Fluorescence applications in medicine

• Why do we use fluorescence?
• Extreme absolute sensitivity: single molecule level
• Ultra-high temporal resolution and speed of response
• High spatial resolution, possibility of imaging of an individual molecule on a surface or within the cell
• Response recorded at a distance: extremely sensitive to changes in environment
• Versatility in quite simple experiment
• DNA sequencing
• Diagnostics: rapid and sensitive detection of antigens associated with disease
• Imaging and analysis of biological systems
• Fluorescence parameters:
• Intensity is related to the probability of the event (quantum yield)
• Wavelength describes the energy of the light absorbed or emitted
• Lifetime is the average time between excitation and emission
• Anisotropy (or Polarization) describes the directionality perpendicular to propagation of light
• Fluorescent proteins:
• Green Fluorescent Protein (GFP) isolated from Jellyfish
• Spontaneous chemical reaction of side-chains produces fluorophore in protein core
• Color diversity achieved with fluorescent proteins
• Flow cytometry applications in clinical medicine:
• Immunophenotyping, the diagnosis of new leukemias, leukocyte subtyping, evaluation of Cytotoxic T Lymphocyte subpopulations, etc
• HIV diagnosis (CD4/CD8 ratio)
• Platelet abnormalities such as autoimmune thrombocytopenia purpura
• Diagnosis of red cell disorders such as hereditary spherocytosis (eosine-5-maleimide binding labelling)
• Fluorescence Microscopy:
• Organelle function:

  • Endoplasmic reticulum (DiOC, Dapoxyl)
  • Golgi apparatus (BIODIPY, NBD)
  • Mitochondria (MitoTracker, MitoFluor)
  • Lysosomes (LysoTracker, LysoSensor)
  • Nucleus (Hoechst, DAPI)
• Cell function:

  • Cell division, apoptosis
  • Endocytosis, exocytosis, membrane fluidity, and enzymatic activity
• Applications:

  • Organelle probes for transport, respiration, mitosis, apoptosis, protein degradation, and acidic compartments
• Future directions in fluorescence sensing:
• Sensing on a single molecule level
• Genomics, proteomics and other ‘omics’
• New level of clinical diagnostics and tomography
• Sensors promising to change the society