March 30, 2021

How to design a confocal fluorescent microscope in OpticStudio

How to design a confocal fluorescent microscope in OpticStudio

Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser confocal scanning microscopy (LCSM) is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures (a process known as optical sectioning) within an object. This technique is used extensively in the scientific and industrial communities and typical applications are in life sciences, semiconductor inspection and materials science.

OpticStudio uses the combination of the Sequential and Non-Sequential modes to design such a system. The system is designed in two major parts: from the laser source to the microscope objective, and from the microscope objective to the tube lens and detector.

Confocal fluorescent microscopy is a means of obtaining high resolution 3D images of a sample and is especially useful in the life sciences and in the semiconductor industry. To generate such high-quality results, the microscopes are designed in two major parts: from the laser source to the microscope objective, and from the microscope objective to the detector. In this article we provide a walk-through of the design to show how to accurately model confocal microscopes in OpticStudio. The microscope objective used in this example is the "Microscope Objective 60x" available in Zemax Design Templates (or file example K_007 previously in Zebase), which is available to all editions of Subscription OpticStudio 20.2 or above.

A confocal fluorescent microscope's optical system consists of a laser illumination source, a focusing lens, a collimating lens, a microscope objective, a tube lens, and a detector. These optics are configured in the following orientation:

The purple beam represents the propagation of the laser source. The thicker red beam represents the in-focus fluorescence captured by the detector. The thinner red beam is included to illustrate the purpose of the second pinhole. The first pinhole is placed between the laser focusing and collimating optics. The second pinhole is placed after the tube lens in front of a photodetector. The placement of these pinholes at conjugate points is what makes this design a confocal microscope.

To learn more about how to design the laser focus system, tube lens, convert to non-sequential mode and model the sample, Zemax customers can access the entirety of this Knowledgebase article and the entire series on MyZemax.com. Otherwise, please reach out to Zemax Sales to learn more about OpticStudio.

Author:
Lisa Li

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