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In 1666, Newton observed that the spectrum of colours exiting a prism in the position of minimum deviation is oblong, even when the light ray entering the prism is circular, which is to say, the prism refracts different colours by different angles. This led him to conclude that colour is a property intrinsic to light a point which had, until then, been a matter of debate. From 1670 to 1672, Newton lectured on optics. During this period he investigated the refraction of light, demonstrating that the multicoloured image produced by a prism, which he named a spectrum, could be recomposed into white light by a lens and a second prism. Modern scholarship has revealed that Newton's analysis and resynthesis of white light owes a debt to corpuscular alchemy. In his work on Newton's rings in 1671, he used a method that was unprecedented in the 17th century, as "he averaged all of the differences, and he then calculated the difference between the average and the value for the first ring", in effect introducing a now standard method for reducing noise in measurements, and which does not appear elsewhere at the time. He extended his "error-slaying method" to studies of equinoxes in 1700, which was described as an "altogether unprecedented method" but differed in that here "Newton required good values for each of the original equinoctial times, and so he devised a method that allowed them to, as it were, self-correct." Newton "invented a certain technique known today as linear regression analysis", as he wrote the first of the two 'normal equations' known from ordinary least squares, averaged a set of data, 50 years before Tobias Mayer, the person originally thought to be the oldest to do so, and he also summed the residuals to zero, forcing the regression line through the average point. He differentiated between two uneven sets of data and may have considered an optimal solution regarding bias, although not in terms of effectiveness. He showed that coloured light does not change its properties by separating out a coloured beam and shining it on various objects, and that regardless of whether reflected, scattered, or transmitted, the light remains the same colour. Thus, he observed that colour is the result of objects interacting with already-coloured light rather than objects generating the colour themselves. This is known as Newton's theory of colour. His 1672 paper on the nature of white light and colours forms the basis for all work that followed on colour and colour vision.

From this work, he concluded that the lens of any refracting telescope would suffer from the dispersion of light into colours (chromatic aberration). As a proof of the concept, he constructed a telescope using reflective mirrors instead of lenses as the objective to bypass that problem. Building the design, the first known functional reflecting telescope, today known as a Newtonian telescope, involved solving the problem of a suitable mirror material and shaping technique. Previous designs for the reflecting telescope were never put into practice or ended in failure, thereby making Newton's telescope the first one truly created. Newton grounded his own mirrors out of a custom composition of highly reflective speculum metal, using Newton's rings to judge the quality of the optics for his telescopes. In late 1668, he was able to produce this first reflecting telescope. It was about eight inches long and it gave a clearer and larger image. Newton reported that he could see the four Galilean moons of Jupiter and the crescent phase of Venus with his new reflecting telescope. In 1671, he was asked for a demonstration of his reflecting telescope by the Royal Society. Their interest encouraged him to publish his notes, Of Colours, which he later expanded into the work Opticks. When Robert Hooke criticised some of Newton's ideas, Newton was so offended that he withdrew from public debate. However, the two had brief exchanges in 167980, when Hooke, who had been appointed Secretary of the Royal Society, opened a correspondence intended to elicit contributions from Newton to Royal Society transactions, which had the effect of stimulating Newton to work out a proof that the elliptical form of planetary orbits would result from a centripetal force inversely proportional to the square of the radius vector. In astronomy, Newton is further credited with the realisation that high-altitude sites are superior for observation because they provide the "most serene and quiet Air" above the dense, turbulent atmosphere ("grosser Clouds"), thereby reducing star twinkling.