This article shows how to design a confocal fluorescent microscope in OpticStudio using a combination of the Sequential and Non-Sequential Modes. 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. This article provides a walk-through of the design of the confocal microscope. It also discusses how to build a merit funciton for optimization and how to use the Convert to NSC Group tool to convert system from Sequential Mode to Non-Sequential Mode.

Authored By Lisa Li

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Introduction

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. This article will provide a walk-through of the design to show how to acurrately 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.

System overview

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 photo-detector. The placement of these pinholes at conjugate points is what makes this design a confocal microscope.

Note: While this microscope is not designed to be a scanning confocal fluorescent microscope, a group of laser collimating optics is included in the example design to serve as a reference template for where to modify the design into a scanning confocal microscope.

Designing the laser focusing system

The design for the laser focusing system will first be done in Sequential Mode. The constraints for the example system’s laser portion are as follows:

First create the surfaces needed for focusing optics, selecting any glass(es) for the materials column. Vary only the radii of the lens surfaces. Using the glass substitution template option in the material column is optional.  Apply the default merit function settings, optimizing on minimum RMS spot radius, as shown below. 

Set some preliminary radii before running a local optimization on the lens. Then, if you wish to change the glasses in your system, run a hammer optimization. After you have reached a satisfactory doublet, freeze all variables and add another surface to the end of your system. This surface will act as a dummy surface placeholder for your pinhole.

Put 70 mm between the pinhole and the first surface of the collimating optics. Add the surfaces for a collimating lens to the end of the system. In this example, the lens thickness is 6mm. Then go to the System Explorer and under the Aperture tab, check the box for “Afocal Image Space”. The collimated beam diameter needs to be smaller than the entrance pupil diameter of the objective. Vary and adjust the thicknesses and radii to meet this constraint.

In the merit function wizard, create a new merit function, this time optimizing for minimum RMS wavefront error. Then click okay and optimize. The output light from the system should now be collimated. Add a dummy surface to the system 40mm after the pinhole location to represent the location of the dichroic mirror. 40mm after the pinhole location, add the K_007 objective to the system. The final system should look similar to the following:

Designing the tube lens

To design the tube lens, begin by opening a new file and opening Zemax Design Templates and opening "Microscope Objective 60x" (or file example K_007 previously in Zebase, see the Help Files section The Libraries Tab > Design Templates Group > Design Templates for more details). Add a 1mm thick glass plate 40mm after the last objective element to model the decenter introduced by the dichroic mirror in the full system. Use the tilt/decenter icon to tilt the 1mm thick glass surface by -45 degrees about x.

Add four lenses to the end of the system to become the tube lens elements. Put 40mm between the dichroic mirror and the first surface of your tube lens and then decenter the tube lens elements in the y-axis to accommodate the shift introduced by the plate. Insert a surface after the tube lens coordinate break and enter a fixed thickness of 1mm.

The system constraints:

Fields:

Vary the radii and thicknesses of each surface and set the material solve type to substitute. Your final optimized system should resemble the following.

Conversion to Non-Sequential Mode

Open the file for the first system. Select File...Convert to NSC Group, and ensure that all boxes in the Convert To NSC Group window are checked. Press OK. 

In the System Explorer, change the Non-Sequential settings to the following.

Insert a Source Ellipse object type in front of the system. Set both the X and Y Half Width parameters to 1.350 mm. Set the Wavenumber to 1, so that the source only emits 488 nm light.

Insert a Standard Surface object type at Surface 5 referenced to Surface 4. Adjust its Z Position so that it sits at the focal point of the focusing lens system. Then, insert a Standard Lens object at Surface 9, referenced to Object 8 so that it sits 40mm away from the second surface. This is your dichroic mirror. Tilt the mirror about X by -45 degrees.

Next, use the Modify Reference Object button to reference each part of the K_007 objective to the object before it with the first surface of the objective referenced globally.

Tilt the first objective surface by 90 degrees about x and move it -40mm in the Y-axis. Maintain the same global Z-position as the dichroic mirror. Then use Modify Reference Object to reference it to the dichroic mirror.

Next, incorporate the tube lens system into the Non-Sequential file. Convert only the tube lens surfaces from Sequential Mode. In the Non-Sequential Component Editor, use the Modify Reference Object to reference each tube lens to the lens before it. Then, highlight all surfaces and right click to select “Copy Objects.” Paste these objects into the end of your total non-sequential system. Correct the reference objects of the tube lenses so that they are all relative to the lens before. The first tube lens should be referenced to the first lens of the microscope objective by changing the reference object number. Decenter the tube lens in the y-axis and set the tilt about Z in the first tube lens to be 180 degrees. Then, use the Modify Reference Object button to reference the tube lens to the dichroic mirror. The tilt about x should be -45 after this step.

Next, coat each lens surface in the system with the AR coating. For the dichroic mirror, set the front surface (facing the objective) to be coated with FLUORESCEIN and the back surface to be coated with the ideal coating I.99. FLUORESCEIN reflects 488nm and passes 521 nm and is not included in the default coating file. It must be added manually by the user. The data for the Fluorescein coating is included in the downloads below.

Finally, add two objects to the end of the system. Make one type a Standard Surface and the other a Detector Color. The Standard Surface material should be set to material type Absorb to model a pinhole. Distance this pinhole to be conjugate to the previous pinhole in the system. Place the detector close behind the pinhole to capture the image.

Modelling the sample

There are three methods that can be used to model the sample of the fluorescent microscope: by source volume rectangle object, by a backlit slide, and by the photoluminescence feature. The final downloadable non-sequential file has the three methods for modelling samples laid out as configurations one, two, and three respectively.

The first (shown above) is to create a small source volume rectangle the approximate size of the beam diameter on the sample plane. Set it to output light at the fluorescing wavelength and to have it radiate light in all directions. This model demonstrates the effect of the second pinhole on the sharpness of the image.

A backlit slide can also be used as a second method. Place an image at the sample plane as a Slide object. Then, use a Source Rectangle object to backlight the slide. This model is used more to demonstrate the image contrast for the system.

Finally, the photoluminescence feature can be used to model the sample seen by the microscope. Add any volume object (volume rectangle, standard lens, volume sphere, etc.) to the system behind the last microscope objective surface where the collimated laser beam is focusing. Make the object’s cube dimensions comparable to the size of the object you will be looking for. In this system, volume sphere objects with a radius of 10 microns were the target size. Under object properties inside the volume physics window, select the model “Phosphors and Fluorescence Data” and enter the spectral data of your fluorescent material into the window.  Spectral data for absorption and emission are required. Then a spectrum for quantum yield must be included.

The example system here is fluorescein in ethanol as a model. Set the fluorescent volume’s Material to model the properties of ethanol. At the sodium d-line, the index of ethanol is 1.36168 and the Abbe number is 59.35.

This spectral data was acquired from the Oregon Medical Laser Center online database provided by Dr. Scott Prahl.

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