|Plaque outside Antoni van Leeuwenhoek's old house in Delft|
The dark age of microbiology existed in the years preceding Antoni van Leeuwenhoek's most famous microscopic study of Delft's canal water and the investigative work of his contempories, Robert Hooke and Athanasius Kercher (who was most likely the first human to witness microscopic life). In these days we had recognised the effects of what we later called microbes but we had little evidence of what was causing them, for who could not wonder what induced a feverish, spluttering epidemic of 'flu? Nor who could not wonder what was controlling the geochemical processes occurring across the Earth? We just did not have the tools then to probe their world any further in any scientific way.
Before the use of lenses to magnify our surroundings, many had speculated about the existence of invisible, miniature creatures that could spread from person to person and cause disease but there was no substantial evidence. Yet fast forward 200 years after Anthonie and contemporaries first glimpsed protozoa, bacteria and fungi and we find Pasteur, Koch and Jenner pursuing their work that led to the establishment of germ theory, the life-saving process of sterilization and vaccination.
Perhaps this work would never have been done were it not for the earlier exploration of the microscopic world. Good science requires good observation and for this - in microbiology at least - we need good microscopes. But the problem now is that viruses and the molecular processes going on within cells are too small for standard techniques to observe well enough. We can no longer rely upon the techniques that van Leeuwenhoek pioneered.
Viruses - awesome nanomachines
A mature, infectious virus particle that has just been released from an infected host cell can be thought of as a complex - yet highly regulated - nanomachine. The cell on the other hand may be considered a nano-fort. Each component of the virus, whether it is a protein, lipid or nucleic acid, interact with each other to achieve the number one goal: take over a new fortress.
To achieve such a molecular feat, each virus must survive in the harsh extra-cellular environment, bind to a cell (analogous to the fort's outer walls) and cross through this barrier and into the relatively safe inner sanctum, the cytoplasm. Although the story doesn't end there and even inside the virus has to navigate itself around deadly host defences. How the virus regulates each step in this cascade of entry is the subject of intense research (if you can stop this you can stop infection). But one issue of this is that it occurs on such dramatically minuscule scales. This is where new microscopic techniques come into their own.
Overcoming the diffraction barrier
In a paper recently published in Science (Chojnacki, et al 2012 - not open-access, sorry), a group used a particular kind of super-res fluorescence microscopy (the kind that can overcome the diffraction limit) known as: 'STED', or Stimulated Emission Depletion microscopy to observe the HIV virus at unprecedented resolution.
This group was interested in how HIV is able to infect our cells through the action of its protein machinery located within each 125nm in diameter virus particle. By using STED imaging on the HIV glycoproteins (these are the 'spikes' the virus using to bind and enter new host cells) they observed something that hadn't been seen before: a mature, fully developed particle contained a single cluster of glycoproteins aimed to latch on to an unsuspecting cell whereas an immature virus would contain two or three. Using a number of mutant virus and in vitro models they show that this interesting change in distribution of viral glycoproteins co-ordinates virus/cell binding and hence entry. They show this behaviour is regulated by the proteolysis of internal HIV proteins.
They would never have been able to observe this clearly important phenomenon were it not for these high resolution techniques. Who knows what other biological processes are hiding from us in plain sight.