Design of Low-Cost Fluorescence and Brightfield Microscopes
Step 1: Overview of Fluorescence Microscopy
To understand the basic concepts of fluorescence microscopy, imagine a dense forest at night with trees, animals, shrubs, and other forests living in it. If you shine a flashlight into the forest, you will see all these structures and it is difficult to imagine specific animals or plants. Assuming you are only interested in seeing blueberry shrubs in the forest. To achieve this, you need to train fireflies to only be attracted to blueberry bushes, so that when you look at the forest, only the blueberry bushes will light up. You can say that you use fireflies to mark blueberry bushes, so that you can see the blueberry structure in the forest.
In this analogy, forests represent the entire sample, blueberry shrubs represent the structures you want to visualize (such as specific cells or subcellular organelles), and fireflies are fluorescent compounds. The situation of illuminating a shooter's flashlight alone without fireflies is similar to a bright field microscope.
The next step is to understand the basic functions of fluorescent compounds (also known as fluorophores). Fluorescent clusters are actually small objects (at the nanoscale) designed to connect specific structures in the sample. They absorb light of a narrow range of wavelengths and re emit light of another wavelength. For example, a fluorophore can absorb blue light (i.e. the fluorophore is excited by blue light) and then re emit green light. Usually, this is summarized by excitation and emission spectra (as shown in the figure above). These charts display the wavelengths of light absorbed by the fluorophores and the wavelengths of light emitted by the fluorophores.
The microscope design is very similar to a regular bright field microscope, with two main differences. Firstly, the light illuminating the sample must be of the wavelength that excites the fluorescent group (for the example above, the light is blue). Secondly, the microscope only needs to collect the emitted light (green light) while blocking blue light. This is because blue light is everywhere, but green light only comes from specific structures in the sample. To block blue light, microscopes typically have something called a long pass filter that allows green light to pass through without blue light. Each low-pass filter has a cutoff wavelength. If the wavelength of light is greater than the cutoff wavelength, it can pass through a filter. Therefore, the name is' Long Distance Pass'. Shorter wavelengths are blocked.
Step 2: Use optical modeling microscope
This is an additional step in designing microscopes based on basic principles. There is no need to build a fluorescence microscope, so if you don't want to delve deeper into optics, you can skip it.
Both bright field and fluorescence microscopes can be modeled using ray optics devices. The basic premise of ray optics is that the behavior of light is similar to that of light propagating away from a light source. When you look around the room, you will see the sunlight outside the window or the light brought by the light bulb. Then the light is absorbed or reflected by objects in the room. Some reflected light will direct it towards your eyes. If an object is illuminated, you can imagine that every point on the object emits light in all directions (as shown in the picture above). The lens, like the lens in our eyes, focuses light onto a point so that we can see objects. Without a lens, light continues to propagate outward and does not form an image.
