It all starts in New York City in the summer of 1927 with the birth of Marvin Lee Minsky. Following his high school education (and a brief stint in the US Navy during WW II), he went on to gain a BA in mathematics from Harvard, and a PhD in the subject from Princeton before joining MIT in 1958. He founded the MIT Computer Science and Artificial Intelligence Laboratory is Professor of Electrical Engineering and Computer Science. In Isaac Asimov’s autobiography, he admitted that there were only two people he considered to be more intelligent than he was, one was Carl Sagan, and the other was Minsky. In addition, Minsky was also an advisor on the movie ‘2001: A Space Odyssey’.

The need to see neurons

In Minsky’s memoir on inventing the confocal scanning microscope, he explained that his home was always full of prisms, optics and lenses- his father was an ophthalmologist. At a young age, he started taking optical systems apart and rebuilding them. At both Harvard and Princeton, he studied biology, neurophysiology and neuroanatomy in addition to his mathematical studies. At the time, not much was known about how brains function and how nerve cells were connected. Minsky admits to being frustrated by this lack of understanding and although the shapes of many nerve cells were visible, what was needed was a three dimensional ‘wiring diagram’. However, with the imaging systems at the time (and given the sheer density of the nerve cells of the brain), it was impossible to distinguish such neural networks due to the scattering of light. Minsky knew that what was needed was an instrument capable of optical sectioning to eliminate the out of focus light.

Zirconium arcs and a military surplus radar

Whilst studying at Harvard, he was fortunate enough to be given a room in the physics laboratory with permission to use and buy whatever equipment he needed. Minsky designed his symmetrical microscope with an objective lens and a pinhole at either side of the specimen to eliminate the scattered and out-of-focus light. When he made his invention, there were no lasers suitable to illuminate the specimen with the intensity of light needed, so he used zirconium arcs instead which was a time-consuming process- each point scan taking up to 10 seconds. The final image was produced on a military surplus radar scope which he had acquired.

The next design problem he faced was whether to move the specimen or move the optics for the scanning procedure. Minsky admitted that the prospect of moving two tiny pinholes simultaneously daunted him, so he decided to keep the optics in a fixed position and move the stage instead. He machined all of the parts for the prototype himself, spending months in the machine shop at Harvard- the skills he learnt during this time also helped him to design and build a robotic hand and arm 10 years later.

1955: The first patent for a scanning microscope

Were it not for the fact that his brother-in-law was a patent attorney, Minsky’s invention may never have been documented at all. Minsky admitted that he didn’t keep notes or write down what he was working on! On November 18th 1955, he sent a patent letter entitled ‘Double focussing stage scanning microscope’ and the confocal was born.

1966-1967: A Nipkow disc confocal

In 1966, a patent was filed by two Czechoslovakian scientists, Mojmir Petran and Milan Hadravsky for a tandem-scanning microscope. This confocal used a Nipkow Disc. Briefly, this is a mechanical spinning disc which has a spiral pattern of thousands of individual pinholes drilled in it. The effect of the Nipkow disc is that thousands of points on the specimen are illuminated simultaneously. This was actually the first commercially available confocal scanning microscope being sold by a small company in Czechoslovakia and by Tracor-Northern in the US. The first scientific paper describing the instrument and its uses was published in the journal Science in 1967. However, the instrument only worked well for the brightest of specimens.

1969: The first laser scanning confocal

The first true laser scanning confocal microscope was designed and built by M. David Egger and Paul Davidovits from Yale University who published a paper on their findings in Nature in 1969. This used a 633 nm He-Ne laser and opposed to the earlier confocal systems (and the next one in the time-line) the sample remained stationary and was illuminated by the movement of an objective lens.

1977-1979: Naming the confocal

It wasn’t until 1977 that the term ‘confocal’ was used to describe such microscopes in a publication on theoretical analysis in ‘Optica Acta: International Journal of Optics’ by two scientists from Oxford, Colin J. R. Sheppard and A. Choudhury. The term ‘confocal’ means ‘having the same or common focus’. There is another claimant to using the term ‘confocal microscope’ for the first time- a Dutch physicist called G. Fred Brakenhoff developed a laser scanning microscope in 1979. He published his findings in the Journal of Microscopy and it seems to be the first time the term Confocal Scanning Light Microscope (or ‘CSLM’) was used.

Most of these early confocals proved to be too inconvenient for biological applications being both too slow and too sensitive to vibrations.

The 1980’s: Time to buy!

In 1982, a company called ‘Oxford Optoelectronics’ (since acquired by Bio-Rad) offered the first commercially available stage scanning CSLM which was connected to a computer (the ‘SOM-25’). This was commercialised from a design by a team based at Oxford University.

In 1986, at the Medical Research Council Laboratory of Molecular Biology in Cambridge, a team of scientists were busy working inside a tent made from World-War II blackout material. Inside the tent, a new prototype CSLM capable of scan speeds in excess of 4000 lines per second was being developed. This instrument used a galvanometer-driven mirror for frame-scanning and a rotating polygonal mirror for line scanning. The line scanning mirror was proving to be problematic and producing chromatic aberrations when green and red fluorophores were used. The polygon was subsequently replaced with two oscillating galvanometer mirrors. Further developments were made to allow adjustable scanning as well as dealing with the chromatic aberration and the patent for the design has since been used by Bio-Rad for all of their confocal point-scanning systems. The team had approached other leading microscope manufacturers including Zeiss and Leica, but this proved to be unfruitful. However, a commercial agreement was soon concluded with Bio-Rad and the ‘MRC 500’ was launched to an excited audience in 1987.

It wasn’t long until the other main microscope manufacturers were developing and building commercially available instruments, either based upon the MRC 500, or in parallel to this microscope. The development of the confocal microscope continues and in the years since the MRC 500 was launched we have seen major advancements including multi-photon techniques and the ability to image 90 separate colours in a single sample. It is an exciting and rapidly expanding field- watch this space!

AUTHOR: Martin Wilson