The Falck-Hillarp Method
Long before the modern era of selective and sensitive antibodies that have allowed virtually any protein of choice to be visualized immunohistochemically, the monoamine histofluorescence method developed by Bengt Falck and Nils-Åke Hillarp in the early 1960s [1-3] (Falck and Torp 1962 , Falck et al 1962, see below for pdfs), provided for the first time the opportunity to visualize these neuronal systems with high sensitivity and specificity in the fluorescence microscope. Falck was at the time a young docent in the Department of Histology at Biskopsgatan 5 in Lund (in the brick-covered annex building that still stands on this site). Together with his former mentor Nils-Åke Hillarp he had spent several years trying out various protocols for catecholamine visualization. Here is, in his own words, a description of the critical steps that led to the final break-through :
“ We had observed that the monoamines were easily dislocated from their cellular stores, which is disastrous for their localization, and we tried to prevent this by treating sections of freeze-dried tissue with gaseous reagents. Formaldehyde (FA) was found to be the reagent of choice. FA vapor generated from a saturated FA solution induced a norepinephrine fluorescence in adrenal medullary cells whose intensity was almost beyond belief compared to what was known then. Our greatest dream, however, was not fulfilled: to prove the transmitter function of monoamines by visualizing them in nerve cells. A first study of the reaction mechanism was reported in the paper that is the topic of this Citation Classic. We found that the catecholamines reacted readily when enclosed in a dry protein film. In parallel studies of tissues, it became clear that the catecholamines and serotonin could form intensely fluorescent compounds in freeze-dried specimens exposed to dry FA gas – that is under conditions of such dryness that any dislocation could be avoided. It was a fantastic day when we saw the first neurons emitting a brilliant fluorescence due to their content of norepinephrine. We began to dream about all the fields that should – and did! – open up in neurobiology.”
The rest, as they say, is history: This was the first microscopic method that allowed sensitive visualization of a neurotransmitter within nerve cells, and it were to dominate the field of catecholamine research for more than two decades, until it was replaced in the early 1980s by the more versatile immunohistochemical methods. Although subsequent refinements in histochemical and tracing techniques have provided correspondingly greater sensitivity and precision, the basic organization originally described by Annica Dahlström and Kjell Fuxe in 1964  stands to this day . The groups in Lund and Stockholm (where Hillarp moved in 1962 as chairman of the Histology department at the Karolinska Institute) flourished and generated a whole generation of neuroscientists that have played a central role in Swedish neuroscience research, not least in Lund.
The original Falck-Hillarp method was based on the exposure of freeze-dried tissue to formaldehyde vapor, allowing dopamine and noradrenaline to be converted to iso-quinoline molecules that emit a yellow-green fluorescence in the microscope. Later modifications, notably the glyoxylic acid method (where the fluorophore formation is carried out in an auto-catalyzed reaction in aqueous solution; Lindvall and Björklund 1974, refs 7,8), provided improved sensitivity and precision which allowed the detection of the dopamine and noradrenaline containing axons and axon terminals in great detail (see figure).
The drawback of these methods were that they required special equipment, and also specialized skills, to give optimal results. For this reason, and in contrast to the widespread use of immunohistochemistry today, full and effective use of the histofluorescence techniques remained a privilege of a few specialized laboratories.
1. Falck and Torp A (1962) New evidence for the localization of noradrenalin in the adrenergic nerve terminals. Med. exp. 6, 169-172.
2. Falck B (1962) Observations on the possibilities of the cellular localization of monoamines by a fluorescence method. Acta physiol. scand. 56, Suppl. 197
3. Falck, B. et al. (1962) Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem. 10, 348-354
4. Falck, B (1991) Shining Monoamines Illuminate Own Function. Citation Classic in Current Contents, December 2, 1991
5. Dahlström, A. and Fuxe, K. (1964) Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons. Acta Physiol. Scand. suppl. 232, 1-55
6. Björklund, A. and Dunnett, S.B. (2007) Dopamine neuron systems in the brain: an update. Trends Neurosci. 30, 194-202
7. Lindvall, O.and Björklund, A. (1974) The glyoxylic acid fluorescence histochemical method: a detailed account of the methodology for the visualization of central catecholamine neurons. Histochemistry 39:97-127
8. Lindvall, O., Björklund, A.: The organization of the ascending catecholamine neuron systems in the rat brain as revealed by the glyoxylic acid fluoresccnce method. Acta Physiol. Scand. 1974, Supplement 412