The energy emitted from certain materials, when irradiated with a specific wavelength of light, can be detected as visible light due to energy reaction. In microscopy, the phenomenon of fluorescence, as described by George Gabriel Stokes, an Irish physicist, in 1843, led to the discovery of the epi-fluorescence microscope.
It is based on the foundation that the longer wavelength of the emitted light than the exciting light characterizes fluorescence. In fluorescence microscopy, the visible light in the microscope’s eyepieces is not emitted from the light source but from the fluoresced or fluorescing sample in its original form.
Epi-fluorescence microscopy, abbreviated as EFM, is based on the method of fluorescence microscopy and is also known as wide-field fluorescence microscopy. Commonly used in all fields of life sciences, it has become a convenient tool in cell biology, research, and medical fields to identify and determine cellular structures and components with high contrast and specificity.
The images produced by EFM provide a deepened understanding and knowledge of the locations of cell structures, dynamics, gene expressions, and cellular interactions. Primarily, the epi-fluorescence microscope excites the sample by passing the excitatory light through the objective lens and onto the specimen.
The fluorescence in the specimen produces emitted light, which is then directed to the detector by the same objective lens used for excitation. As the majority of the excitation light passes through the specimen, only reflected excitation light, along with emitted light, reaches the objective, resulting in an enhanced signal-to-noise ratio.
A supplementary filter positioned between the objective and the detector can be used to eliminate any remaining excitation light from the fluorescent light, thereby producing images with high contrast and resolution compared to conventional fluorescence microscopy.
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Principle of Epi-Fluorescence Microscopy
Epi-fluorescence microscopy is based on the principle of fluorescence where an intense light source is used to stimulate a fluorescent molecule instead of using visible light to illuminate samples. This fluorescent molecule is known as a fluorophore. The fluorophore takes in photons, causing electrons to move to a higher energy state. As the electrons return to their original state, they release light of a longer wavelength. The emitted light is then separated from the initial excitation light using a filter, resulting in an enlarged image of the specimen under examination. The use of fluorescence enables the specific targeting of the object of interest. When the excitation light is filtered out, only the fluorescent objects of interest are visible through the emitted fluorescence
To briefly describe the working principle, when multispectral light from a light source is passed through the excitation filter, a wavelength-selective bandpass filter, a specific wavelength of light in the ultraviolet or visible spectrum is generated. As the selected wavelength of light travels through the filter, they are reflected from the dichromatic mirror to the object exposing the sample to intense light. The emitted light collected by the object passes through the dichromatic mirror if the sample glows and then gets filtered by an emission filter to block the unwanted excitation wavelengths. A detector like a CCD camera receives the collected light and visualizes the image.
Parts of Epi-Fluorescence Microscope
The major optical components of epi-fluorescence microscopy are briefly described below:
Light Source
A high-intensity light source capable of emitting a wide spectrum of wavelengths is necessary. Commonly used light sources in epi-fluorescence microscopes are Tungsten-Halogen bulbs, Mercury and xenon arc, Metal Halide such as Chroma Technologies-Photofluor, Zeiss-Illuminator HXP 120, Light emitting diodes (LEDs), and Monochromator.
Objective Lens
Light is transmitted to the sample through the objective lens to visualize the image. The brightness of the objective lens depends on the numerical aperture of the lens and magnification, which should be taken into account while choosing the appropriate objective. It is preferred not to use the phase contrast objectives with the epi-fluorescence microscopy as the phase ring hinders the fluorescence emission light. However, DIC objectives with this technique are considered a good combination with the removal of the DIC prism and analyzer from the light path.
Filters
This component is used to filter out specific wavelengths of excitation and emission light. In epi-fluorescence microscopy, an excitation filter, dichroic mirror or beamsplitter, and emission filter are used. The excitation filter selects specific wavelengths of excitation light and the emission filter or barrier filter selects specific wavelengths of emission light, by blocking excitation light. The dichroic mirror carries out the separation of excitation light and emission light, reflects shorter excitation light wavelengths, and transmits longer emission light wavelengths. Filters and mirrors designed with soft coatings are considered to produce high-resolution images.
Cameras
High-resolution images of the samples are recorded with the use of Charge-coupled device (CCD)-based cameras in epi-fluorescence microscope. This device is capable of capturing images at high speed with high sensitivity. However, the use of color cameras should be avoided as they capture images with low resolution and sensitivity. Similarly, electrons multiplied (EMCCD) cameras are considered useful for high-speed events and applications.
Sample Preparation for Epi-Fluorescence Microscopy
Thick samples (=10µm deep) are generally viewed and observed using the Epi-fluorescence microscope. The intense lighting and activation of molecules beyond the focal point may result in images having a significant background signal compared to other illumination methods like TIRF and HILO. The procedure for sample preparation is explained below:
- The cells grown in vitro are fixed on the coverslips by appropriate incubation. Different types of proteins are incubated in specific solutions at different temperatures.
- Phosphate buffer saline (PBS) is used three times to rinse the culture.
- 0.2% Triton X-100 is used for 5 minutes to permeabilize cells at room temperature.
- Again, it is rinsed with PBS for three times.
- To prevent drying, the coverslips are placed in a humid environment by placing the wet Kimwipes in the incubation chamber.
- The sample is then incubated with 100 μl of 20% goat serum for 1 hour at room temperature to avoid nonspecific binding.
- The primary antibody is diluted in 5% goat serum at a dilution of 1:100, however, the concentration could be lowered or increased based on the antibody used.
- Next, the coverslips are incubated with 100 μl of primary antibody at room temperature for an hour or at 4°C overnight.
- The coverslips with the sample are rinsed thrice with PBS.
- A fluorophore-conjugated secondary antibody in 5% goat serum diluted at 1:500 is stored in the dark. The concentration of the antibody should be tested for specificity and minimal nonspecific labeling.
- Then, the coverslip is incubated with 100 μl of secondary antibody for an hour at room temperature. Avoid incubating the sample overnight to prevent nonspecific binding.
- Again, it is rinsed with PBS for three times.
- An equivalent 10 μL of mounting media with an anti-fade agent such as ThermoFisher is placed on the microscope slide. The coverslip is then inverted onto the slide and pressed down gently avoiding air bubbles using a cotton swab.
- The coverslips are then sealed with a sealant such as a mixture of Vaseline, Lanolin, and Paraffin in a ratio of 1:1:1. The specimen is preserved using mounting media that cures and hardens. It can be stored for weeks at 4°C in the dark.
Operating Procedure of Epi-Fluorescence Microscopy
The operating procedure of the epi-fluorescence microscopy is similar to that of fluorescence microscope which is described below in stepwise manner:
- The lights in the operating room are turned off and the mercury lamp is turned on. Wait until 15 minutes for the microscope to provide maximum brightness.
- The filter is selected based on the fluorescein used in the sample.
- The slide containing the sample solution is placed on the stage and secured with the clips.
- Adjust the focus using the fine and coarse knobs to view the image clearly.
- To the microscope, attach the camera eyepiece in order to capture the images of the sample.
- If the microscope is used for 30 minutes, switch the microscope off by switching the focus off from the control box. Then, the mercury lamp is turned off.
- Unplug the epi-fluorescence microscope and place it safely with the protective cover.
Applications of Epi-Fluorescence Microscopy
- Epi-fluorescence microscope is used as an important tool in cell biology, microbiology, pathology, biochemistry, and neurosurgery.
- It is used to observe and study structures, localization, dynamics, and functions of cellular components as well as live cells and cellular activities in real time.
- It is widely used in the identification and observation of microorganisms.
- Moreover, it is used for monitoring and quantification of specific molecules in immunoassays.
- It is used to identify, examine, and study disease conditions and impurities.
Examples of Epi-Fluorescence Microscopes
Some examples of Epi-fluorescence microscopes are as follows:
OMAX M837 Epi-Fluorescence Microscope
- It offers high-quality performance and advanced features.
- It can include a super speed 18MP USB 3.0 digital CMOS camera.
- It has 4 high-quality fluorescence objectives (FLUOR 4x, 10x, 40x(S), 100x(S,Oil)), an EPI-fluorescent assembly, a 100W HBO power supply for mercury lamp, and a double-layer mechanical stage.
- It can used for advanced research in biology, microbiology, genetics, cytology, hematology, immunology, etc.
DM5000 B Automated Upright Epi-Fluorescence Microscope
- It is used for live cell analysis and cell morphology studies.
- It features a coded 7x nosepiece, manual z-focus, fully automated transmitted light axis, and automated 5x or 8x fluorescence axis.
- Dedicated software together with the motorized fluorescence axis make the Leica DM5000 B ideal for advanced fluorescence applications involving fixed cells, live cells, and tissues.
- It has basic four colour wide-field image capture of fluorescent and pathology stained samples via monochrome and colour CCD cameras.
Leica DM RA2 Microscope
- The Leica DM RA2 motorized microscope can be brightfield, fluorescence, DIC, phase contrast, and darkfield.
- This is a highly customizable microscope, and you can configure this microscope to meet specific needs.
- The built-in mag changer has 1.0x, 1.25x, and 1.6x positions.
- There is a motorized 4-position turret – we can add filters and a fluorescence illuminator.
- The motorized nosepiece can hold up to 7 objectives.
- The motorized stage allows to move the sample, the stage can be swapped for a standard XY stage.
BSM500FL Stereo Epi-Fluorescence Microscope (Nikon SMZ800 Clone)
- It can be used for the 3D observation of samples such as living organisms using fluorescence methods such as green fluorescence protein (GFP), red fluorescence protein (RFP) or Texas Red, Dapi/Hoechst, as well as photo resistant semiconductor wafers and printed circuit boards (PCB).
- Perfect microscope for Zebrafish, embryo development studies, and Vet Labs.
- Good for Drosophila, C. elegans epi-fluorescent Imaging, macroscopic pictures of bacterial colonies, semi-conductor, and microfluidic chips.
- Excellent Image Quality & Optical Performance with Infinity Parallel Optical System.
- Comfortable Operation with Ergonomic Design Principle.
- With LED light for both incident and transmitted illumination, Providing even illumination and life expectancy can reach 6,000 hours.
Advantages of Epi-Fluorescence Microscopy
- With the use of fluorescent markers, this technique produces images of target structures with a high amount of specificity.
- It is possible to achieve multiplexing with the use of various fluorophores with unique excitation and emission spectra.
- It supports the production of optical slices of the fluorescence samples.
Limitations of Epi-Fluorescence Microscopy
- This technique of imaging has limitations in localizing molecules and does not enable the analysis of 3-dimensional spatial data, as it collects out-of-focus light.
- A standard compound microscope with epi-fluorescence illumination cannot distinguish or separate two objects that are closer than 200 nm to each other.
- Photobleaching and phototoxicity can hinder the observation of the cell samples.
- Autofluorenecence and fluorescence in the background can diminish the image quality and contrast losing fine details of the sample.
Precautions and Safety Considerations using Epi-Fluorescence Microscopes
- Avoid looking into the light source as it may reflect UV radiation to the eyes.
- Keeping records of mercury lamp usage is important as it causes hazards and risk of contamination when used beyond its lifetime.
- Avoid frequent switching of the mercury lamp on and off. It must be switched on for at least half an hour before switching it off.
- Avoid using an epi-polarization set when a mercury lamp is being used.
- The fluorescence shutter must be closed when the specimen is not visualized to prevent photobleaching.
- It is significant to cool the cameras to 0°C or less for the exposure duration when biological samples are to be visualized.
Conclusion
Significant advancements in the use of light sources, optics, filters, and cameras in microscopes have enabled researchers and scientists to study the intricate details of cells, molecules, and microorganisms in recent years.
The wide range of highly specific immuno-fluorescence agents, cellular markers, sensors, and fluorescent proteins illuminates the detailed architecture and complex biochemical pathways that govern cellular function.
In life sciences and research, epi-fluorescence microscopy has played a crucial role in the visualization of cellular compartments and specific cellular markers. Due to the enhanced specificity and sensitivity of image output, this technique allows the observation of delicate tissues such as primary neurons, through immuno-fluorescence imaging and live-cell imaging.
It has become an essential tool in research laboratories in the field of life sciences as it produces high-resolution quantitative images when coupled with restorative image deconvolution.
References
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