Confocal microscopy

Chapter focusing on confocal microscopy. A confocal microscope is one in which the illumination is confined to a small volume in the specimen, the detection is confined to the same volume and the image is built up by scanning this volume over the specimen, either by moving the beam of light over the specimen or by displacing the specimen relative to a stationary beam. The chief advantage of this type of microscope is that it gives a greatly enhanced discrimination of depth relative to conventional microscopes. Commercial systems appeared in the 1980s and, despite their high cost, the world market for them is probably between 500 and 1000 instruments per annum, mainly because of their use in biomedical research in conjunction with fluorescent labelling methods. There are many books and review articles on this subject ( e.g. Pawley ( 2006) , Matsumoto( 2002), Wilson (1990) ). The purpose of this chapter is to provide an introduction to optical and engineering aspects that may be o f interest to biomedical users of confocal microscopy

Helium ion microscopy and energy selective scanning electron microscopy – two advanced microscopy techniques with complementary applications

Both scanning electron microscopes (SEM) and helium ion microscopes (HeIM) are based on the same principle of a charged particle beam scanning across the surface and generating secondary electrons (SEs) to form images. However, there is a pronounced difference in the energy spectra of the emitted secondary electrons emitted as result of electron or helium ion impact. We have previously presented evidence that this also translates to differences in the information depth through the analysis of dopant contrast in doped silicon structures in both SEM and HeIM. Here, it is now shown how secondary electron emission spectra (SES) and their relation to depth of origin of SE can be experimentally exploited through the use of energy filtering (EF) in low voltage SEM (LV-SEM) to access bulk information from surfaces covered by damage or contamination layers. From the current understanding of the SES in HeIM it is not expected that EF will be as effective in HeIM but an alternative that can be used for some materials to access bulk information is presented

Fluorescence antibunching microscopy

Breaking the diffraction limit in microscopy by utilizing quantum properties of light has been the goal of intense research in the recent years. We propose a quantum superresolution technique based on non-classical emission statistics of fluorescent markers, routinely used as contrast labels for bio-imaging. The technique can be readily implemented using standard fluorescence microscopy equipment

Condensate Phase Microscopy

We show that the phase of a Bose-Einstein condensate wave-function of ultra-cold atoms in an optical lattice potential in two-dimensions can be detected. The time-of-flight images, obtained in a free expansion of initially trapped atoms, are related to the initial distribution of atomic momenta but the information on the phase is lost. However, the initial atomic cloud is bounded and this information, in addition to the time-of-flight images, is sufficient in order to employ the phase retrieval algorithms. We analyze the phase retrieval methods for model wave-functions in a case of a Bose-Einstein condensate in a triangular optical lattice in the presence of artificial gauge fields.Comment: 6 pages, 3 figures, article + supplement, version accepted for publication in Physical Review Letter

Helium Ion Microscopy

Helium Ion Microcopy (HIM) based on Gas Field Ion Sources (GFIS) represents a new ultra high resolution microscopy and nano-fabrication technique. It is an enabling technology that not only provides imagery of conducting as well as uncoated insulating nano-structures but also allows to create these features. The latter can be achieved using resists or material removal due to sputtering. The close to free-form sculpting of structures over several length scales has been made possible by the extension of the method to other gases such as Neon. A brief introduction of the underlying physics as well as a broad review of the applicability of the method is presented in this review.Comment: Revised versio

Quantum differential ghost microscopy

Quantum correlations become formidable tools for beating classical capacities of measurement. Preserving these advantages in practical systems, where experimental imperfections are unavoidable, is a challenge of the utmost importance. Here we propose and realize a quantum ghost imaging protocol able to compensate for the detrimental effect of detection noise and losses. This represents an important improvement as quantum correlations allow low brightness imaging, desirable for reducing the absorption dose. In particular, we develop a comprehensive model starting from a ghost imaging scheme elaborated for bright thermal light, known as differential ghost imaging and particularly suitable in the relevant case of faint or sparse objects. We perform the experiment using SPDC light in microscopic configuration. The image is reconstructed exploiting non-classical intensity correlation rather than photon pairs detection coincidences. On one side we validate the theoretical model and on the other we show the applicability of this technique by reconstructing a biological object with 5 micrometers resolution

Diffractive oblique plane microscopy

Imaging of neuronal activity with fluorescent indicators is an important technique in neuroscience. However, it remains challenging to record volumetric image data at fast frame rates and good resolution. One promising technique to achieve this goal is light sheet microscopy (LSM), but the right angle configuration of the excitation and imaging system limits its application. Oblique plane microscopy (OPM), a variant of LSM, circumvents this limitation by exciting oblique planes and detecting the image through the same microscope objective lens. So far, these techniques have relied on the use of high numerical aperture (NA) detection objective lenses, which limits their field of view. Here we present an OPM technique that allows for the use of low NA objective lenses by redirecting the light with the help of a diffraction grating. The microscope maintains a micrometer-scale lateral resolution over a large addressable imaging volume of 3.3×3.0×1.0  mm3. We demonstrate its practicality by imaging the whole brain of larval and juvenile zebrafish

Scanning Quantum Dot Microscopy

Interactions between atomic and molecular objects are to a large extent defined by the nanoscale electrostatic potentials which these objects produce. We introduce a scanning probe technique that enables three-dimensional imaging of local electrostatic potential fields with sub-nanometer resolution. Registering single electron charging events of a molecular quantum dot attached to the tip of a (qPlus tuning fork) atomic force microscope operated at 5 K, we quantitatively measure the quadrupole field of a single molecule and the dipole field of a single metal adatom, both adsorbed on a clean metal surface. Because of its high sensitivity, the technique can record electrostatic potentials at large distances from their sources, which above all will help to image complex samples with increased surface roughness.Comment: main text: 5 pages, 4 figures, supplementary information file: 4 pages, 2 figure