Inverted Microscope – Principle, Procedure, Parts, Uses

What is an Inverted Microscope?

An inverted microscope is literally an inverted microscope. The lights and lenses are positioned above the specimen stage and the objective lenses are below. This allows the user to critically observe the samples from below; this is important because many living specimens and cells to be observed are in petri dishes or glass vials. One can never have an open sample above with a microscope.

Thus, observing a sample in situ, without disturbance, provides a means for the cell researcher, medical deviants and application specialists, as well as industrial quality assurance. For example, in a research application, the inverted microscope impacts the quality of cell culture in nutrient solutions, for example, while in an industrial application, the inverted microscope ensures the quality of thick materials or liquid processes.

The first known application of the inverted microscope came from American chemist J. Lawrence Smith in the 19th century, in the 1850s, when he hypothesized that an inverted microscope would help study things that wouldn’t flatten out on a slide. From there, the technology evolved to include phase contrast and fluorescence technology. Without the inverted microscope, which requires the study of microscopic life in situ, many cancer therapies and IVF wouldn’t exist. It’s a common reversal of perspective that changes everything.

Inverted Microscope Definition

An inverted microscope is a specialized light microscope where the light source and condenser are positioned above the specimen stage, while the objectives and turret are below the stage, allowing for the viewing of specimens from a bottom-up perspective. This design is opposite to that of conventional microscopes.

Principle of Inverted Microscope

An inverted microscope works on the same principles as an upright optical microscope. Both use light rays to project an image onto a slide that can then be viewed in a magnified manner. The unique aspect of an inverted setup is simply the positioning of the components through which a user views the device and where the associated parts are located. An inverted microscope has the parts reversed. For example, the light source and condenser are positioned above the slide as if it were a stage, shining down onto the slide and up onto the specimen.

Additionally, the condenser lens provides a more focused beam of light onto the specimen positioned on the large, stable stage. The objective lenses sit below the stage, absorbing the light projected upward from below, allowing it to magnify the image, which the mirror then directs to the eyepiece lens for viewing. This is a fantastic way to view cells from the bottom of a Petri dish type cell culture device, but arguably even better with the ibidi polymer and glass coverslips. So this is a microscope suitable for viewing living cells/samples in their natural state.

Parts of Inverted Microscope

Inverted Microscope has these following parts;

1. Base– Heavy bottom section that stabilizes the microscope.
2. Arm– Vertical support connecting the base to the upper components.
3. Stage – Flat platform where slides or samples are placed; often includes clips.
4. Objective lenses– Magnifying lenses (e.g., 4x, 10x, 20x) mounted below the stage.
5. Condenser – Focuses light onto the sample, located beneath the stage.
6. Light source– LED or halogen lamp positioned above the stage (shines downward).
7. Coarse/fine focus knobs– Adjusts stage height for sharpening the image.
8. Eyepieces (oculars) – Top lenses you look through; typically 10x magnification.
9. Diaphragm– Controls light intensity passing through the sample.
10. Stage controls– Knobs to move the sample horizontally for positioning.
11. Filter holder – Slot for adding optical filters to enhance contrast.
12. Trinocular head (optional) – Third port for attaching a camera or display.
13. Phase contrast components (optional) – Specialized rings for live cell imaging.
14. Power switch/controls– Buttons to adjust light brightness or turn the device on/off.
15. Accessory ports– Openings for adding external devices like monitors or cameras.

Operating Procedure of Inverted Microscope

1. To turn on your microscope light, plug it in and turn on the power switch, usually at its base. To view a slide, place it on the stage and make sure it is directly aligned with the objective lenses under the stage.
2. Make sure your slide does not move with the clips or supports on the stage, these will ensure your slide stays in position.
3. Look through the eyepieces and first turn the coarse focus knob for a general focus adjustment.
4. Use the fine adjustment knob to bring the image into focus for personal viewing.
5. If the images appear too dark or too bright, adjust the brightness control knob for an appropriate light intensity.
6. Use the stage control knobs to move the images left, right, up and down. If necessary, rotate the turret to increase or decrease the magnification.
7. Use the iris diaphragm in the light path below the stage, which opens and closes to increase and decrease light intensity for optimal viewing.
8. Use filters or adjust the phase contrast setting if your slides are phase contrast or stained for optimal viewing.
9. When finished, dim the light, turn off the microscope, and unplug it if not in use.
10. Carefully remove slides from the stage; use lens paper to clean the stage and objective to avoid scratches.

Types of Inverted microscope

Two main types of inverted microscopes include:

1. Biological Inverted Microscopes—made for observing live cells or organisms at the bottom of their respective cultivation vessels. The inverted configuration allows the researcher to see the cells without disturbing the specimen as one might do when peering in from above. Used in the fields of cell biology, live-cell imaging, and microbiology.
2. Metallurgical Inverted Microscopes—made for observing polished metal specimens and ores and more. Inverted microscopes provide access to microstructure from the bottom up, as the specimen sits large and heavy atop the stage. This is beneficial in many industrial applications and materials sciences.

Uses of Inverted Microscope

• Observing living cells or tissues in culture dishes without disturbing their growth environment.
• Studying liquid-based samples like blood, water, or suspensions settled at the bottom of containers.
• Analyzing thick or irregularly shaped specimens that cannot fit under a traditional microscope.
• Monitoring cell behavior in real-time during experiments, such as drug testing or toxicity studies.
• Examining organisms in aquatic environments, like plankton or microorganisms in petri dishes.
• Facilitating micromanipulation techniques, such as injecting cells or handling embryos in fertility research.
• Inspecting industrial materials, electronics, or metal surfaces from below for quality control.
• Teaching labs where multiple users can view samples in open containers without slide preparation.
• Tracking chemical reactions or crystallization processes in open vessels over time.
• Supporting geological studies by examining rock fragments or mineral samples in solution.

Advantages

• Natural observation: View live cells in original containers.
• Accommodates large samples, ideal for industrial applications.
• Reduces contamination by keeping lenses below the stage.
• Compatible with advanced imaging techniques like fluorescence.
• Minimizes sample preparation with direct vessel observation.
• Enhances image quality using phase-contrast or DIC methods.
• Protects delicate samples from accidental damage.
• Provides images in true orientation for easy analysis.

Limitations

• Higher cost compared to upright microscopes
• More complex design, may require more maintenance
• Not all models support certain imaging techniques
• Container thickness can affect image quality
• Image orientation can be confusing due to inverted design
• Limited penetration depth for thick samples
• Sensitive to vibrations, affecting image stability

Inverted vs Upright Microscope

Precautions of Inverted Microscopy

• Handle the microscope carefully to avoid damage
• Use soft, lint-free cloths for cleaning lenses
• Operate on a stable, vibration-free surface
• Keep the microscope out of direct sunlight
• Use only compatible accessories
• Follow regular maintenance schedules
• Ensure proper training before use
• Wear protective equipment like gloves and safety glasses

Inverted Microscope Free Worksheet

Inverted microscope Images

Quiz

FAQ

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
6. https://en.wikipedia.org/wiki/Inverted_microscope
7. https://microbenotes.com/inverted-microscope/
8. https://www.microscopemaster.com/inverted-microscope.html
9. https://ibidi.com/content/212-inverted-and-upright-microscopy
10. https://www.biocompare.com/25969-Inverted-Microscopes/
11. https://www.microscopeworld.com/t-inverted_microscopes.aspx