The Digital Inverted Microscope is equipped with advanced video and image processing and is best for precise laboratory cell analysis and live-cell imaging and recording. It can magnify various specimens with a total magnification between 100X to 400X. 

The construction of this microscope is the reverse of the normal microscope, where its components are placed in inverted order. The condenser lens points downwards towards the specimen stage, while the turret and the objective face upwards from below the stage. However, its working principle is similar to upright microscopes. Both focus light rays on the specimen and form an image viewed by the objective lenses. The condenser lens focuses light on the specimen stage placed on a larger stage, and the objective (below the stage) collects the light from the condenser, magnifies images, and sends it to the ocular lens. This ocular lens reflects the light through a mirror, and cells can be viewed easily through the bottom part of the cell culture apparatus. 

The digital inverted microscope is useful for observing specimens under more natural conditions, such as a tissue culture flask or Petri dish since samples don’t have to be prepared on glass slides. 

Apparatus 

The Digital Inverted Microscope comes with a total magnification between 100X and 400X. It is connected with a display screen or microscope camera to view the image of the specimen under observation. Also, it has a double-layer mechanical stage (with horizontal and vertical adjustable knobs) and three-sized petri dish trays (86mm × 129.5mm, 34mm × 77.5mm, and 57mm × 82mm in size), allowing you to observe different samples. With an inbuilt 2.0 USB interface, the microscope can transfer data at high speed. 

The LWD infinity plan monochromatic objective and LWD infinity plan phase contrast objective are perfect for both black and white and colored image viewing. The 22mm WF10X (Optional 11mm WF16X) eyepiece is (30mm diam and 10mm parfocal distance) fitted in a 45° inclined eyepiece tube with an interpapillary distance of 48~75mm. The 0.35M to 14M hardware resolution ideally presents fine details in the sample. 

With the 9W LED light source, you can also control the brightness depending upon how much is required for sample observation. 

Technical Protocol 

Place the glass container containing the specimen on the center of the microscope’s stage. Turn the 10X objective. Adjust the light control knob to get proper illumination. Adjust the interpapillary distance through the eyepieces. Also, adjust the position of condenser light control and diaphragm aperture diaphragm to get satisfactory illumination. To obtain the sharp image from the specimen, adjust the fine/coarse focus. Use the vertical thumbscrew if the lamp needs to be adjusted and the horizontal thumbscrew to adjust the centering of the light. Connect the camera to the eyepiece tube. Connect the camera to a laptop or computer to view the specimen on the screen.

Applications 

The Digital Inverted Microscope is used for various research, including fungal cultural diagnoses such as Phytophthora spp in cultures, observation of nematodes (Vermiform nematodes), and in the observation of living microbial cells in the tissue cultures placed in Petri dishes and flasks. Some other applications of the digital inverted microscopes include; 

Detection of Early Growth of Mycobacterium tuberculosis in MODS Culture

The MODS is a rapid, sensitive, and inexpensive method used to detect the early growth of Mycobacterium tuberculosis (MTB). The detection of tuberculosis and multi-drug resistant tuberculosis requires an inverted light microscope as it needs an image in the MODS culture to be viewed from below. The visualization of MTB is done in liquid media using 24-well cultured plates. The microscope visualizes the characteristics of the growth of MTB and presents rapid and accurate detection of the sample. Using the digital inverted microscope in the detection overcomes two problems put forth by the conventional upright light microscope. Firstly, it facilitates the condensation on the plate lid, and cultural medium obstruction and interference get cleared when viewing from the bottom (that gets obscured when viewing from above). Secondly, the objective-bottom (where the MTB colonies aggregate) is greater than the upright light microscope and thus helps in clear magnification of the image.  

Live Cell Imaging

The digital inverted light microscope is used in live-cell imaging using an aluminum cell culture incubator on its stage. The incubator effectively maintains the temperature at 37°C and provides focal stability of an image for 20-25 min after equilibrium. Rines, Thomann, Dorn, Goodwin, and Sorger (2011) performed live-cell imaging of yeast and studied dynamic cellular processes like cell division, growth, and morphogenesis. Hickey, Swift, Roca, and Read (2004) studied live-cell imaging of fungal hyphae and analyzed its molecular dynamics and organelle at high spatial resolutions at a single-cell level. 

Precautions 

1. Never touch the glass part of the microscope while studying/observing the specimen.
2. Ensure to unplug and turn off the microscope. 
3. Avoid disassembling the microscope to avoid doing any damage to it.
4. Do not place combustibles near the bulb to prevent fire.   
5. Keep the microscope at the temperatures between 0°C-40°C/32°F-104°F, with an 85% of maximum humidity. 
6. Do not keep the microscope in direct sunlight. 
7. Keep the microscope on a sturdy and level surface for clear and proper observation. 

Strengths and Limitations 

Strengths 

Cell observation through a digital inverted microscope has greatly widened cellular and molecular biology knowledge. It comes with a wide stage to allow the convenient study of specimens in glass tubes, Petri plates, or flasks and thus is ideal for studying live cells from the bottom of the cell culture apparatus. It is used to study cells in large amounts of the medium. This also allows the user to study cells in original vessels compared to upright microscopes that require the samples to be prepared on glass slides. 

Another benefit is that it keeps the specimen uncontaminated by preventing contact with the objective lens. Therefore, the sterility of the sample is maintained. The samples can also be observed over a longer period. Furthermore, the need for staining the specimen is eliminated as it creates different shades of brightness to enhance the image viewing experience. 

Limitations 

These microscopes require high optical quality as the image is viewed through a thick Petri plate compared to an upright light microscope. Another limitation is its high cost.

Summary 

1. The digital inverted microscope is used for precise laboratory cell analysis.
2. It views the sample in an inverted manner compared to a conventional upright light microscope. 
3. It is available connected with a display screen or microscope camera.
4. The total magnification between 100X and 400X allows precise and clear magnification. 
5. The microscope is best for live-cell imaging and Mycobacterium tuberculosis observation. 
6. The microscope allows the sample to be observed longer than upright microscopes since the specimen can be kept in its original glass container. 

References 

1. Child, J. M. (1972). International Specifications for Technical Manuals. Journal of Technical Writing and Communication, 2(3), 189–197. https://doi.org/10.2190/hw02-644g-ekdm-7hua
2. Oktaviyanthi, R., & Agus, R. N. (2021). Guided Worksheet Formal Definition of Limit: An Instrument Development Process. AL-ISHLAH: Jurnal Pendidikan, 13(1), 449–461. https://doi.org/10.35445/alishlah.v13i1.483
3. Zimic, M., Velazco, A., Comina, G., Coronel, J., Fuentes, P., Luna, C. G., Sheen, P., Gilman, R. H., & Moore, D. A. J. (2010). Development of Low-Cost Inverted Microscope to Detect Early Growth of Mycobacterium tuberculosis in MODS Culture. PLoS ONE, 5(3), e9577. https://doi.org/10.1371/journal.pone.0009577
4. Rines, D. R., Thomann, D., Dorn, J. F., Goodwin, P., & Sorger, P. K. (2011). Live cell imaging of yeast. Cold Spring Harbor protocols, 2011(9), pdb.top065482. https://doi.org/10.1101/pdb.top065482.
5. Hickey, P. C., Swift, S. R., Roca, M. G., & Read, N. D. (2004). Live-cell imaging of filamentous fungi using vital fluorescent dyes and confocal microscopy. Methods in microbiology, 34, 63-87. https://doi.org/10.1016/S0580-9517(04)34003-1