Use of dyes to increase phase contrast for biological holographic microscopy

Holographic microscopy is an emerging biological technique that provides amplitude and quantitative phase imaging, though the contrast provided by many cell types and organelles is low, and until now no dyes were known that increased contrast. Here we show that the metallocorrole Ga(tpfc)(SO_3H)_2, which has a strong Soret band absorption, increases contrast in both amplitude and phase and facilitates tracking of Escherichia coli with minimal toxicity. The change in phase contrast may be calculated from the dye-absorbance spectrum using the Kramers–Kronig relations, and represents a general principle that may be applied to any dye or cell type. This enables the use of holographic microscopy for all applications in which specific labeling is desired

Microscopic Object Classification through Passive Motion Observations with Holographic Microscopy

Digital holographic microscopy provides the ability to observe throughout a volume that is large compared to its resolution without the need to actively refocus to capture the entire volume. This enables simultaneous observations of large numbers of small objects within such a volume. We have constructed a microscope that can observe a volume of 0.4 µm × 0.4 µm × 1.0 µm with submicrometer resolution (in xy) and 2 µm resolution (in z) for observation of microorganisms and minerals in liquid environments on Earth and on potential planetary missions. Because environmental samples are likely to contain mixtures of inorganics and microorganisms of comparable sizes near the resolution limit of the instrument, discrimination between living and non-living objects may be difficult. The active motion of motile organisms can be used to readily distinguish them from non-motile objects (live or inorganic), but additional methods are required to distinguish non-motile organisms and inorganic objects that are of comparable size but different composition and structure. We demonstrate the use of passive motion to make this discrimination by evaluating diffusion and buoyancy characteristics of cells, styrene beads, alumina particles, and gas-filled vesicles of micron scale in the field of view

Qualification of Fiber Optic Cables for Martian Extreme Temperature Environments

Means have been developed for enabling fiber optic cables of the Laser Induced Breakdown Spectrometer instrument to survive ground operations plus the nominal 670 Martian conditions that include Martian summer and winter seasons. The purpose of this development was to validate the use of the rover external fiber optic cabling of ChemCam for space applications under the extreme thermal environments to be encountered during the Mars Science Laboratory (MSL) mission. Flight-representative fiber optic cables were subjected to extreme temperature thermal cycling of the same diurnal depth (or delta T) as expected in flight, but for three times the expected number of in-flight thermal cycles. The survivability of fiber optic cables was tested for 600 cumulative thermal cycles from -130 to +15 C to cover the winter season, and another 1,410 cumulative cycles from -105 to +40 C to cover the summer season. This test satisfies the required 3 times the design margin that is a total of 2,010 thermal cycles (670 x 3). This development test included functional optical transmission tests during the course of the test. Transmission of the fiber optic cables was performed prior to and after 1,288 thermal cycles and 2,010 thermal cycles. No significant changes in transmission were observed on either of the two representative fiber cables subject through the 3X MSL mission life that is 2,010 thermal cycles

Imaging technologies and strategies for detection of extant extraterrestrial microorganisms

There is no reductionist definition of life, so the way organisms look, behave, and move is the most definitive way to identify extraterrestrial life. Life elsewhere in the Solar System is likely to be microbial, but no microscope capable of imaging prokaryotic life has ever flown on a lander mission to a habitable planet. Nonetheless, high-resolution microscopes have been developed that are appropriate for planetary exploration. Traditional light microscopy, interferometric microscopy, light-field microscopy, scanning probe microscopy, and electron microscopy are all possible techniques for the detection of extant micro-organisms on Mars and the moons of Jupiter and Saturn. This article begins with a general discussion of the challenges involved in searching for prokaryotic life, then reviews instruments that have flown, that have been selected for flight but not flown or not flown yet, and developing techniques of great promise for life detection that have not yet been selected for flight

Holographic microscopy for 3D tracking of bacteria

Understanding when, how, and if bacteria swim is key to understanding critical ecological and biological processes, from carbon cycling to infection. Imaging motility by traditional light microscopy is limited by focus depth, requiring cells to be constrained in z. Holographic microscopy offers an instantaneous 3D snapshot of a large sample volume, and is therefore ideal in principle for quantifying unconstrained bacterial motility. However, resolving and tracking individual cells is difficult due to the low amplitude and phase contrast of the cells; the index of refraction of typical bacteria differs from that of water only at the second decimal place. In this work we present a combination of optical and sample-handling approaches to facilitating bacterial tracking by holographic phase imaging. The first is the design of the microscope, which is an off-axis design with the optics along a common path, which minimizes alignment issues while providing all of the advantages of off-axis holography. Second, we use anti-reflective coated etalon glass in the design of sample chambers, which reduce internal reflections. Improvement seen with the antireflective coating is seen primarily in phase imaging, and its quantification is presented here. Finally, dyes may be used to increase phase contrast according to the Kramers-Kronig relations. Results using three test strains are presented, illustrating the different types of bacterial motility characterized by an enteric organism (Escherichia coli), an environmental organism (Bacillus subtilis), and a marine organism (Vibrio alginolyticus). Data processing steps to increase the quality of the phase images and facilitate tracking are also discussed

ELVIS: A Correlated Light-Field and Digital Holographic Microscope for Field and Laboratory Investigations

This is the first of two articles on the Extant Life Volumetric Imaging System (ELVIS) describing a combined digital holographic microscope (DHM) and a fluorescence light-field microscope (FLFM). The instrument is modular and robust enough for field use. Each mode uses its own illumination source and camera, but both microscopes share a common objective lens and sample viewing chamber. This allows correlative volumetric imaging in amplitude, quantitative phase, and fluorescence modes. A detailed schematic and parts list is presented, as well as links to open-source software packages for data acquisition and analysis that permits interested researchers to duplicate the design. Instrument performance is quantified using test targets and beads. In the second article on ELVIS, to be published in the next issue of Microscopy Today, analysis of data from field tests and images of microorganisms will be presented

Approaches to distinguishing bacteria from mineral particles in microscopic imaging

Biologists use microscopy as the gold standard for identification of the presence of microbial life in terrestrial environments. Even when DNA analysis is used to identify strains and genetic population diversity, microscopic imaging is typically used to verify the presence of live organisms and obtain information about their sizes and morphologies. With identified microbes, a variety of microscopic techniques, particularly fluorescence imaging, are used for identification of features such as proteins, lipids, and nucleic acids. For identification of microbial life in situ, the challenge is extended to include discrimination of potential live cells from non-living objects of similar size. We present here a survey of analyses that can be made from microscopic images to make this distinction. The techniques include measurement of refractive index, Brownian motion, gravitational settling, autofluorescence, induced fluorescence, and induction of various forms of taxis

Imaging technologies and strategies for detection of extant extraterrestrial microorganisms

There is no reductionist definition of life, so the way organisms look, behave, and move is the most definitive way to identify extraterrestrial life. Life elsewhere in the Solar System is likely to be microbial, but no microscope capable of imaging prokaryotic life has ever flown on a lander mission to a habitable planet. Nonetheless, high-resolution microscopes have been developed that are appropriate for planetary exploration. Traditional light microscopy, interferometric microscopy, light-field microscopy, scanning probe microscopy, and electron microscopy are all possible techniques for the detection of extant micro-organisms on Mars and the moons of Jupiter and Saturn. This article begins with a general discussion of the challenges involved in searching for prokaryotic life, then reviews instruments that have flown, that have been selected for flight but not flown or not flown yet, and developing techniques of great promise for life detection that have not yet been selected for flight

Action on obesity: Comprehensive care for all. Report of a Royal College of Physicians, London, working party

Control tumble Originally published in Optics Letters on 01 September 2015 (ol-40-17-4114