Multi-photon microscopy: seeing more by imaging less.

Abstract

Since the earliest microscopes of Leuwenhoek and Hooke in the 17th century, optical designers have strived to maximize a key performance characteristic of each new instrument model—that of contrast. Put simply, contrast is how well we can discern useful information about a microscopic specimen from unwanted background and noise. Resolution (the ability to accurately measure features) and sensitivity (the ability to detect features) are intimately related to contrast. The Rayleigh criterion for resolution describes how far apart two tiny objects must be so that are clearly contrasted from the darker space between them. Sensitivity defines the faintest feature that can be contrasted from the background. Why is this seemingly philosophical discussion important? The answer is clear; we can improve both sensitivity and effective resolution by improving contrast between what we want to see and what we don’t. Scanning optical microscopes in general (23) deliver improved contrast in a number of different ways. A regular film or digital camera, mounted on a conventional microscope, takes a snapshot or a complete picture of the desired scene at the press of a button. The laser-scanning (optical) microscope (LSM) is quite different. The picture is built up in stages, rather as an artist might construct a work on canvas. The scene or field-of-view is surveyed, and the color and brightness of different regions are assessed and then represented by appropriate amounts of paint applied in small strokes, dots, etc. Surveying in the LSM is achieved by scanning a focused beam of laser light through the sample. One or more photodetectors are used to measure the brightness, color, or other properties at various points, and the resulting values are used to “paint” each corresponding point (or pixel) on a display screen. The final part of this analogy is the most important. The painter does not make an exact copy of the scene on the canvas but makes an interpretation according to her artistic tendencies. While the LSM is somewhat more impartial, it too has a tendency to inexactitude. This is called the microscope’s “response”, and since we are building-up our picture pointby-point, we can easily engineer our microscope’s “point-response” to get the kind of pictures we want. More precisely, we can increase the contrast of selected features or parts of the specimen.