How does a fluorescent microscope work?
In a fluorescence microscope, the light path is quite distinct from a regular light microscope due to the need for specific excitation and filtering of light. Here's a breakdown of the light's journey:
Light Source
The journey begins with a powerful light source, often a xenon arc lamp or a high-powered LED. This light source emits a broad spectrum of white light, containing many different colors (wavelengths).
Epi-illumination
(Traditional Left, Epi-Illumination Right)
What does Epi-Illumination mean?
Objective lens is used to focus the excitation light onto a sample and collect emission light.For fluorescence microscopy this means that the user is not exposed to the excitation light.
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Excitation Filter
The light then passes through an excitation filter. This filter acts like a colorful gatekeeper, only allowing a specific range of wavelengths to pass through. This chosen range should match the absorption peak of the fluorophore being used in the experiment.
Dichroic Mirror
The filtered light encounters a dichroic mirror, which acts like a clever traffic cop for light. It reflects the specific excitation wavelengths coming from below down onto the sample. This is the light that will excite the fluorophores.
Exciting Fluorophores
The reflected excitation light reaches the sample. If the fluorophore's absorption range matches the chosen excitation wavelength, the light gets absorbed by the fluorophore, causing it to become excited.
Fluorescence Emission
In an excited state, the fluorophore doesn't stay put for long. It releases the absorbed energy almost immediately, but in a special way – by emitting light of a longer wavelength (lower energy) compared to the absorbed light. This emitted light is what makes the fluorophore "glow."
Emission Filter
The emitted fluorescent light travels back up through the objective lens. Here, another filter, the emission filter, comes into play. This filter blocks any remaining excitation light (reflected by the sample) and allows only the specific fluorescence emission wavelengths from the fluorophore to pass through.
Reaching the Detector
Finally, the filtered fluorescent light reaches the eyepieces for visual observation or a detector (like a camera) for image capture. This allows researchers to see only the glowing signal from the fluorophores, highlighting the specific structures or molecules they are interested in within the sample.