We usually use dedicated fluorescence microscopy to observe fluorescence, but due to the high price, not every laboratory can have fluorescence microscopy. Therefore, we propose a simple, low-cost design scheme for fluorescence microscopy so that other laboratories that require fluorescence observations but lack equipment can refer to imitation. At the same time, we specially designed a blade cutting module to realize the integration of leaf cutting and fluorescence observation, saving space and easy carrying. The main components of the hardware module are eyepiece, objective lens, filter, excitation light source. The shell of the device is a box made of an acrylic panel, which is divided into three layers, with eyepiece and filters, objectives and slides. Three-layer acrylic plates are fixed in position with three stainless steel screws and some nuts.

4.1 Principle interpretation

The principle of partial fluorescence microscopy is shown in Figure 1, where the excitation light passes through filter 1 to obtain the desired blue light of 450-490nm. Since the beam splitting mirror can only pass through light with a wavelength greater than 510nm, the blue light is refracted to the objective part through the beam splitting mirror and illuminated to the object through the objective mirror. The fluorescence excited by the object is reflected back to the beam separator 2 through the objective lens. Because the excited fluorescence wavelength is greater than 510nm, the required special green fluorescence signal between 520 and 560nm can be obtained through the beam separator 2 and then through the filter 3.

Our hardware principle is similar. Since the required excitation light wavelength is 488nm and the filter wavelength is 400-750nm, we can directly filter the excitation light to observe the special fluorescence signal emitted by the fluorescent material. So our basic hardware architecture is shown in Figure 2.

4.2 Structural description

The eyepiece, the objective, the filter, and the slide layer inside the box are a 3mm thick acrylic plate. The acrylic plate in each layer had three holes with a miniature electric drill, and was fixed up and down with three 30cm long stainless steel screws and some nuts. The screw allows the acrylic plate to be mounted parallel in the middle of the black box, and the nut is not only fixed, but it also can be used to manually adjust the distance between the eyepiece, objective, filter, and slide to find the clearest observation point. Considering the filtering wavelength of the filter, we placed the filter under the eyepiece, namely between the eyepiece and the objective lens. When the excitation light is connected to the power supply (220V AC), illuminating the blade of the slide with excitation light, the fluorescence from the blade can be observed through the eyepiece.

4.3 Operating principle of simple microscope

Since the magnification of the eyepiece is 10 times, the objective magnification is 40 times, the eyepiece focal length is 25mm, and the objective focal length is 0.48mm, we adjusted the position between the nut of the upper and below the nut on the screw, so as to observe the image more clearly.

There is a self-made blade cutting table, which is used for the convenient cutting of the leaf samples, which is the characteristic design of the device. It consists of sponge, cylindrical blade, spring hinge, press clip, two scale stickers. When not in use, remove and put it in the vacant area in the corner of the black box, and take out the splicing on the removable board, saving space and easy to use. The assembled appearance of each component is shown in Figure 3.

The use method is as follows:

We usually use dedicated fluorescence microscopy to observe fluorescence, but because of the high price, not every laboratory can have fluorescence microscopy. To address this issue, we provide a simple, low-cost design protocol for fluorescence microscopy so that other laboratories that require observation of fluorescence but lack equipment can refer to imitation to make a simple device.

At the same time, the laboratory can imitate and make our blade cutting module, so as to realize the integration of blade cutting and fluorescence observation, saving space and easy to carry.