Fluorescent staining uses fluorescent dyes or labeled antibodies to tag specific biomolecules, cells, or structures, enabling their visualization and localization under a fluorescence microscope. It increases contrast and sensitivity for detecting proteins, nucleic acids, organelles, or pathogens, supports quantitative analysis, multiplexing, live‑cell imaging, diagnostics, and helps study cellular processes, interactions, and disease-related changes.

A smear test for TB (sputum smear microscopy) examines a patient’s sputum under a microscope after special staining to detect acid-fast bacilli (Mycobacterium tuberculosis). It’s quick and inexpensive, uses early-morning sputum samples (usually two to three), and helps identify contagious pulmonary TB. It’s less sensitive than culture or molecular tests, so negative smears may need further testing.

The fluorescent test for TB is fluorescence microscopy of sputum smears stained with auramine‑rhodamine (auramine‑O). Under a fluorescence or LED microscope, mycobacteria glow bright against a dark background, giving faster, more sensitive detection than conventional Ziehl–Neelsen staining. It’s used for rapid screening; positive smears need culture or molecular testing (e.g., NAAT) for confirmation and drug‑resistance assessment.

Fluorescence microscopy is used to visualize and track specific molecules, organelles, and structures in cells and tissues by labeling them with fluorescent dyes or proteins. It enables study of protein localization, gene expression, cell signaling, and dynamic processes in live or fixed samples, aids diagnostic detection of pathogens and abnormal cells, and supports quantitative, high-sensitivity imaging including co-localization and single-molecule analyses.

An example of a fluorescent stain is DAPI (4′,6‑diamidino‑2‑phenylindole). DAPI binds strongly to A–T rich regions of DNA and is widely used to label cell nuclei in fluorescence microscopy. When excited with ultraviolet light, it emits a bright blue fluorescence, enabling clear visualization of nuclear morphology and reliable cell counting in fixed or permeabilized samples.

Fluorescence occurs when a molecule absorbs light and an electron is excited to a higher energy state; after internal vibrational relaxation, the electron returns to the ground state by emitting a photon of lower energy (longer wavelength). Efficient fluorescence needs rigid, conjugated structures that favor radiative decay over non‑radiative processes. Quantum yield and the environment (solvent, pH, temperature) affect intensity and wavelength.