Confocal microscopes are known for optically removing the out-of-focus information (i.e., blur) in each transverse section of the sample’s volume, providing a more accurate three-dimensional image of thick microscopic samples than widefield microscopes. In this work, we have designed and evaluated a confocal microscope using off-of-shelf optical components from Thorlabs’ catalog, one of the major optical manufacturers. The design and evaluation have been implemented using Zemax OpticStudio, the standard optical system design software. Here, we have also reported a practical protocol for building a confocal microscope using sequential mode.

BACKGROUND OF CONFOCAL MICROSCOPY

A confocal microscope is composed of two subsystems: the illumination and detection systems. Figure 1 shows the optical configuration of a confocal system. The monochromatic light emitting from a laser source is collimated and focused onto the three-dimensional sample by a microscope objective lens. Although the focused beam illuminates all parts of the sample within the illumination cone, the small region surrounding the beam’s focus receives the highest illumination energy. Assuming that the illumination wavelength is within the excitation band of the fluorescent dye, the sample’s region stained by those fluorophores are activated, emitting fluorescent light (e.g., light with a longer wavelength than the illumination wavelength). In traditional confocal microscopes, the detection system is composed of the microscope objective lens, the tube lens, a pinhole (e.g., an optical aperture), and a photomultiplier as a detector. The fluorescent light scattered by the sample is collected by the microscope objective lens and focused on the pinhole thanks to the tube lens. The photomultiplier is the ideal photometric detector for this imaging modality based on its speed and sensitivity. Experimentally, the photomultiplier is placed right after the pinhole to collect the maximum amount of light. As previously mentioned, the feature of confocal microscopy is its optical sectioning capability (i.e., the optical removal of the out-of-focus information). This capability is a consequence of the fact that the object focal plane of the objective lens is conjugated with a pinhole plane. Therefore, parts of the sample outside the in-focus plane (e.g., object focal plane of the objective lens) are not conjugated with the pinhole (e.g., image plane), reducing the probability of fluorescent light detection. In other words, point-based detection using the pinhole is responsible for providing optical sectioning capability. On the other hand, parts of the sample outside the in-focus plane receive a smaller illumination density, reducing the probability of fluorescent emission and photobleaching of the samples.

[Fig. 1. Optical configuration of a Confocal Microscope. The key feature of the point-based illumination and detection.]

Here we provide a practical protocol for building a confocal microscope using off-of-shelf optical components from Thorlabs’ catalog, one of the major optical manufacturers. The design and evaluation have been implemented using Zemax OpticStudio, the standard optical system design software. In our design, we have two main changes to Figure 1. The first one is the replacement of the dichroic mirror by a beam splitter. Dichoric mirrors reflect and transmit light at two different wavelengths (e.g., color). For example, a longpass dichroic mirror with a cutoff wavelength of 490 nm reflect light with wavelength shorter than 480 nm towards the microscope objective lens in the illumination system and transmit wavelength larger than 500 nm towards the tube lens in the detection system. Conversely, beam splitters are optical elements that split incident light into two beams regardless of the wavelength. Although a beam splitter is not the desired optical element to build an experimental confocal microscope due to the loss of energy, it allows us to analyze the performance of the confocal microscope at different wavelengths without replacing the dichroic mirror. The second change is replacing the microscope objective lens with a condenser lens for design simplicity.

Let us start building a confocal microscope. The Zemax design of the microscope objective lens and tube les are found on Thorlabs’ website. The first element of the confocal microscope to insert is the beam splitter (Surface 2 in Fig. 2), which reflects the incident beam down at a 90o angle. Remember that the beam splitter separates the incident beam in two directions: one beam is transmitted through the beam splitter, and the second is reflected as a mirror does. The beam splitter was created by inserting a foldable mirror surface after the first element (a dumb surface, Surface 1 in Fig. 2) in our system with an angle of 45o.

[Fig.2. Two-dimensional optical layout of the confocal microscope in Zemax OpticStudio.]

The second element in our system is an aspheric condenser lens (Surface 3 in Fig. 2), which replaces the microscope objective lens. Without any loss of generality, we select the AC1210U-A Thorlab’s aspheric condenser lens with a diameter of 12 mm, a focal length of 10.5 mm, and a numerical aperture (NA) of 0.54. The AC1210U-A lens has a focal spot size equal to 3.953 µm at a wavelength of 633 nm. The third element of the confocal microscope is a flat mirror. This mirror mimics a perfect thin fluorescent sample, reflecting the light back into the condenser lens. Since the confocal microscope is a point-based illumination, the mirror should be located at the focal plane of the condensing lens. Based on the manufacturer’s datasheet, the focal plane is placed at a distance equal to 6.725 mm distance from the last surface of the lens. The diverging light reflected by the mirror is collimated by the condenser lens, generating a collimated plane wave after the condenser lens. A collimated plane wave is such a wave whose size does not change, generating a point source at infinity. The collimated plane wave meets the tube lens (TTL200-A, Thorlabs), which is Surface 5 in Fig. 2. The separation between the condenser lens and the tube lens is irrelevant since the wave is collimated. Nonetheless, we ensure that the distance was enough so that the tube lens is after the beam splitter in Fig. 2. Since the tube lens is illuminated by a collimated plane wave, focusing the light into a point at its image plane. Based on the manufacturer’s datasheet, the image plane is located at 152.008 mm to the last surface of the tube lens. In an actual experiment, the image plane is the plane to set the pinhole. It is important to mention that the tube lens is shown as a black box in Fig. 2. We have chosen this lens to optimize the performance of the confocal microscope. This type of tube lens is known for its careful design, which contains multiple optical surfaces to compensate the optical aberrations further.

Zemax OpticStudio software allows the evaluation of the performance of any optical system using different metrics, including the analysis of the spot diagram, the aberrations present at each surface of the system, and the impulse response and its reciprocal Fourier transform. All these analyses are available on the Analyze tab of the software. Without any loss of generality, we have evaluated the performance of the proposed confocal microscope by analyzing the aberrations through the Seidel, the system’s frequency response through the Modulation Transfer Function (MTF), and the variation of the spot size. First, let us start with the Seidel diagram, which provides information about the aberrations up to the third order. In other words, Zemax OpticStudio informs us about the following aberrations: spherical aberration, coma, astigmatism, field curvature, distortion, axial color, and lateral color. A more detailed information of these optical aberrations can be found in (Dereniak, 2008). Based on the Seidel diagram, we found that spherical and axial color aberrations were the only ones present in the system. Spherical aberration is present when the outer parts of an optical element do not focus the light rays at the same axial plane, generating a blurred image. On the other hand, axial color aberration is a type of chromatic aberration caused by the fact that light rays focus on different axial planes based on wavelength/color. In other words, if the material of the optical element presents normal dispersion, blue light always focuses on planes before red light. The aspheric condenser lens introduced these aberrations. However, since the light passes through the condenser lens twice (e.g., forward in the illumination system and reverse in the detection system), these aberrations cancel each other, not affecting the final performance of the confocal microscope (e.g., at the image plane there are not aberrations distorting the confocal system).

[Fig.3. System’s MTF for different wavelengths.]

The second metric used to analyze the performance of the confocal microscope was the MTF plot. This curve informs us how the information is transferred through the imaging system. In particular, we can find which and how much the spatial frequencies are distorted by the system. In other words, the MTF plot provides a quantitative measurement of the image contrast versus the spatial frequency. A key feature of Zemax OpticStudio software is that we can plot the system’s MTF and compare it with the theoretical limit (known as the diffraction limit), providing us with another way to visualize if our system is distorted by aberrations. Although we cannot identify the type of aberrations by observing the MTF plot, as a general rule, the lower the area under the curve of the MTF plot, the higher the number of aberrations. Figure 3 shows the MTF plot of the confocal microscope for different wavelength (blue curve in Fig. 3) and its comparison with the diffraction limit (black curve in Fig. 3). As we can see, both blue and black curves overlap for the different wavelength, meaning that the system does not present any aberration. Observing Fig. 3, we have estimated the maximum spatial frequency passing through the confocal microscope. This maximum spatial frequency is known as the cutoff frequency, the first frequency at which the MTF value is null. The cutoff frequency of the confocal microscope is wavelength dependent, being 130 lp/mm for violet color (λ = 0.4 μm), 98 lp/mm for green color (λ = 0.515 μm), and 77 lp/mm for deep red color (λ = 0.655 μm). The higher the value of the cutoff frequency, the higher the resolution of the confocal microscope, being able to discriminate finer details. This behavior is already known in Optics; the same optical instrument has a better resolution capability using purple-blue color light than red.

[Fig.4. Performance of the confocal microscope by comparing the spot size at different wavelengths]

Finally, we have evaluated the spot diagram of our confocal system, see Fig. 4. The spot diagram shows the image produced by our confocal system assuming that the object was a point source (e.g., the spot of light). Aberrations such as astigmatism and coma are instantly viewed on the spot diagram since they produce asymmetric images. Figure 4 shows that the spot diagrams for the different wavelengths are symmetrical, confirming that astigmatism and coma aberrations are not present. This result already agrees with the conclusion provided by the Seidel diagram. From Fig. 4, we can see that the spot size is wavelength dependent. The root-mean-square (RMS) radius is: 1.015 μm for purple color (λ = 0.400 µm), 1.138 µm for green color (λ = 0.515 µm), and 1.339 µm for deep red color (λ = 0.655 µm). Note that there is a variation of 0.329 μm within the visible spectrum.

Conclusions

We have implemented a confocal microscope using off-of-shelf optical components and evaluated its optical performance in terms of the aberrations, the spot size, and the MTF plot. Because confocal microscopes are typically used for multicolor imaging, it is important to ensure that the design is invariant to different wavelengths within the visible range. To simplify the design of the microscope, we replaced the dichroic mirror with a beam splitter. Note that another alternative exists to build a confocal microscope for inexperienced users of Zemax OpticStudio. This alternative is the design of a confocal microscope following an inline configuration. In other words, no dichroic mirror and beam splitter are considered, and we insert all the elements in a sequence. If one follows this configuration, the microscope objective lens must be insserted twice and in reverse order since the light passes through it twice. In the proposed confocal microscope, we have also replaced the microscope objective lens with an aspheric condenser lens. The latest change would reduce the cost of the experimental system since the cost of the proposed aspheric condenser lens is $32.61 versus $1,2667 for a Nikon microscope objective lens with the similar NA (N20X-PF, Thorlabs). The results shown here demonstrate the effectiveness of our design in comparison with the theoretical expectation. We have also shown that the proposed confocal microscope is quite robust to the change of the wavelength, showing a slight variation of 0.329 μm in the spot size from 400 nm to 655 nm. The small variation ensures the use of the same pinhole size for multicolor imaging without affecting the performance of the experimental confocal microscope. Future work should be focused on a more detailed comparison of the system’s performance using an aspheric condenser lens, including experimental results since no reported system has used it. This work allows undergraduate students interested in Optical Engineering to increase their knowledge in optical system design using the Global Academic Program of Zemax (e.g., free license for academia). In the event of insufficient funding to implement an optical laboratory, Zemax OpticStudio also allows undergraduate students to become familiar with different imaging systems, providing them with a hands-on tool to design and analyze optical systems.

References

Dereniak, E. L., and Dereniak, T. D. (2008). Geometrical and Trigonometric Optics. Cambridge: Cambridge University Press.

Zemax files

Download the different files for the ZEMAX confocal microscope
file.ZDA
file.ZMX

Funding

This research undergraduate experience is part of the NSF CAREER award (grant number 2042563).

Credits

The spectrometer is implemented using the Global Academic Program (e.g., free license for academia) of Zemax OpticStudio version 22.2.1.

Citation

If using this design for publication, please kindly cite the following: E. Costello, L. Hamilton, and A. Doblas, “Design and evaluation of a confocal scanning microscope using off-of-shelf optical components by means of Zemax OpticStudio optical design software,” QuaesitUM Undergraduate Research Journal, under review (2023).

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