Introduction 

The trinocular inverted microscope is a high-level microscope designed for research institutes, health and medical units, and universities to observe cultured living cells. It is similar to the digital inverted microscope as it also has two eyepieces, but it includes an additional eye tube that connects to the microscope camera for video documentation and imaging. Moreover, unlike traditional microscopes, its light source, condenser, objectives, and turret are in inverted positions. The objectives and turret are placed below the stage, while the condenser and light source is above the stage. It is ideal for observing cell cultures without previous preparation, quality control, biologics production, and growth and activity monitoring. 

The trinocular inverted microscope’s phase-contrast feature emphasizes internal structures and outlines of the cell to meet high-level research needs. It is ideal for viewing specimens in various sample containers like Petri dishes, cultivation bottles, and microplates. With a total magnification between 100X and 400X and a 45° inclined eyepiece, the user can easily study the specimen under observation in detail. 

Apparatus 

The trinocular inverted microscope comes with an easy-to-adjust double layer 112 × 79 mechanical stages with horizontal and vertical adjustable knobs, three sizes Petri dishes, and LWD infinity plan achromatic objectives. The additional eye tube allows the user to connect the microscope with a display screen or camera to view the image of the specimen under observation. 

Additionally, a quadruple nosepiece, 10X/0.25 (20X 40X optional) phase contrast objective, 10X/0.25,20X/0.40, 40X/0.60 BF objective, coaxial fine/coarse focusing with 2 um focusing, and 11mm effective distance, frosted blue filter, and 9W LED brightness control are present, making it ideal for high-level magnification and detailed observation of specimens. 

Training Protocol 

Analyze if the microscope has all the parts in place for proper operation. Check if the condenser is set to provide you with appropriate illumination. Adjust the lens paper next to the iris diaphragm and move its field and the lamp focusing control. Make sure all the optics are well-adjusted to the microscope. 

Turn on the microscope. Center your specimen on the mechanical stage. Use the objective lens to examine your specimen. Connect the eye tube to the camera to view the image or record a video of the specimen.

Applications 

The trinocular inverted microscope is ideal, stable, and ergonomic for examining specimens in multi-well plates, cell cultures, and larger specimen containers. It is used in various laboratory studies to study live-cell imaging of cancer cells and others. Some other applications of the trinocular inverted microscope include; 

Studying the Combined Effect of Electromagnetic field and Low-level Laser on Human Adipose tissue-derived Mesenchymal Stem Cells

Nurković et al. (2017) used the trinocular inverted microscope to examine the effect of the electromagnetic field and low-level laser on human adipose tissue-derived mesenchymal stem cells. In the experiment, subcutaneous adipose tissue cells were isolated from six persons between 21 and 56 years. They were subjected to the EMP for 7 days, once a day for 30 min through a magnetic cushion surface. The frequency was kept at 50 Hz, 3 mT of intensity, 3 J/cm2 radiation energy, 808 nm wavelength, 200 mW power output, and 0.2 W/cm2 power density, but was applied for only 5 mins once a day for 7 days. On the other hand, non-exposed cells were also placed under the same culture conditions. After seven days of this treatment, the cells were examined for cell viability, morphology, and proliferation. The result was a higher number of EMF-treated hATMSCs. The increase in cell surface area and fractional dimensions were also observed. 

There is enormous evidence that regenerative medicine combines stem cells, nanomaterials, and biomaterials, growth factors to repair damaged organs and tissues. Also, the asymmetrical cell division of stem cells provides differentiation of cells into multiple lineages and self-renewal- the start of tissue engineering. These biotechnological and biomedical breakthroughs have evolved the need to use a Trinocular inverted microscope to develop innovative and effective methods to promote recovery and restore function after tissue injury. 

Studying the chitin-based scaffolds

Chitin-based scaffolds are derived from Aplysina aerophobia – a cultivated marine demosponge. Mutsenko et al. (2017) examined chitinous scaffolds under a trinocular inverted microscope. They were discovered as a supported and cytocompatible attachment, proliferation, and growth of human mesenchymal stromal cells in vitro. The growth of hMSCs on the Chitinous scaffolds increases the metabolic activity and double the normal/natural metabolic activity. This indicates the increased cell numbers, namely adipogenic, chondrogenic, and osteogenic lineages. A. aerophobia is the main source of Chitinous scaffold that forms the basis for many hMSCs-based tissue engineering strategies. 

Precautions 

1. Do not touch the lens and hold the microscope with two hands.
2. The eye tube is delicate to handle; thus, carefully connect it to the camera. Read the user manual before connecting the eye tube for image capturing and video recording.  

Strengths and Limitations 

Strengths

The Trinocular inverted microscope allows users to take pictures and videos of the cultures to examine in further detail. Additionally, the biggest advantage of this microscope is that it is versatile to use since it comes with a three-position trinocular facility. Thus, it is great for bright field photographic purposes providing 20% visual and 80% of the photosystem. 

Limitations 

The trinocular inverted microscope is more complex to use than upright microscopes. It demands great professionalism from the user to carefully examine the specimen. 

Summary 

1. The trinocular inverted microscope comes with 2 eyepieces and a third eye tube for a better viewing experience. 
2. The third eye tube allows the microscope to be connected to a camera for image capturing and video recording, which adds to its versatility in using varying laboratory studies. 
3. It can be used to study live-cell imaging of cancer cells, the combined effect of EMF and low-level lens on biological processes, and study the chitin-based scaffolds, etc.
4. The only limitation to this microscope is that it is expensive and is complex to use for beginner users. 

References 

1. Nurković, J., Zaletel, I., Nurković, S., Hajrović, Š., Mustafić, F., Isma, J., Škevin, A. J., Grbović, V., Filipović, M. K., & Dolićanin, Z.  . (2017). Combined effects of electromagnetic field and low-level laser increase proliferation and alter the morphology of human adipose tissue-derived mesenchymal stem cells. Lasers in medical science, 32(1), 151–160. https://doi.org/10.1007/s10103-016-2097-2 
2. Mutsenko V., Bazhenov V., Rogulska O., Tarusin N., Schütz K.,  Brüggemeierd S., Gossla E., Akkineni, A. R., Meißner, H., Lode, A., Meschke, S., Ehrlich, A., Petović, S., Martinović, R., Djurović, M., Stelling, A. L., Nikulin, S., Rodin, S., Tonevitsky, A., Gelinsky, M., … & Ehrlich H. (2017 ). https://pubmed.ncbi.nlm.nih.gov/28347785/. International Journal of Biological Macromolecules, 104 (Pt B),  1966-1974. https://doi.org/10.1016/j.ijbiomac.2017.03.116
3. Anka, P. (2018). Uses of Transmission Electron Microscope in Microscopy and its Advantages and Disadvantages. International Journal of Current Microbiology and Applied Sciences, 7(05), 743–747. https://doi.org/10.20546/ijcmas.2018.705.090