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.

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.


For decades, the Kohler epi Illumination system has been an industry standard. This design intentionally obscures the performance of illumination components in the epi light path, resulting in an inability to visualize how illumination components interact together from within the microscope. Advanced microscopy techniques increasingly rely on a complex relationship of components in the epi light path of a microscope. These components need to be properly coupled, focused, aligned and free of light path obstructions to provide maximum illumination performance. 

After more than a century of microscope evolution, the CaliCube now provides users with the ability to monitor all of these critical parameters together. Unlike a Bertrand lens, which provides a view of the illumination plane in rear of the objective, the CaliCube offers a view of the illumination plane as it appears in the epi illumination path. This greatly improves the ability of users to monitor problems that frequently compromise microscope performance.

Laser Illumination

Laser beam profilers are used to evaluate bench top laser illumination systems. Laser illuminated microscope systems obscure laser illumination for various reasons, including safety. The CaliCube, like a Laser beam profiler, provides an image of the laser illumination as it appears in the illumination plane, enabling users to evaluate performance characteristics such as position, focus, intensity, jitter, beam wander, beam diameter, beam quality and beam divergence, without exposing users to safety issues. These issues can now be reported to service experts without costly service visits, while minimizing the down time of expensive equipment.  

Microscope imperfections

Most microscopes have rotating filter turrets with tolerances that can lead to filter cube registration problems. Each position in a filter cube turret will often have a unique alignment bias, leading to small changes in image centration. Some of these turret positions may have similar orientations while others may have significant offsets. One effect of this tolerance shows up when a multilayer image is created using more than one filter cube. The multiple cube orientations leads to field diaphram misalignments and image shifting among the image layers captured. The CaliCube can be used to measure orientation variations between each turret location and determine which of those locations offer the best options for colocalized imaging. Additionally, advanced imaging systems that rely on precise filter cube alignment performance can use this information to help resolve offset related problems.

The montage above is a collection of CaliCube images taken in each turret position from three randomly chosen microscopes. Four common problems are revealed.

The top series of images show three significant problems. The obvious movement of the center of the image from one picture to another is the first issue. This clearly demonstrates a significant filter turret alignment issue. The second is the collector lens has a significant amount of debris on it, that will reduce the throughput and contrast of the light source. The third issue is the rough edge along the left side of each image. This rough edge is the result of the mounting surface of the microscope not being perpendicular to the light path. It shows up cropping the side of the image. In this case, the user complained of being unable to properly align the lamp and assumed they were doing something wrong. Significant defects such as these are not uncommon, even in high end research microscopes.

The second series of images again shows quite a bit of movement from one filter position to the next, indicating significant filter turret variations.  Additionally the liquid light guide coupler was defective, such that right side of image is distorted and shows lots of shading.

The third set of images, were captured with a wider field CMOS camera. There are no distortions in the image from alignment issues and the centration of each image, as indicated by the proximity of the illumination circle with respect to the red line, is similar in each turret position, with the exception of an approximately 3% offset for the first position. 

Simply inserting a CaliCube into each position in a filter turret and monitoring the shift in image centration provides users with the ability to measure these turret offsets. Once measured, turret locations can be selected to help users improve repeatability.

Light source degradation and standardization

The illumination intensity of fluorescent bulbs and LEDs in microscopes often deteriorate faster than expected. The CaliCube offers the ability to:

  • Monitor the nonlinear decline in light intensity
  • Index that decline with future data
  • Make intelligent decisions about replacing required parts to ensure optimal performance

Liquid light guide coupler performance

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The CaliCube is the first and only device that enables users to evaluate the quality and efficiency of this critical component. A large number of light guide couplers are available for users to choose from today. Choosing a proper light guide coupler can have a significant effect on widefield performance. The CaliCube is the perfect product for evaluating coupler performance. Whether choosing a coupler with a fixed field size or properly configuring an adjustable coupler, the alignment and size of the coupler illumination field can be clearly observed with a CaliCube. This provides users with the ability to optimize this component for their application. Coupler alignment problems (shown in the second series of images above) also contribute to a significant percentage of illumination problems and are capable of reducing the illumination throughput by more than 50% and creating serious shading issues. 

Liquid light guide performance

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A microscope's widefield performance is dependent on a properly functioning liquid light guide. The task of efficiently transmitting light from a light source to the microscope can be easily compromised. The recent incorporation of liquid light guides into standard widefield microscope design has offered microscope users an unprecedented choice of convenient and powerful light sources, facilitating a historic leap in performance. Unfortunately, that performance can be significantly compromised by poor positioning of the cable, particle obstructions at the tips of the light guide, bubbles inside the light guide and premature degradation. Most users are unaware of the performance degradation in their light guides even if they are properly connected, simply because they can not view a coupled light guide from within a microscope. The CaliCube is a calibration device that enables users to simply view and measure light guide performance so users can detect problems as they arise. the CaliCube provides the crucial information needed to resolve these issues without the need for expensive and time-consuming professional help.

Arc lamps

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Although not widely used, High pressure Mercury and Xenon illumination systems have a short life span that can vary enormously, depending on the conditions of use. The Calicube provides quantifiable data making it possible for users to avoid prolonged or premature bulb replacement. 
The replacement of Mercury and Xenon arc lamps also requires a tedious alignment procedure. Many users count on professionals to do this when changing bulbs. The Calicube provides a clear view of the arc so alignment can be performed with the aid of the imaging system or with the use of remote assistance, facilitating a more precise and easier alignment procedure.

Tool for Learning

The CaliCube is an excellent instructional aid, revealing those elements involved in the illumination path that would not be visible otherwise. Students can now visualize the relationship between the various components and adjustments in the illumination path and the effect they have on the resulting image. 

The total picture

The sum effect of all of the contributing epi light path components can now be imaged together. Epi illumination performance can be properly monitored and quantified as never before, simply by routinely monitoring an image captured with the CaliCube. As components degrade over time, this important data can be used to explain the consequential change in values or non-uniformities that end up in the data. Informed decisions can then be made to create performance thresholds that are meaningful to users. Lab and Core facility managers can use the CaliCube to provide quantifiable performance standards for their equipment.