3. TYPES OF MICROSCOPE

3.2. FLUORESCENCE MICROSCOPE WORKING PRINCIPLES

A fluorescence microscope works by exciting a fluorescent dye (fluorophore) in a specimen with high-energy, short-wavelength light, causing it to emit lower-energy, longer-wavelength light. An excitation filter selects the specific wavelength from the light source, which is then reflected by a dichroic mirror onto the specimen. The emitted fluorescent light is then transmitted back through the same dichroic mirror and an emission filter, which blocks any remaining excitation light, before being detected by a camera or the observer's eye. 
Step-by-step working principle
  1. Excitation: 
    Light from a specialized light source (like a mercury vapor lamp or laser) passes through an excitation filter that selects a specific wavelength. 
  2. Reflection: 
    The filtered excitation light is reflected by a dichroic mirror (or "dichroic beamsplitter") at a 45° angle towards the objective lens. 
  3. Illumination: 
    The objective lens focuses the excitation light onto the fluorophore-labeled specimen. 
  4. Emission: 
    The fluorophore absorbs the excitation light and emits light at a longer wavelength (a different color). 
  5. Transmission: 
    This emitted light travels back through the objective lens and the same dichroic mirror, which is now designed to transmit the longer wavelength of the emitted light. 
  6. Filtering: 
    The emitted light then passes through an emission (or "barrier") filter that blocks any residual excitation light, allowing only the desired fluorescent light to reach the detector. 
  7. Detection: 
    The filtered fluorescent light is then captured by a detector, such as a camera or the observer's eye, to form a bright, colored image of the specimen against a dark background