light path. 

Human sourced errors including poorly coupled or maintained components can significantly affect microscope performance while remaining invisible to users. These include:

  • Dirt or residue from a spill on internal and external optics
  • Improperly attached optical components such as cables and couplers.
  • Misaligned diaphragms
  • Poorly positioned filter wheel or shutter 
  • Poorly positioned epi modulators such a polarizers

Now, both manufactured and human sourced errors can easily be diagnosed and monitored using the newly developed CaliCube. The CaliCube provides a comprehensive diagnostic image of the epi illumination plane in a microscope that not only contains critical information about the illumination input, but more importantly how that illumination input integrates with your microscope.

Advanced imaging applications utilize sophisticated illumination algorithms in the conjugate illumination plane to achieve a wide range of performance objectives. Moreover, the ability to monitor and quantify the performance of any epi illuminated application is essential for standardization. The following advanced imaging applications can benefit from this ability:

  • Light Sheet Microscopy (SPIM)
  • Super Resolution Microscopy
  • Structured Illumination
  • DLP, Galvanometer, LCOS directed Laser Systems
  • Laser Tweezers
  • Laser Ablation
  • FLIM
  • TIRF
  • FRET
  • FRAP
  • Polarimetry
  • LED  and Liquid Light guide coupled live cell imaging

The CaliCube provides users and developers with the unprecedented ability to simply monitor the expected output of these illumination devices by relaying a diagnostic image of the epi light source as it appears in the conjugated illumination plane of a microscope.

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The schematic diagrams demonstrate how a CaliCube can function in a wide range of epi illuminated applications.

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The example above demonstrates how a CaliCube can reveal centration issues resulting from filter turret and optical misalignments.

The advanced epi illumination systems listed above, rely on numerous components in the epi illumination path of a microscope. CaliCube users have the ability to discover and monitor tolerance and human error issues that can distort illumination and compromise performance. Once systems are optimized, the CaliCube can be used to document the illumination status of a microscope, prior to an experiment. Simple comparative performance procedures can be created between commonly equipped microscopes or with the same microscope over a period of time to ensure performance standards. 

Lab and Core facility managers, tasked with optimizing the value of expensive microscope equipment, now have the ability to simply optimize and monitor the performance of any microscope. Routinely generated CaliCube images can be used prior to an experiment to document equipment status and ensure users of the highest performance standards.

One simple tool provides users with the ability to monitor the many components that risk degrading a high performance microscope system.