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Introduction to Fluorescence Microscopy

Fluorescence microscopy, initially explored by August Köhler in 1904, has evolved into a cornerstone technique in biomedical research and clinical pathology. The development of this technique over the years by various researchers has expanded its applications in studying the localization of molecules and specific sequences of DNA. This article discusses the contributions of key scientists and the advancements in fluorescence microscopy.

• Fluorochrome Attachment by Albert Coons (1941): lbert Coons made a significant stride in 1941 by pioneering the technique of attaching a fluorochrome to an antibody. This innovative approach laid the foundation for later advancements in fluorescence microscopy. Coons' work marked the inception of using fluorescent molecules in biological studies.
• Fluorescein Isothiocyanate (FITC) Immunofluorescence (1950): Continuing the legacy, Albert Coons, in collaboration with N.H. Kaplan, introduced the fluorescein isothiocyanate (FITC) immunofluorescence technique in 1950. In this method, a fluorescent molecule is covalently bonded to an immunoglobulin, designed to target a specific protein. FITC immunofluorescence has since become a powerful tool for localizing molecules within the field of light microscopy.
• Fluorescence in Situ Hybridization (FISH) by B.J. Trask (1991): In 1991, B.J. Trask introduced the fluorescence in situ hybridization (FISH) technique, enabling the specific labeling of DNA sequences. This method paved the way for the precise identification of genetic material within cells, revolutionizing genetic research.
• Discovery of the "Green Fluorescent Protein" (GFP) by D.C. Parsher et al. (1992): D.C. Parsher and colleagues made a groundbreaking discovery in 1992 by cloning the gene responsible for the "green fluorescent protein" (GFP) derived from a chemiluminescent jellyfish. This innovation served as the basis for genetic engineering techniques, where the GFP gene is fused with a host gene of interest and incorporated into the host genome. The resulting protein produced by the cell emits fluorescence, enabling the tracking of specific cellular components.
• Advancements by Rodger Tsien: The work initiated by D.C. Parsher was extended by Rodger Tsien, who expanded the range of fluorescent colors and adapted the technology to a diverse set of biological applications. Tsien's contributions broadened the utility of GFP and other fluorescent proteins, allowing researchers to explore a multitude of biological phenomena with precision and versatility.Evolution of Fluorescence Microscopy

The collective efforts of these scientists have driven the evolution of fluorescence microscopy, enhancing its utility as a powerful and flexible tool in various areas of scientific research. From the initial attachment of fluorochromes to antibodies to the cloning of fluorescent genes, these developments have revolutionized the field and continue to shape the future of biological and medical investigations.

The following table summarizes the key milestones in the development of fluorescence microscopy:

Applications of Fluorescence Microscopy

Fluorescence microscopy offers a wide array of applications in various scientific fields. These applications can be categorized into distinct divisions, each contributing to our understanding of biological and medical processes.

Autofluorescence

• Definition: Autofluorescence refers to the intrinsic ability of certain biological specimens to fluoresce when exposed to specific wavelengths of light.
• Natural Phenomenon: Some substances, such as those listed in Table 12.1, exhibit autofluorescence without the need for additional staining.
• Challenges: Autofluorescence can pose challenges in fluorescence microscopy as it may interfere with the desired fluorescence signals and affect image quality.

Fluorescent Stains

• Early Adoption: M. Haitinger pioneered the staining of histological specimens with fluorescent dyes back in 1933.
• Common Histological Stains: Many traditional histological stains exhibit fluorescence properties. A selection of these stains is listed in Table 12.2.
• Selective Tissue Staining: Various fluorescent dyes are employed to selectively stain specific components within tissues, resulting in what is known as secondary fluorescence. This approach enhances the visibility of certain tissue structures.

Immunofluorescence

• Inception by Coons and Kaplan: Albert H. Coons and N. H. Kaplan were the pioneers in attaching fluorescent dyes to antibodies, creating a revolutionary technique known as immunofluorescence.
• Localization of Antigens: Immunofluorescence allows the precise localization of antigens within tissue sections, making it invaluable in the fields of biology and medicine.
• Biological Implications: This technique finds application in diverse biological and medical contexts, such as mapping the chromosomal localization of genes with specific defects.

Fluorescence microscopy continues to play a pivotal role in various scientific disciplines, offering powerful tools for the visualization and study of biological specimens, molecular interactions, and cellular structures. These diverse applications illustrate its significance in advancing our understanding of the natural world.

Table 12.1: Examples of Autofluorescent Biological Compounds

Table 12.2: Examples of Histological Stains Exhibiting Fluorescence

Fluorescence Unveiled: A Review

Fluorescence is a fascinating optical phenomenon displayed by certain substances, characterized by the emission of light of one color when illuminated by light of a different color. This phenomenon can be dissected into two distinct categories: fluorescence and phosphorescence, each defined by the duration of light emission following exposure.

• Fluorescence: When the emitted light manifests within one millionth of a second after light exposure, it falls under the category of fluorescence. This rapid response results in a shorter emission duration.

• Phosphorescence: In contrast, phosphorescence refers to luminescence where light emission occurs over an extended period, exceeding one millionth of a second.

Stokes' Law: A key characteristic of fluorescence is that the emitted light typically exhibits a longer wavelength than the light that initially excites the substance. This color shift is encapsulated by Stokes' law, which explains this intriguing relationship. For instance, when fluorescein isothiocyanate (FITC) is excited by blue light, it emits green light. Similarly, rhodamine isothiocyanate, when excited by green light, emits red light.

Fluorescent substances are characterized by several fundamental features:

• Absorption Spectrum: They have a range of wavelengths at which they efficiently absorb light, known as their absorption spectrum.

• Emission Spectrum: Correspondingly, they emit light across a range of wavelengths, forming their emission spectrum.

Stokes' Shift: The difference between the absorption maximum and emission maximum in the spectra of a fluorescent substance is defined as the Stokes' shift. It provides insights into the color transition experienced during fluorescence.

Overlap in Spectra: It's important to note that the absorption and emission spectra of a fluorescent substance are not discrete; they exhibit overlapping regions.

Maximal Fluorescence Intensity: For any fluorescent substance, the intensity of fluorescence is most pronounced when the excitation light aligns with the wavelength of the absorption maximum. This alignment results in the highest level of light emission.

Fluorescence plays a pivotal role in a wide array of scientific and technological applications, enabling the exploration of molecular interactions, biological processes, and material properties. Understanding the intricacies of fluorescence, including Stokes' law and the interplay between absorption and emission spectra, is essential for leveraging this phenomenon effectively in research and practical endeavors.

Two Flavors of Fluorescence Microscopes

Fluorescence microscopy is a versatile technique, and it comes in two main varieties: diascopic fluorescence and episcopic fluorescence. Each type has its own optical setup and set of advantages and disadvantages.

Diascopic Fluorescence:

• Historical Origins: Diascopic fluorescence microscopy has a historical pedigree dating back to K. Reichert and O. Heimstadt in 1911. In this approach, transmitted light is utilized for observation, and it's well-suited for autofluorescent specimens.
• Optical Configuration: The optical setup for diascopic fluorescence involves several key elements. First, the illumination source's light passes through an excitation filter, which selects the appropriate wavelengths. Then, it proceeds through a dark-field condenser to illuminate the specimen, effectively eliminating most of the excitation light from the imaging side. The fluorescent light emanating from the specimen travels through the objective lens and is further refined by a barrier filter (emitter), which selects only the desired fluorescent wavelengths.
• Advantages: Diascopic fluorescence has its strengths. It produces excellent dark field images, enhancing contrast. This method accommodates various objective lenses, even for ultraviolet (UV) excitation, provided the numerical aperture (NA) is lower than that of the condenser. It excels at generating bright images at lower magnifications.
• Disadvantages: However, this technique is not without drawbacks. Aligning high NA dark-field condensers can be challenging, and thicker glass slides may pose alignment issues. Specimens need to be transparent, limiting its applicability. Furthermore, it tends to illuminate a large area of the specimen, which can be problematic for rapidly fading fluorescence.

Episcopic Fluorescence:

• Dominant Modern Approach: Episcopic fluorescence microscopy has become the dominant modern approach, enabled by the invention of the dichroic mirror (chromatic beam-splitter) by E.M. Bromberg in 1953.
• Optical Configuration: In episcopic fluorescence microscopy, the excitation light is directed from above the specimen through the objective lens. The optical setup features high numerical aperture objectives at their full aperture, delivering superior expected resolution and brighter images at higher magnifications. Köhler illumination is achieved by employing a field iris in the episcopic optical system, limiting excitation to the area viewed by the objective lens. The invention of the epi-illumination filter cube by J. S. Ploem in 1970 simplified the interchange of filter combinations with the episcopic apparatus. Additionally, it's straightforward to combine the fluorescent image with a transmitted light image of the specimen.

Essential Components of Fluorescence Microscopy

Fluorescence microscopy relies on the precise selection and manipulation of light wavelengths. To achieve this, several critical components, including filters, are utilized. These filters play a fundamental role in enabling the fluorescence phenomenon to be observed.

Filters in Fluorescence Microscopy:

1. Excitation Filter: An excitation filter is employed to select specific wavelengths of light from an appropriate light source. These wavelengths should correspond to the maximum absorption region of the fluorescent dye. By using this filter, fluorescence is triggered within the specimen.

2. Emission Filter: On the other hand, an emission filter serves the purpose of passing the fluorescent wavelengths generated by the specimen while blocking the excitation wavelengths. This enables the isolation and observation of the fluorescence signal without interference from the excitation light.

   

Types of Filters:
Several types of filters are utilized in fluorescence microscopy, each characterized by the wavelengths and intensity of light they transmit. The most prominent types include:

• Colored Glass Filters: These are basic filters made of colored glass that selectively transmit certain wavelengths of light. While they are simple, they may have limitations in terms of precision.

• Interference Filters: Considered the superior choice for fluorescence microscopy, interference filters are constructed by depositing multiple layers of carefully chosen dielectric materials onto a glass substrate. These materials possess different refractive indices, and the interactions between them result in selective transmission of specific wavelengths. This precise design allows for highly accurate filtering.

• Dichroic Mirrors (Chromatic Beam-Splitters): Dichroic mirrors, also known as chromatic beam-splitters, have specific applications in fluorescence microscopy. They were first introduced by E. M. Bromberg in 1953. These mirrors can reflect light of shorter wavelengths than their cut-off frequency, while transmitting light of longer wavelengths. They have distinct reflection and transmission characteristics tailored to the requirements of fluorescence microscopy.

Evolution of Filter Technology:
Filter technology in fluorescence microscopy has undergone significant advancements. For instance, in 1969, J. Rygaard and W. Olsen developed a specialized interference filter for FITC, optimizing the selectivity of filter characteristics. J. S. Ploem further refined this technology by introducing the filter cube, illustrated in Figure 12.5, which simplifies the interchange of filter combinations using interference filters, making fluorescence microscopy even more versatile.

Dry Dark Field Condensers

1. Maximum NA: Dry dark field condensers have a maximum numerical aperture (NA) of 0.95. This limits the acceptance angle for the incident light, affecting the intensity and quality of illumination.

2. Objective Limitation: When using dry dark field condensers, it is essential to pair them with objective lenses having a numerical aperture (NA) of less than 0.75. This combination ensures compatibility and optimal image quality.

Oil Dark Field Condensers:

1. Higher NA: Oil dark field condensers are capable of achieving a higher numerical aperture (NA) of up to 1.4. This extended range of NA allows for enhanced control over the illumination, particularly when used with objectives of higher numerical aperture.

2. Objective Compatibility: Oil dark field condensers are suitable for use with objectives having a numerical aperture (NA) of less than 1.1. This compatibility ensures the effective matching of the condenser and objectives.

Illuminators for Fluorescence Microscopy

Fluorescence microscopy relies on illuminators that provide the appropriate light source to excite the fluorochromes used in the specimen. These illuminators must produce light within the absorption region of the specific fluorochromes and provide sufficient intensity for effective excitation. There are various light sources available for fluorescence microscopy, each with its advantages and limitations. Here are some of the common light sources and their emission spectra:

1. Tungsten Halogen Lamps:
   • Emission Spectra: Tungsten halogen lamps can effectively excite fluorochromes like FITC.
   • Advantages: They are commonly used in fluorescence microscopy due to their suitability for specific fluorochromes.
2. High-Pressure Mercury Lamps:
   • Emission Spectra: High-pressure mercury lamps emit radiation in both the UV and visible spectra.
   • Advantages: They are widely used because of their broad spectrum. However, their emission is not continuous, making them unsuitable for some fluorochromes. For instance, they lack spectral lines between 440 and 540 nm.
3. High-Pressure Xenon Lamps:
   • Emission Spectra: High-pressure xenon lamps offer an alternative to mercury lamps, but their emission in the UV range is relatively low.
   • Advantages: They are used when the UV spectrum is not a primary concern.
4. CSI Lamp (Metal-Halide Arc Lamp):
   • Advantages: CSI lamps, also known as metal-halide arc lamps, provide an alternative to high-pressure mercury lamps. They offer a continuous spectrum and are versatile for various fluorochromes and applications.

The choice of illuminator depends on the specific fluorochromes being used, the spectral characteristics required for excitation, and the application's needs. While high-pressure mercury lamps are common due to their broad spectrum, alternative illuminators like high-pressure xenon lamps and CSI lamps provide flexibility and are valuable in scenarios where specific spectral characteristics are needed.

In summary, selecting the appropriate illuminator is crucial in fluorescence microscopy to ensure effective excitation of fluorochromes and reliable imaging results. The choice should align with the spectral requirements of the fluorochromes and the goals of the microscopy experiment.

Microscope Set-Up for Episcopic Fluorescence

The set-up for episcopic fluorescence microscopy involves aligning the microscope components to ensure proper illumination and imaging. This process typically follows Köhler illumination principles. Here are the steps for setting up episcopic fluorescence using a mercury arc lamp:

1. Centering the Arc Lamp:

• Caution: Mercury arc lamps emit UV radiation, so precautions must be taken.
• Begin by focusing on a specimen using a low-power objective lens.
• Remove the objective lens and replace the specimen with a white paper.
• Open the epi-illuminator's field iris and aperture iris fully.
• Place a filter pack in the optical path and open the lamp shutter.
• Use the collector lens focus knob to focus the image of the lamp's electrodes onto the white paper.
• If the lamp image is too bright, try adjusting the field iris, aperture iris, or use a different filter pack.
• One electrode will appear pointed, and the other blunt.
• Use the controls on the lamp housing's side to center the electrode's image in the circle of illumination. These controls affect only the real image of the electrode.
• A fainter image of the electrodes may also be visible, which is a reflection from the lamp's concave mirror. The mirror controls on the rear of the lamp housing adjust both images.
• Ensure that the reflected image of the electrodes is superimposed onto the primary image.
• Some manufacturers may have specific recommendations for the distance between the two images.
• Close the lamp shutter.

2. Focusing the Collector Lens:

• Replace the objective lens and place a fluorescent specimen on the microscope.
• Initially, focus on the specimen using bright field microscopy.
• Open the epi-illuminator's field and aperture irises.
• Focus the lamp housing's collector lens to achieve uniform illumination of the field of view.
• If the illumination remains uneven, adjust the lamp centering screws while observing the specimen until uniform illumination is achieved.

3. Adjusting the Epi-Field Iris:

• Focus on the fluorescent specimen.
• Close the field iris and ensure that a sharp image of its edge enters your field of view.
• Center the field iris using the provided centering controls.
• Open the field iris just out of the field of view.

4. Adjusting the Epi-Aperture Iris:

• Stopping down the aperture iris can help reduce non-specific background fluorescence.
• This adjustment should be made while observing the fluorescent specimen to minimize any impact on the fluorescence from specifically labeled structures.
• Ensure that the aperture iris is fully open when starting work.

Tip: On upright stands, consider lowering the transmitted light condenser or removing it from the stand if it won't be used. This prevents reflected light from the condenser's surface from affecting the fluorescent image and producing a darker background.

Properly aligning the microscope components in episcopic fluorescence is essential for achieving high-quality fluorescent imaging with reduced background fluorescence. Following these steps ensures that the microscope is optimized for fluorescence microscopy applications.