Inverted Fluorescent Microscope

Introduction

An inverted fluorescent microscope uses fluorescence to study specimens, such as living cells at the bottom of a petri dish or tissue culture. It is called “inverted” due to the arrangement of the microscope’s components, which are placed in inverted positions. The objective lenses are located above the stage, while the condenser and the light source are below the stage. The primary purpose of the condenser lens above the specimen stage is to focus light on the specimen. Conduct Science’s Inverted Fluorescent Microscope is equipped with wide-field eyepieces that allow images to be obtained in a wide view field. It also contains plain achromatic objectives that allow a longer working distance of 50 mm. 

Fluorescence microscopy utilizes fluorescent dyes, called fluorophores, that absorb one wavelength of light (the excitation wavelength) and emit the second, longer wavelength  (the emission wavelength). Since most cell molecules are not fluorescent, the experimenter introduces fluorescent labels to be imaged. Therefore, the labels can be targeted to the desired molecules by attaching fluorescently labeled antibodies or genetically encoding a fluorescent protein. This allows the identification of different fluorescent molecules at the same time, which can be detected at extremely low abundance, making it a very powerful technique (Thorn, 2016).

The microscope’s halogen lamp serves as excitation light and illuminates the sample through the same objective that detects the light emission. Interference from the excitation light is eliminated through a fluorescence filter cube that separates light by wavelength, allowing the emitted light to be imaged. 

Apparatus 

The Inverted Fluorescent Microscope has similar components to those of other microscopes, and the only difference is the arrangement of the parts, which are placed in inverted positions. Apart from its similar components such as the stage, objective lens, eyepiece, eyepiece tube, dual-concentric knobs, nosepieces, and condenser lens, it also consists of other elements. A 6V 30W Halogen lamp is present with a brightness control that serves as an excitation lamp that illuminates the sample. It contains an excitation filter that filters all wavelengths of the light source except for the excitation range of the fluorophore under investigation. It also contains an emission filter that filters out the excitation range and transmits the emission range of the fluorophore under investigation. A dichroic mirror is placed between the two filters that serve as a beam splitter. 

Principle

The specimen is lit using a halogen lamp as a light source. When light enters the microscope, it strikes a dichroic mirror, reflecting one range of wavelengths while allowing another to pass through. The ultraviolet light is reflected up to the specimen by the dichroic mirror. The UV light causes fluorescence in the specimen’s molecules and the fluorescent-wavelength light generated is collected by the objective lens. The fluorescent light then goes via a dichroic mirror and the emission filter before reaching the eyepiece and forming the picture of the targeted specimen (Sanderson, Smith, Parker, & Bootman, 2014).

Protocol

• Place the microscope on a solid table and turn on the power button.
• Insert the slide or glass container containing the specimen on the specimen stage.
• Keep the specimen stage stationary.
• Move the objective and condenser lens to fine-tune the focus.
• The specimen’s picture may be seen via the ocular lens or on a computer screen.

Applications

The Inverted Fluorescent Microscope can be used to observe and detect various living cells and microscopic organisms at the bottom of lab vessels like tissue culture flasks and Petri dishes. For example, it can be used to identify Phytophthora spp, nematology extraction specimens such as vermiform, and to see Mycobacterium tuberculosis. Moreover, micromanipulation is also possible with this microscope

Wei et al. (2019) developed a nano-drug of doxorubicin and exosome derived from mesenchymal stem cells to treat osteosarcoma in vitro. They used an inverted fluorescent microscope and flow cytometry to analyze the cellular absorption of Exo-Dox and free Dox by myocardial H9C2 cells since the major side effect induced by doxorubicin is cardiac toxicity. An exosome isolation kit was used to separate the exosome from bone marrow. The exosome and Exo-Dox were characterized using nanoparticle tracking analysis and a transmission electron microscope. 

In a study conducted by Sun et al. (2019), they used an Inverted fluorescent microscope and a hyperspectral microscope to explain the exact mechanism of how Escherichia coli O157:H7 interacts and colonizes fresh cucumber (Cucumis sativus L.). They found out that E. coli O157:H7 occupied the epidermis of cucumbers largely around the stomata without penetrating the interior tissues. After infiltrating the internal tissues, the bacteria colonized the cucumber placenta and vascular bundles (particularly the xylem vessels) and migrated along the xylem vessels. Furthermore, E. coli O157:H7 moved quicker from the stalk to the flower bud than from the flower bud to the branch.

Strengths and Limitations

Strengths

The inverted fluorescent microscope has a large stage to see specimens in glass tubes and Petri plates. Its design allows the observation and analysis of cells in vast quantities of media and cell tissues in their native vessel, bigger than microscopic slides, making it superior to the upright microscope. It has three different specimen holders. It maintains the sterility of the sample since the specimen is not contaminated by contact with the objective lens. The microscope can be used with differential interference contrast and phase-contrast optics to improve picture quality. It allows the connection of a wide range of digital recording devices. The wide-field eyepieces give clear images of up to 2-micron fine focusing, and a large view field is obtained using simple, achromatic objectives with a long working distance (no cover glass).

Limitations

Photobleaching occurs when fluorophores lose their capacity to glow when they are illuminated. Photobleaching reduces the time a sample can be viewed using fluorescence microscopy; it may be reduced by various methods, including the use of more robust fluorophores, reduced light, and the application of photoprotective scavenger chemicals.

Precautions

• Turn off the power during installation, assembly, cable connection/disconnection, and maintenance.

• Wet or spilled substances on the device might cause it to malfunction, overheat, or electrocute you. 

• Hold the product firmly by the bottom front recess and the bottom rear recess while carrying it. 

• Prevent scratches on optical components such as lenses and filters because scratches can diminish the focus of the microscopic image. 

• Dispose of contaminated slides according to your facility’s regular procedures to eliminate biohazard concerns. 

• Place the microscope in an area with a temperature range of 0 to 40°C with a relative humidity of 85% or less. 

• Avoid physical shocks and vibrations while handling the goods. Even little physical shocks have the potential to degrade the accuracy of targets.

Summary

• The inverted fluorescent microscope has the objective lenses above the stage and the condenser and the light source below the stage.
• Its consists of similar components to other microscopes but also includes a halogen lamp, a dichroic mirror, an excitation filter, and an emission filter.
• The large stage allows specimens to be seen in glass tubes and Petri plates. Moreover, its design allows the observation and analysis of cells in vast quantities of media and cell tissues in their native vessel, bigger than microscopic slides, making it superior to the upright microscope. 
• It can be used to observe and detect various living cells and microscopic organisms.

References

1. Wei, H., Chen, J., Wang, S., Fu, F., Zhu, X., Wu, C., Liu, Z., Zhong, G., & Lin, J. (2019). A Nanodrug Consisting Of Doxorubicin And Exosome Derived From Mesenchymal Stem Cells For Osteosarcoma Treatment In Vitro. International journal of nanomedicine, 14, 8603–8610. https://doi.org/10.2147/IJN.S218988

2. Sun, Y., Wang, D., Ma, Y., Guan, H., Liang, H., & Zhao, X. (2019). Elucidating Escherichia Coli O157:H7 Colonization and Internalization in Cucumbers Using an Inverted Fluorescence Microscope and Hyperspectral Microscopy. Microorganisms, 7(11), 499. https://doi.org/10.3390/microorganisms7110499

3. Sanderson, M. J., Smith, I., Parker, I., & Bootman, M. D. (2014). Fluorescence Microscopy. Cold Spring Harbor Protocols, 2014(10), pdb.top071795. https://doi.org/10.1101/pdb.top071795

4. Thorn, K. (2016). A quick guide to light microscopy in cell biology. Molecular Biology of the Cell, 27(2), 219–222. https://doi.org/10.1091/mbc.e15-02-0088

‌