Low-cost fluorescence and bright field microscope design
Fluorescence microscopy is a method used to visualise the
In this guide, I will review the basics of fluorescence microscopy and how to build three different low-cost fluorescence microscopes. These systems typically cost thousands of dollars, but there have been some recent efforts to make them more accessible. The designs I present here use a smartphone, dSLR, and USB microscope. All of these designs can also be used as 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, bushes, and other living forests. If you shine a torch into the forest, you will see all of these structures and have a hard time visualising specific animals or plants. Suppose you are only interested in seeing blueberry bushes in the forest. To do this, you train the firefly to be attracted only to blueberry bushes, so that when you look at the forest, only the blueberry bushes light up. You could say that you use fireflies to mark blueberry bushes so that you can see blueberry structures in the forest.
In this analogy, the forest represents the entire sample, the blueberry bushes represent the structures you want to visualise (e.g. specific cells or subcellular organelles), and the fireflies are fluorescent compounds. Illuminating the torch alone without the fireflies is similar to bright field microscopy.
The next step is to understand the basic function of fluorescent compounds (also known as fluorophores). Fluorophores are actually small objects (nanoscale) designed to connect specific structures in a sample. They absorb a narrow range of wavelengths of light and re-emit another wavelength of light. For example, a fluorophore may absorb blue light (i.e. the fluorophore is excited by blue light) and then re-emit green light. This is usually summarised by the excitation and emission spectra (above). These diagrams show the wavelength of light absorbed by the fluorophore and the wavelength of light emitted by the fluorophore.
The microscope design is very similar to that of a normal brightfield microscope, with two main differences. Firstly, the light illuminating the sample must be at the wavelength that excites the fluorophore (for the example above, the light is blue). Secondly, the microscope only needs to collect the emitted light (green light) while blocking the 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 usually have something called a long-pass filter that lets green light pass through without blue light. Each long-pass filter has a cut-off wavelength. If the light has a wavelength longer than the cut-off wavelength, it can pass through the filter. Hence the name, "long pass". Shorter wavelengths are blocked.
Step 2: Modelling the microscope with optical optics
This is an additional step to the basic principles of microscope design. There is no need to build a fluorescence microscope, so you can skip it if you don't want to delve into optics.
Both brightfield and fluorescence microscopes can be modelled using ray optics. The basic premise of ray optics is that light behaves similarly to light travelling away from a light source. When you look around a room, you see light from sunlight outside a window or from a light bulb. The light is then absorbed or reflected by objects in the room. Some of the reflected light causes it to be directed toward your eyes. If the object is illuminated, you can imagine each point on the object emitting light in all directions (above). The lens, like the lens in our eyes, focuses the light to a point so we can see the object. Without a lens, the light continues to travel outward and does not form an image.
So how do we make optical systems that magnify small objects? In order to understand the design, you really only need to know two equations: the thin lens imaging and magnification equations:
1/f = 1/si + 1/so
M = -si/so
f is the focal length of the lens. A shorter focal length means the lens has more focusing power.
The same is true for object distance; the distance between the lens and the object (e.g. a tree).
si is the image distance; the distance between the lens and where the image is formed
M is magnification; how large the image is relative to the object. For microscopes, we want to increase the magnification.
For a full tutorial on the thin lens equation, check out this Khan Academia video. In the gif above, you can see that the distance the object moves closer to the lens increases the image distance, which increases the magnification. The vertical line with two arrows indicates the lens.
