The macromolecules and organelles within cells are not permanent and need to get replaced over time. To do this, cells need to both produce new components and to degrade the old ones. The degradation of the bigger cellular structures depends on a process called autophagy. During autophagy, the cell envelopes the to-be-destroyed organelle and uses lysosomes to digest it down to its basic components (amino acids, sugars, lipids, etc.). Unfortunately, lysosomes cannot completely degrade all the substances the organelles are made of, and this defect results over time in the formation of lipofuscin. In cells that are actively dividing, lipofuscin is diluted through the generations, but in long-lived cells that do not divide anymore - like neurons and cardiomyocytes - it continuously accumulates in cytoplasmic granules, and ultimately determines the apoptotic death of the cell. For these reasons, lipofuscin is generally known as the “age-pigment” and its accumulation is the most prominent cytological manifestation of the ageing process.
Lipofuscin shows a broad spectrum of autofluorescence that varies among different tissues. Within the brain, lipofuscin shines mostly yellow light when excited at 330 to 370 nm. It exhibits a broad, gently-sloping, emission spectrum that covers wavelengths between 480 and 660 nm, with an emission maximum between 540 and 570 nm.
When imaging tissue with heavy Lipofuscin fluorescence together with Amytracker 520 or Amytracker 540, we recommend to use a confocal laser scanning microscopy with availability of various options for excitation and emission. When emission is collected in four channels (blue: 400-480nm, green: 480-600 nm, yellow: 600-650 nm, red: 656-700 nm), it is possible to excite at 405 nm or 488 nm. Excitation at 405 nm has the advantage that lipofuscin will localise close to the nuclei, when a DAPI counterstain is used. Amytracker fluorescence would not be very bright due to excitation far from the excitation maximum. When excitation is performed at 488 nm, nuclei won’t be visible, but Amytracker fluorescence will be much brighter, which would allow to omit Lipofuscin fluorescence completely by modulating brightness or laser power. (Image: Fresh Frozen tissue section from temporal lobe tissue with AD pathology, fixed in-ice cold EtOH and labelled with Amytracker 520 and DAPI. Left: 405 nm excitation Right: 488 nm excitation, scale: 50 µm)