Introduction: Confocal Microscopy
As the need to visualize and study scientific samples is increasing, the enabling techniques have kept pace with them. Microscopy is one such technique that came into being some 430 years ago and has evolved tremendously over time. From its use in cell biology, genetics, and forensics to microbiology and medicine, the different types of microscopy techniques have proved their potential and power from time to time.
One such powerful form is “Confocal Microscopy”. When students are first introduced to this topic, they tend to get curious about its evolution from the simple light microscope. They wish to learn to handle and utilize it for their lab experiments. Teachers are also enthusiastic to educate their students about the advancements of this microscopy and its utilization in research labs of both academia and industry.
Till this point, everything looks fancy about the confocal microscopy technique. But the complications arise when the functioning, setup, sample preparation, practical handling, and more know-how of the techniques are taught. Since there are many microscopes that have their specific functions and role, students are overwhelmed with the details taught in class.
To make the subject easier for both students and teachers, we have compiled the intricacies of the subject in this article. A deeper understanding of how a confocal microscope works, what happens when a laser beam scans a sample, or how this microscopy works for even live and thick sample sections are some questions that students can find interesting to deal with.
Read on to learn more about how you can ease the process of explaining the confocal microscopy technique to students in your next class. We try to highlight all the issues encountered by students when dealing with this microscopy and its handling techniques. We also list some practical solutions to solve the same. By the end, we’ll convince you why a virtual lab simulation will prove useful not only for your students but also for you as an educator to deliver concepts more efficiently.
3 reasons why Confocal Microscopy can be tricky to teach or learn
There are 3 reasons why students are overwhelmed by the topic of Confocal Microscopy techniques. Acknowledging these issues is the first step toward making the topic more approachable.
1. Having no idea about the different parts of the confocal microscope
Since students mostly use a ‘simple’ or ‘compound’ light microscope, they are usually aware of the parts of that microscope. With no idea of how a confocal microscope has evolved over a light microscope, they aren’t able to distinguish the different parts of this one. Plus, practical lab handling of microscopy at high school and university/college level usually involves hands-on training on light microscopes only. Due to the higher costs of confocal, fluorescence, and electron microscopes, they are rarely available on academic campuses for student use.
2. Failure to comprehend so many details
The confocal microscopy technique is a complex one as compared to the light microscopy technique. Having no idea of why lasers are used in this microscopy technique rather than a simple bulb or being oblivious as to what role a pinhole serves could leave most of the students in a dilemma. Not knowing the meaning of “stacking”, “out-of-focus light”, “unfocused background”, “horizontal optical sections”, “reconstruction of 3D structures”, “Z-stacking”, “invasive versus non-invasive techniques”, “photo-multiplier tube (PMT)” and many more of such complicated terminologies can trouble any student in the first encounter.
3. Lack of practical handling and demonstrations
Since owning one or multiple confocal microscopes can pierce a hole in the pockets of academic institutes, usually, they are not available in research facilities. This makes the topic a theoretical lesson to cram for students. Also, even if a confocal microscope is available, high school students are not allowed to handle it very often. This lack of practical handling and demonstrations in class kills the curiosity of the students to learn new techniques. Educators are further troubled due to a paucity of virtual teaching tools in the Edtech market. There aren’t any interesting and interactive visuals or videos or simulations available for teachers to make their classes more engaging and free-flowing for students.
5 ways to make Confocal Microscopy a more approachable topic to understand
To address the complications encountered while teaching the Confocal Microscopy technique, educators can engage the under-listed solutions in their classes. These can clarify many instrumental aspects of the techniques and their operation. Not only can they make teaching easier for educators like you but will also make lessons clearer and easier to assimilate for your students.
1. Simplify the basics of Confocal Microscopy
It’s of prime importance that the basics of confocal microscopy are clear to the students. As a first measure, educators should talk in depth about the different parts of the confocal microscope and explain their importance in the overall build of the instrument.
You can begin with the few examples listed below.
Talk about the up gradation of the lighting system: While a traditional light microscope has an incandescent tungsten-halogen bulb for illuminating the sample, a confocal microscope uses lasers for illuminating the sample. This laser serves as an intense source of monochromatic light (single, pure wavelength of light) which is very bright and enables better visualization of the sample than a halogen bulb.
Discuss the importance of the pinhole: Confocal microscope has a special “pinhole” that helps in the spatial filtering of the analysis volume. This filter enables us to gather information only from the ‘focal plane of our interest’.
Similarly, you can explain the role of other parts of the instrument (PMT, microscope stage, dichroic mirror, etc) to your students. This will clarify the subject and might generate most questions making the class on confocal microscopy livelier.
2. Explain the underlying principles and untangle data processing
After explaining the different parts of confocal microscopy, it's time you explain to your students how these different parts are assembled and work in conjunction with each other. You’ll be able to see the benefits of Step 1 now.
Assembling the instrument and imagining the path of light becomes a cakewalk once students are clear of the roles of the different parts.
You can use colorful illustrations and flowcharts to navigate your students through the path of light as it is emitted through a laser source, to dichroic mirrors (usually 2), across the sample exciting the fluorophores (dye) in the sample. Then trace the path of the emitted fluorescence that is descanned by the same two dichroic mirrors and finally reach the pinhole aperture. Lastly, make your students see how the light pinhole aperture rejects all the extra light from the different planes except the plane of interest. Finally, a photomultiplier tube that acts as a detector measures just light from one plane after all of the out-of-focus light has been eliminated.
You can further explain how PMTs translate the photons (light energy) to photoelectrons (electrical signal) used for final detection.
Figure: An interactive gif from the Confocal Microscopy simulation from Labster demonstrating how the image is generated using PMTs in the confocal microscope. Full simulation is available for University / College classes.
3. Clarify new concepts rather than just introducing the terminologies
Since sample preparation of confocal microscopy is different from light microscopy, teachers should explain each step with utmost care and detail. The fluorophores used in tagging the sample for visualization using a confocal microscope are a novel concept for students.
Educators can work on the following subtopics to clarify how fluorescence works in this technique.
Idea of fluorescence
Fluorescence versus Luminescence versus Phosphorescence
Wavelengths of light
Absorbance and Emission spectrum
Natural source of fluorophores
4. Exemplify the use in real life
Students must be educated about the real-world applications of techniques when they are trained to master any technique. This is bound to deepen the interest in the subject and can draw more effort when learning it.
If a confocal microscope is available in your school or institute, you should use different types of cell and tissue samples to exemplify the different uses of confocal microscopy.
There is a range of fields where this microscopy has proved its potential, some of them are:
Confocal microscope’s ability to enhance the ‘signal-to-noise ratio’, ‘step-up the chances of comprehensively examining thick sample sections’, ‘generation of in-depth visualization using stacking and Z-sectioned images’, and ‘exclusivity to adjust the microscope’s magnification electronically’ make this technique highly useful across a variety of fields.
Figure: An interactive gif from the Confocal Microscopy simulation from Labster demonstrating how confocal microscopy can be used to detect the presence of different disease-causing microbes in the plant leaf samples. Full simulation is available for University / College classes.
5. Use virtual lab simulations
Since confocal microscopy is a broad topic with many intricacies involved in its functioning and assembly, it can sometimes be difficult to demonstrate all of it in a single class. Even though educators and teachers are passionate to demonstrate these aspects, many times they fall short due to the non-availability of the instrument or consumables in the labs.
Since this microscope is a state-of-the-art facility, students are often not permitted to handle it on their own. This deprives them of practical handling and learning of the technique. Teachers trying to make this subject interesting for their students can switch to the Confocal Microscopy simulation option from Labster.
We overcome all the issues of practical handling by bringing this lively interactive simulation which improves the way your students understand the assembly, functioning, and handling of a confocal microscope.
With virtual laboratory simulations, educators can ease their process of lecture delivery as well as lab handling sessions. You can make more insightful points as students are rendered with better picture options. The 3D simulations help them better understand the intricacies of operating this instrument. Our interactive Confocal Microscopy simulation along with gamification elements ensure that your students follow what you are trying to convey in the class. By using this way of active and immersive teaching, our virtual learning platform takes an advent in the field of Science to make the upcoming scientists thorough with the “basics of their respective subjects”.
Figure: An interactive gif from the Confocal Microscopy simulation from Labster demonstrating the path of light in a confocal microscope. Full simulation is available for University / College classes.