Inverted microscopes are light microscopes whose objectives are below the stage – where examining samples are placed. It is the opposite of traditional compound microscopes, where the objectives are above the stage.
The placement enables users to examine samples from the bottom rather than the top, allowing them to observe the specimen in its container and work on it while being observed.
Components and their Functions
Inverted microscopes contain all typical components of the upright design. And the following are the key components of an inverted microscope:
- The light source provides the light that illuminates the specimen on the stage. The light is projected to the objective lenses, creating an image of the specimen.
- The condenser is a collection of optical lenses that focuses the light from the source and projects it onto the specimen.
- Objectives are optical lenses that capture and focus the reflected light, creating a magnified image of the specimen called the real intermediate image. They are removable and arranged on a rotational nosepiece that users could adjust to select desirable ones.
- A stage is where the specimen is placed for microscopic examination. It has controllers that move the objectives and height of the stage, adjusting the focus area. The stage is often equipped with removable inserts that fit various containers or microscope slides. Some inverted microscopes may have a specialized stage or environmental chamber that creates a specific condition for the specimen during the examination.
- Eyepieces or oculars are optical lenses that magnify the real intermediate image produced by the objectives and condenser, allowing users to observe and examine the image of the specimen.
Modern inverted microscopes typically have two oculars with a display screen and camera connected to one ocular. A trinocular inverted microscope has an eyepiece tube, which specifically connects to a monitor and camera.
Advantages of Inverted Microscope
The placement of the objectives under the stage gives users better access to the specimen, thus providing these practical advantages:[1,2]
- Direct observation of cells or specimens in the culture vessel without preparing a section or microscope slide.
- Direct access to the specimen while it is being imaged, allowing users to view or capture the images of the specimen in real-time.
- An environmental chamber or a specialized stage can be constructed to create an artificial environment that influences or directly manipulates the specimen.
- Higher stability while working with the microscope because of the shift of the center’s mass closer to the bench.
- Reduced risks of an objective being damaged by an unintentional collision between the objectives and specimen.
Limitations of Inverted Microscope
The placement of the objectives, on the other hand, is also a practical weak point in maintenance. This is particularly the case when working with oil immersion objectives, which can dribble from the lenses onto the objectives into the objective heads, unbeknownst to the user.
Along the same line, dried immersion oils, buffer, and dust can stick to the objectives, damaging the lenses and other components attached to them.[1,2] Thus, an inverted microscope requires more maintenance efforts and technical skills from users.
Applications of Inverted Microscope
Inverted microscopes are especially popular for life science, clinical research, and routine diagnosis.
1. Cell Cultures
Inverted microscopes are go-to’s for works involving live-cell imaging and cell cultures.
- With an inverted microscope, cells can be microscopically observed throughout the culturing period with minimal disturbance without destroying the sterility.
- Users can examine cells in the culturing vessels in real-time without preparing multiple cell cultures for each observation point, reducing the costs and preparation time.
- Examining cells in their culture vessel allows users to gain an impression closer to natural events. This way, cells can adhere and stretch out onto the surface of the culture vessel as they naturally do.
- Besides, there is no need to miniaturize the sample surface area and volume, so the overall changes of the cells can be observed and recorded without having to sample them as you would do for microscopic slides.
- Another added benefit is the possibility of recreating an artificial condition from an environmental chamber or a specialized stage. Thus, the cells can respond to the simulated condition with minimal manipulation while simultaneously imaged.
- Thus, the images obtained of cells examined under an inverted microscope should reflect events closest to the natural settings in real-time.
Another major advantage of examining cells using inverted microscopes is the accessibility to the cells during the imaging. With necessary add-on equipment, inverted microscopes are suitable for works involving micromanipulation such as microinjection, in vitro fertilization (IVF), and cell or tissue ablation.
Factors to Consider When Purchasing an Inverted Microscope
When buying an inverted microscope, it is essential to understand the intended applications. Here are some factors to consider when buying an inverted microscope.
1. Light source
The light source generates the light that illuminates the specimen. Hence, the information you expect from microscopic images is dependent on the wavelength and brightness of the light generated.
Most inverted microscopes possess an internal light source with adjustable brightness. Some models have an option that allows users to connect to an external light source so that a functional feature can be added later on.
Typical examples of light sources:
- Light-Emitting Diodes (LED) are often found in contemporary inverted microscopes. They can generate white light of exceptional and reliable quality. Nonetheless, the generated light contains more blue spectrum in the cool white shade; thus, LED lights can appear more washed-out to users familiar with light generated from halogen bulbs.
- Halogen bulbs were the most popular light source before LED technology, emitting a stable white light that shifts towards yellow as they age, thus, affecting the specimen’s color. The main limitation of halogen bulbs is that they require high temperatures, generating substantial heat that could inadvertently affect the specimen.
- Mercury bulbs produce white light with an occasional blue tint, which can be corrected with a layer of phosphor coating. Their lifespan is significantly shorter than LED and halogen bulbs, and they must be disposed of as hazardous waste.
2. Optical Lenses and Condensers
Optical lenses in inverted microscopes are objective and ocular lenses. Together with the condenser, objectives create the real intermediate image of the specimen. The real image is reflected and amplified by ocular lenses directly to observers or on a display screen.
An essential characteristic of objectives is magnification, which is noted in the objectives by the letter “x,” which follows the magnification number. Another important characteristic is the numerical aperture (NA), designated as numbers following the magnification.
Objectives are usually removable from the nosepiece. Air objectives produce lower magnification, typically 5x, 10x, 20x, and 40x.
Higher magnifications (such as 60x and 100x) are achieved by oil immersion objectives, requiring immersion oil for imaging. The required oil must have the same refractive index (RI) value as oil immersion objectives.
Similar to objectives, magnification is the most important characteristic of ocular lenses, but they are typically available only in one magnification, as indicated on the lenses.
Together with the diaphragm, the condenser filters and focuses the light from the source. For the most optimal image resolution, the numerical aperture value of the condenser should be equal to those of the objectives. Otherwise, the condenser’s NA value should be greater than the objective’s value to create an evenly illuminated field.
3. Microscopic Techniques
In addition to magnification and resolution, microscopic techniques are crucial in obtaining high-quality pictures from an inverted microscope.
Brightfield is usually the default microscopic technique
Brightfield microscopy is the conventional, default microscopic technique. Inverted microscopes with a typical light source, objectives, and ocular lenses can perform brightfield microscopy.
Darkfield microscopy requires a darkfield condenser to create microscopic images with dark background
In contrast to brightfield microscopy, the darkfield technique creates a black instead of white background. It is suitable for observing the external structure of colored samples that are somewhat transparent. This is achieved by a darkfield condenser, which partially blocks the light source, illuminating the specimen from the side.
Phase-contrast microscopy uses specialized lenses and components
In phase-contrast microscopy, the specimen is lit from the sides to enhance the contrast of the sample. The so-called phase objectives, plate, and condenser annular create phase-contrast images added to the objectives and condenser.
Phase-contrast microscopy is a popular technique for the live imaging of cell cultures in their natural condition, including the internal structures of the cells. For example, the trinocular inverted microscope, and trinocular phase-contrast digital microscope include phase-contrast objectives and a condenser with the phase-contrast device.
4. Additional features
Lastly, inverted microscopes can also have additional features, such as an eyepiece tube linked to a camera or monitor, allowing microscopic pictures to be taken. Another example is a color filter placed between the diaphragm and condenser to adjust the light’s color.
Inverted microscopes are compound light microscopes that look into a specimen from below. Users can acquire microscopic images of cell cultures directly from the culture vessels in real-time.
Unlike the traditional upright design, users can access the top of the specimen while being examined to perform micromanipulation techniques.
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- Lawlor, D. Introduction to Light Microscopy: Tips and Tricks for Beginners, Springer Nature Switzerland AG, 2019.
- Murphy D.B. and Davidson M.W. Fundamentals of Light Microscopy and Electronic Imaging, 2nd edition, Wiley-Blackwell, 2013.