Inverted Fluorescent Microscope – Principle, Protocol, Parts, Uses

What is Inverted Fluorescent Microscope?

Ever peeked into the world of living cells? Scientists use a nifty tool called an inverted fluorescent microscope for this. Unlike regular microscopes, this one flips the script—literally. Imagine the lenses sitting above the sample and the light source shining from below. This upside-down setup makes it perfect for studying cells chilling at the bottom of a petri dish or growing in lab cultures. No need to flip the dish—just slide it under and start observing.

Here’s how it works: A bright halogen lamp beams light upward, hitting a special mirror that acts like a bouncer for wavelengths. It reflects UV light (the kind that makes things glow) onto the sample. When the UV hits molecules tagged with fluorescent dyes, they light up like tiny neon signs. This glow gets scooped up by the lenses above, letting researchers spy on everything from DNA to proteins.

The real magic? Those fluorescent dyes, or fluorophores. They’re like microscopic highlighters, sticking to specific parts of a cell. When hit with UV, they soak up the energy and re-emit it as a different color. This lets scientists track multiple molecules at once—even if they’re super scarce.

Oh, and that halogen lamp? It’s multitasking. Not only does it kickstart the fluorescence, but it also lights up the whole sample for basic viewing. To avoid confusing the glow with the original light, a filter cube steps in. It blocks the harsh UV and only lets the gentle fluorescent colors through, creating a crisp, vivid image.

In short, this microscope is a backstage pass to the hidden light show inside living cells. By flipping the design, it makes studying life in action simpler, clearer, and way more colorful.

Principle of Inverted Fluorescent Microscope

An inverted fluorescence microscope works with light like any fluorescence microscope for fluorescence excitation and emission; it simply makes it easier to view biological samples, especially living cultures, from an inverted position. The inverted fluorescence microscope is designed to capture images of materials from the bottom of culture vessels, where a standard microscope would send the optical path in the opposite direction. See below for more details on the two systems.

The halogen bulb produces light in the white visible spectrum, meaning it is part of the visible spectrum, which is then filtered through the microscope optics. However, when it enters the microscope, it encounters a dichroic mirror, a wavelength-specific mirror that reflects certain wavelengths and lets others pass through. UV or just below visible light is reflected upward through the objective lens located below the sample stage. This bottom-up orientation is useful because the objective can see the cells permanently attached to the bottom of the culture dishes without accidentally touching or disturbing what it is looking at. As UV light bathes the sample, it comes into contact with fluorescent molecules, called “fluorophores,” bound to different parts of the cell.

Each of these clusters absorbs the intense photons and emits at longer, less energetic wavelengths. For example, a fluorophore that captures blue (488 nm) emits green (510 nm). The objective also sees this emission simultaneously because its numerical aperture is built to collect these types of light interactions. The other part of the light returning through the dichroic mirror also acts to separate the fluorescence and any residual excitatory light. The dichroic mirror allows the longer wavelengths, associated with fluorescence, to pass through and reflects the shorter wavelengths associated with excitation.

Finally, an additional emission filter cuts off any nonspecific light or autofluorescence detected by the emission detector. Only specific wavelengths are allowed to enter the detection device. The amplified fluorescence is directed to the eyepiece or a CCD camera to produce a magnified version of the sample. This magnified version, filled with spatially resolved fluorescent signals, allows researchers to visualize in real time (e.g., how they move, how they exchange ions). Some more advanced configurations even offer computer programs to evaluate the results, from longitudinal measurement of fluorescence intensity to z-stacking imaging for 3D reconstructions.

Parts of Inverted Fluorescent Microscope

1. Light Source – Halogen or LED lamp used to shine light into the sample.
2. Condenser Lens – Focuses the light onto the sample.
3. Specimen Stage – Location of the sample (usually a petri dish or culture flask) that is also easily transportable.
4. Objective Lenses – Located below the stage, these positioned lenses provide magnification.
5. Nosepiece – Location of the objective lenses. Allows rotation between different magnifying lenses.
6. Eyepieces (ocular lenses) – Located above the stage, these are the lenses through which the magnified, super-focused image is viewed.
7. Filter Cube Holder – Holds the fluorescence filters that determine the excitation and emission wavelength.
8. Dichroic Mirror – Reflects specific wavelengths back onto the sample and transmits the emitted fluorescence to the eyepieces or camera.
9. Fluorescence Filter Sets – Consisting of an excitation and emission filter. Together with the dichroic mirror, they isolate specific wavelengths for fluorescence imaging.
10. Focusing mechanism – The device used to focus the sample. There are coarse and fine focusing adjustments.

Protocol of Inverted Fluorescent Microscope

1. Preparation

• Turn on the microscope and light source.
• Warm up the lamp for 10-15 minutes if using a mercury lamp.
• Ensure the sample is prepared and labeled with fluorescent dyes.
• Place the sample on the stage.
• Dim the room lights to reduce interference.
• Ensure the microscope is stable to avoid vibrations.

2. Microscope Setup

• Choose the appropriate objective lens for magnification.
• Set the light path to eyepieces or camera.
• Select the appropriate filter cube for the fluorophore.
• Ensure the excitation and emission wavelengths match.

3. Sample Observation

• Use coarse and fine focus knobs to bring the sample into focus.
• Switch to fluorescence mode and adjust focus.
• Adjust excitation light intensity to avoid photobleaching.
• Optimize camera or software settings for exposure, gain, and contrast.
• Capture images or record videos.
• Save images in the desired format.

4. Post-Observation Steps

• Turn off the fluorescence light source.
• Clean the microscope and stage with appropriate materials.
• Remove the sample and clean any spills.
• Power down the microscope and equipment.
• Cover the microscope to protect it from dust.

5. Data Analysis

• Use image analysis software to process and analyze images.
• Quantify fluorescence intensity or other parameters as needed.

Applications of Inverted Fluorescent Microscope

• Live cell imaging for studying cell behavior and interactions
• Tissue analysis for examining tissue sections and architecture
• Microbial studies for observing microorganisms in culture
• Fluorescent labeling for visualizing specific cellular components
• Industrial applications for quality control and material analysis

Advantages of Inverted Fluorescent Microscope

• Large stage for observing specimens in various vessels
• Maintains sterility by minimizing specimen contamination
• Multiple specimen holders for different sample types
• Enhanced imaging with techniques like DIC and phase-contrast
• Easy compatibility with digital recording devices
• Wide-field eyepieces for clear, detailed images

Limitations

• Higher cost compared to upright microscopes
• Limited availability from fewer manufacturers
• Difficulties observing specimens through thick glass vessels
• Risk of photobleaching with prolonged light exposure

Precautions of Inverted Fluorescent Microscope

• Handle the microscope carefully to avoid damage
• Clean lenses with soft, lint-free cloths
• Ensure stable and vibration-free surface for operation
• Keep the microscope away from direct sunlight
• Use compatible accessories only
• Follow regular maintenance and calibration schedules
• Operate the microscope after proper training
• Wear protective equipment like gloves and safety glasses

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