Microscopy: A Very Short Introduction QUOTES

1 " With the advent of nanotechnology, microfabrication has produced novel manmade constructs called metamaterials which exhibit entirely new properties in terms of their effect on light, effects which are not found in conventional materials, or even in nature itself. Early in the 21st century, a chance observation showed that an ultrathin layer of silver on a flat sheet of glass would act like a lens, and from this point, the development of the ‘perfect’ or ‘superlens’ began, with the theoretical possibility to image details such as viruses in living cells with a light microscope, bypassing Abbe’s diffraction limit. Metamaterials have been produced that make this possible, as they have a property previously unimagined in optics, and not found in nature, which is a negative refractive index. "

― , Microscopy: A Very Short Introduction

2 " Long before the idea of scanning specimens with a small spot of light produced confocal light microscopy, the idea of using a small spot of electrons to scan surfaces had been around for as long as electron microscopy itself. A surface demarcates the boundary of a solid, and is the site of interaction with the surrounding environment, from a ball bearing to a living cell. In the mechanical world, adhesion, friction, wear, and corrosion are all dependent upon surface properties. The smooth surface of a ball bearing is crucial in the reduction of friction, but its efficiency may well be compromised by wear or corrosion. "

― , Microscopy: A Very Short Introduction

3 " Pulsed lasers produce incredibly short bursts of electromagnetic energy. For example, a pulsed femtosecond laser produces a flash of light that lasts for femtoseconds to a picosecond (a picosecond is one trillionth of a second, a femtosecond is one thousandth of a picosecond), instantly followed by another (and so on). These lasers brought about the possibility of exciting fluorophores with two photons of only half the necessary energy, but they need to arrive almost simultaneously to generate the ejection of a photon. Infrared pulsed lasers penetrate living tissue more effectively, with the advantage that fluorescence is achieved from much deeper in the tissue than normal fluorescence, where the depth of penetration is limited by multiple light scattering events. Multiphoton microscopy (mainly two photon in practice, but also feasible as three or more photons) allows imaging from as deep as a millimetre (one thousand micrometres), an improvement of several hundred micrometres over fluorescence confocal microscopy. A second advantage of two photon excitation is that it forms as a single spot in the axial plane (z axis) without the ‘hourglass’ spread of out of focus light (the point spread function) that happens with single photon excitation. This is because the actual two photon excitation will only occur at the highest concentration of photons, which is limited to the focal plane itself. Because there is no out of focus light, there is no need for a confocal pinhole, allowing more signal to reach the detector. Combined with the increased depth of penetration, and reduced light induced damage (phototoxicity) to living tissue, two photon microscopy has added a new dimension to the imaging of living tissue in whole animals. At the surface of a living brain, remarkable images of the paths of whole neurons over several hundred micrometres can be reconstructed as a 3D z section from an image stack imaged through a thinned area of the skull in an experimental animal. Endoscopes have been developed which incorporate a miniaturized two photon microscope, allowing deep imaging of intestinal epithelium, with potential to provide new information on intestinal diseases, as most of the cellular lining throughout our gut is thin enough to be imaged in this way. So far a whole range of conditions including virtually all the cancers of the digestive tract as well as inflammatory bowel disease have been investigated, reducing the need for biopsies and providing new insights as to the nature of these conditions. "

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4 " The advent of low temperature scanning EM led to a study by Bill Wergin and colleagues from NASA in which they collected samples from different types of snow cover found in the prairies, taiga (snow forest), and alpine environments. With snow depths up to a metre, various layers occurred in which the crystals underwent a change in their microscopic shape from the original freshly fallen crystals, to the development of flat faces and sharp edges. It is this metamorphosis of lying snow that determines the likelihood of avalanches, which can be predicted from the crystal structures at various depths. Although scanning EM (electron microscopy) is hardly available as a routine assay in distant mountain regions, this work helped in the use of microwave radiology investigation of the snow water equivalent in the snow pack, as large snow crystals scatter passive microwave more than small crystals. Smaller and more rounded crystals of snow do not interlock, and can slide more easily over each other, increasing the risk of avalanches. "

― , Microscopy: A Very Short Introduction

5 " At one end of the scale, astronomers search the heavens for new information about the universe, whilst at the other end, microscopists chase atoms and molecules to study defects in crystals or the basic processes of life. These investigations may be separated by more than twentyfold orders of magnitude, but are nevertheless driven by the same insatiable curiosity of the human psyche to explore beyond the vision of our own eyes. "

― , Microscopy: A Very Short Introduction

6 " From its earliest days, scanning EM proved to be a source of images that everybody could relate to, regardless of a microscopic or indeed even a scientific background. From early images showing great detail of everyday objects and animals, for example the edge of a scalpel or razor blade or the multiple compound eyes of a spider, the extra information provided by the high magnification was instantly apparent, grasping the attention of the general public in a way that transmission EM images did not (Figure 19). Today, images of bacteria, stem cells, and tumour cells are a regular sight in TV news, documentaries, newspapers, and magazines, usually brightly coloured. False or pseudo-colouring of scanning EM images is useful for highlighting specific features, as well as increasing the overall impact, which can sometimes be a little on the garish side. "

― , Microscopy: A Very Short Introduction

7 " So far we have considered the effects of varying the type of illumination, so at this point we can sum up how one specimen can be imaged in four separate ways. In a conventional microscope with bright field illumination, contrast comes from absorbance of light by the sample (Figure 7a). Using dark field illumination, contrast is generated by light scattered from the sample (Figure 7b). In phase contrast, interference between different path lengths produces contrast (Figure 7c), and in polarizing microscopy it is the rotation of polarized light produced by the specimen between polarizer and analyser (Figure 7d). This is ‘converted’ into an image that has colour and a three dimensional appearance by the use of Wollaston prisms in differential interference microscopy. For virtually any specimen, hard or soft, isotropic or anisotropic, organic or inorganic, biological, metallurgical, or manufactured, there will be a variety of imaging modes that will produce complementary information. Some of the types of light microscopy we have looked at above have direct parallels in electron microscopy (Chapter 4). "

― , Microscopy: A Very Short Introduction

8 " Our best ability to see detail with the eye is to perceive two strands of human hair that are separated by a hair’s width. This is the limit of our resolution—the ability to see detail, measured by the distance at which two points are still distinct. Magnification without increased resolution merely produces a larger image with no increase in detail. Amazing as our own eyes are, they are poor compared with those of an eagle, where the resolution is eight times as good, enabling it to spot a rabbit at a distance of two miles. "

― , Microscopy: A Very Short Introduction

9 " It has been estimated that a career electron microscopist who spends his working days preparing, sectioning and staining, observing, and recording biological material will get through the equivalent of one cubic millimetre of tissue in a forty-year working lifetime. "

― , Microscopy: A Very Short Introduction

10 " Modern scanning probe instruments will often provide several different modes within the same instrument, including aspects of light microscopy such as near field optical scanning microscopy (NOSM) and micro tools for nanofabrication such as micro-writing devices (nanolithography), indentation probes providing exact positioning and force control, all in a specimen chamber in which both the temperature and gaseous environment can be precisely controlled. This type of scanning probe microscopy has made it possible to investigate a surface phenomenon termed surface plasmon polaritons (SPPs for short), which are surface electromagnetic waves that propagate between the interface of a metal and a dielectric (insulator). More explanation of SPPs would require a VSI on surface physics, but suffice it to say, the scanning plasmon near field microscope has made it possible to work towards practical exploitation in the applications of SPPs (which make it possible to ‘package’ light in smaller quantities than ever before) in optics, data storage, solar cells, chemical cells, and biosensors. "

― , Microscopy: A Very Short Introduction