Thin Coverslip Research Articles

Optimization of Sample Preparation Using Glass Slides for Spectral Pathology.

The clinical translation of Fourier transform infrared (FT-IR) microspectroscopy in pathology will require bringing this technique as close as possible to standard practice in pathology departments. An important step is sample preparation for both FT-IR microspectroscopy and pathology. This should entail minimal disruption of standard clinical practice while achieving good quality FT-IR spectral data. In fact, the recently described possibility of obtaining FT-IR spectra of cells placed on glass substrates brings FT-IR microspectroscopy closer to a clinical application. We have now furthered this work in order to identify two different types of lung cancer cells placed on glass coverslips. Two types of sample preparation which are widely used in pathology, cytospin and smear, have been used. Samples were fixed with either methanol, used in pathology, or formalin (4% paraformaldehyde) used widely in spectroscopy. Fixation with methanol (alcohol-based fixative) removed lipids from cells causing a decrease in intensity of the peaks at 2850 cm−1 and 2920 cm−1. Nevertheless, we show for the first time that using either type of sample preparation and fixation on thin glass coverslips allowed to differentiate between two different types of lung cancer cells using either the lipid region or the fingerprint region ranging from 1800 cm−1 to 1350 cm−1. We believe that formalin-fixed cytospin samples would be preferred to study cells on thin coverslips using FT-IR microspectroscopy. This work presents a clear indication for future advances in clinical assessment of samples within pathology units to gain a deeper understanding of cells/tissues under investigation.

Ratiometric oxygen probes with a cell-penetrating peptide for imaging oxygen levels in living cells

We investigated the photophysical and intracellular properties of two newly designed molecule-based ratiometric oxygen probes, 7DEAC-R8-BTQphen (R8) and 7DEAC-R12-BTQphen (R12), to evaluate their suitability for intracellular O2 measurements. R8 and R12 consist of a blue fluorescent coumarin derivative (7DEAC) and a red phosphorescent iridium(III) complex (BTQphen) connected respectively by an octa- or dodecaarginine linker to promote intracellular delivery of the probes. The probes showed oxygen-sensitive dual emissions in acetonitrile, lipid bilayer membrane, and in living cells. Energy state diagrams showing the relaxation processes of R8 and R12 in the excited state were constructed using emission spectra, quantum yield, and lifetime data. Efficient photon absorption at 405 nm due to the 7DEAC chromophore is followed by intramolecular energy transfer to the BTQphen moiety, resulting in 7DEAC fluorescence and BTQphen phosphorescence with an intensity ratio and spectral separation suitable for ratiometric measurements. The validities of O2 concentrations derived from the ratios between the phosphorescence and fluorescence intensities of R8 and R12 were verified in lipid membranes, as well as in solution by phosphorescence lifetime measurements of the BTQphen moiety. The cellular uptake efficiencies of R8 and R12 were significantly enhanced due to their oligoarginine linkers compared to previous probes with oligoproline linkers. These ratiometric oxygen probes allowed visualization of the O2 gradient produced by placing a thin coverslip onto a HeLa cell monolayer.

High-Resolution Fluorescence Measurements Correlated to Cellular Traction Forces

Many cellular mechanosensory functions rely on molecular interactions related to cellular force exertion. Despite the rich biochemical knowledge in this field, the current mechanistic picture - that can be enhanced by high-resolution optical microscopy techniques - is still in its infancy. Here, we describe a simple yet universal approach to combine high-resolution optical microscopy techniques with traction force measurements. Etching 50 µm high spacers next to a micropillar array and inverting the array onto a thin coverslip before imaging enables the use of a short working distance objective. In our setup we use a spinning-disk confocal on an inverted microscope with a 100X, NA 1.4 objective. Our deflection detection resolution per micropillar is below 30 nm, corresponding to a force resolution of 500 pN. We validate our technique by imaging fixed and live mouse fibroblasts with dye-conjugated fibronectin micro-contact printed on the micropillars and immunostaining for paxillin and actin. We observe forces of up to 15 nN on individual pillars using a precise force-deflection relationship calibrated using scanning electron microscopy, tensile testing and finite element analysis. We quantified focal adhesion growth with increasing force of 4 ± 1 nN/µm2 and co-orientation of focal adhesion elongation and force direction within 2 ± 24 degrees. Furthermore, the cells remain viable during overnight live-cell imaging and stay adhered to the substrate. Our novel approach opens up the combined application of traction force measurements and super-resolution optical microscopy techniques.

FNOCT as a fluorescent probe for in vivo localization of nitric oxide distribution in tobacco roots

The nitric oxide-specific fluorescent probe Fluorescent Nitric Oxide Cheletropic Trap (FNOCT) 8a was applied to intact tobacco (Nicotiana tabacum cv. Samsun) roots to detect sites of nitric oxide formation and NO distribution. Three week old tobacco seedlings were gently removed from the sand culture pots with intact roots and transferred to small Petri dishes, whose base was replaced by a thin coverslip. Intact roots were subjected to FNOCT 8a to localize NO-dependent fluorescence in these roots; controls with an exogenous NO donor confirmed the presence and distribution of the probe in the roots. To confirm the NO-dependent fluorescence, roots were incubated with the three different NO scavengers cPTIO {2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-L-oxyl-3-oxide}, methylene blue and sodium diethyl dithiocarbamate (DCC) followed by incubation with FNOCT 8a. Methylene blue and DCC were able to completely quench NO-dependent fluorescence, cPTIO quenched partially. The roots were incubated in the presence of NaNO₂ and NaNO₃, which are substrates for nitrite:nitric oxide reductase (NI-NOR) and plasma membrane-bound nitrate reductase (PM-NR), respectively. The NO-dependent fluorescence was more or less same at the root tips upon treatment with NaNO₂, while the overall fluorescence was reduced in the presence of NaNO. Fluorescence from the living roots was visualized by inverted confocal laser scanning microscope (CLSM) using UV laser (excitation 360 nm and emission 408 nm). A specialized apparatus has been devised by the authors for analysis of intact roots as described in the methods section of this paper. Intact roots were chosen for microscopic observation rather than incised roots to avoid production of NO due to stress or physical injury.

An Improved Chamber for Direct Visualisation of Chemotaxis

There has been a growing appreciation over the last decade that chemotaxis plays an important role in cancer migration, invasion and metastasis. Research into the field of cancer cell chemotaxis is still in its infancy and traditional investigative tools have been developed with other cell types and purposes in mind. Direct visualisation chambers are considered the gold standard for investigating the behaviour of cells migrating in a chemotactic gradient. We therefore drew up a list of key attributes that a chemotaxis chamber should have for investigating cancer cell chemotaxis. These include (1) compatibility with thin cover slips for optimal optical properties and to allow use of high numerical aperture (NA) oil immersion objectives; (2) gradients that are relatively stable for at least 24 hours due to the slow migration of cancer cells; (3) gradients of different steepnesses in a single experiment, with defined, consistent directions to avoid the need for complicated analysis; and (4) simple handling and disposability for use with medical samples. Here we describe and characterise the Insall chamber, a novel direct visualisation chamber. We use it to show GFP-lifeact transfected MV3 melanoma cells chemotaxing using a 60x high NA oil immersion objective, which cannot usually be done with other chemotaxis chambers. Linear gradients gave very efficient chemotaxis, contradicting earlier results suggesting that only polynomial gradients were effective. In conclusion, the chamber satisfies our design criteria, most importantly allowing high NA oil immersion microscopy to track chemotaxing cancer cells in detail over 24 hours.

A dielectrophoretic chip with a roughened metal surface for on-chip surface-enhanced Raman scattering analysis of bacteria.

We present an analysis of the results of in situ surface-enhanced Raman scattering (SERS) of bacteria using a microfluidic chip capable of continuously sorting and concentrating bacteria via three-dimensional dielectrophoresis (DEP). Microchannels were made by sandwiching DEP microelectrodes between two glass slides. Avoiding the use of a metal nanoparticle suspension, a roughened metal surface is integrated into the DEP-based microfluidic chip for on-chip SERS detection of bacteria. On the upper surface of the slide, a roughened metal shelter was settled in front of the DEP concentrator to enhance Raman scattering. Similarly, an electrode-patterned bottom layer fabricated on a thin cover-slip was used to reduce fluorescence noise from the glass substrate. Gram positive (Staphylococcus aureus) and Gram negative (Pseudomonas aeruginosa) bacteria were effectively distinguished in the SERS spectral data. Staphylococcus aureus (concentration of 10(6) CFUml) was continuously separated and concentrated via DEP out of a sample of blood cells. At a flow rate of 1 mulmin, the bacteria were highly concentrated at the roughened surface and ready for on-chip SERS analysis within 3 min. The SERS data were successfully amplified by one order of magnitude and analyzed within a few minutes, resulting in the detection of signature peaks of the respective bacteria.

Novel anti-cholesterol monoclonal immunoglobulin G antibodies as probes and potential modulators of membrane raft-dependent immune functions

Natural autoantibodies against cholesterol are present in the sera of all healthy individuals; their function, production, and regulation, however, are still unclear. Here, we managed to produce two monoclonal anti-cholesterol antibodies (ACHAs) by immunizing mice with cholesterol-rich liposomes. The new ACHAs were specific to cholesterol and to some structurally closely related 3beta-hydroxyl sterols, and they reacted with human lipoproteins VLDL, LDL, and HDL. They bound, usually with low avidity, to live human or murine lymphocyte and monocyte-macrophage cell lines, which was enhanced substantially by a moderate papain digestion of the cell surface, removing some protruding extracellular protein domains. Cell-bound ACHAs strongly colocalized with markers of cholesterol-rich lipid rafts and caveolae at the cell surface and intracellularly with markers of the endoplasmic reticulum and Golgi complex. These data suggest that these IgG ACHAs may serve as probes of clustered cholesterol (e.g., different lipid rafts) in live cells and thus may also have immunomodulatory potential.

Mechanical properties of very thin cover slip glass disk

The biaxial flexural strength, Young’s modulus, Vicker’s microhardness and fracture toughness data for very thin, commercial, soda-lime-silica cover slip glass (diameter, D-18 mm, thickness, T-0.3 mm; T/D ≈ 0.02) are reported here. The ball on ring biaxial flexure tests were conducted at room temperature as a function of the support ring diameter (≈ 10–20 mm) and cross head speed (0.1–10 mm min−1). In addition, the Weibull modulus data were also determined. The Young’s modulus data was measured using a linear variable differential transformer (LVDT) from biaxial flexure tests and was checked out to be comparable to the data obtained independently from the ultrasonic time of flight measurement using a 15 MHz transducer. The microhardness data was obtained for the applied load range of 0.1–20 N. The fracture toughnessK IC data was obtained by the indentation technique at an applied load of 20 N.

A simple test for demonstrating erythrocytic inclusions

In paediatric practice the diagnosis of the ‘absent spleen syndrome' is particularly important in a neonate when associated with congenital cyanotic heart disease, as this finding may militate against any planned surgery. The customary inclusion body stains as well as search for and enumeration of Howell-Jolly bodies is often not sufficiently informative. The test consisted of examination of a dry or alcohol-fixed blood film with a standard microscope. A thin cover-slip is placed over the preparation, with a drop of immersion oil on the cover-slip, and a suitable field brought into view. Next, an inverted × 10 eyepiece is placed beneath the sub-stage condenser, and the eyepiece moved around until the erythrocytes attain a distinct shadowing effect. Next, the sub-stage condenser diaphragm is partially closed and the pits or vesicles may then be seen in approximately 40% of cells in neonates with asplenia. Similar inclusions may be seen in subjects with thalassaemia major after splenectomy. In patients with hereditary spherocytosis after splenectomy the number of pits and vesicles is somewhat less. Inclusion bodies in children with a syndrome of haemolysis associated with prematurity and in subjects with drug-induced haemoiytic anaemia may also be seen with the technique. In such situations pits and vesicles occur in less than 10% of red blood cells.

The irreversibly sickled cell; a determinant of haemolysis in sickle cell anaemia.

Summary. Red cell survival was estimated in a group of adult sickle cell anaemia patients using the 51Cr technique. Counts of irreversibly sickled cells were performed on thin coverslip preparations of capillary blood. These counts are relatively constant within the same individual but show marked variations between individuals. A significant correlation has been demonstrated between the irreversibly sickled cell count and the red cell survival. This correlation does not apply to sickle cell anaemia patients with splenomegaly in whom the irreversibly sickled cell count is always low.

Spermatid Transformation in Orchelimum Nigripes (De Geer)

INTRODUCTION This study gives a detailed account of the metamorphosis of the spermatids in Orchelimum nigripes (De Geer), using only living material. A considerable amount of work has been done on the spermatogenesis and the spermiogenesis of the Orchelimum nigripes using stained material, but in this paper all of the drawings and observations were made upon living tissue. No doubt the earlier reading of the literature did help in recognizing the structures; but the earlier drawings were not again referred to until these were completed. MATERIAL AND METHODS The specimens were collected near Lawrence, Kansas during the later part of the summer; additional specimens were collected near Fredonia, Kansas during the fall. All the material was obtained from the adult individuals. The parts to be examined were removed from the animal and placed on a thin cover slip with a small drop of Belar's nutrient solution. This cover slip was inverted over a hollow-ground slide and the edges of the cover slip were sealed with melted vaseline to prevent any evaporation of the nutrient solution. The mounts were studied with low and high power objectives; and in some cases, the oil immersion was used.

Quantitative Use of Neufeld Reaction with Special Reference to Titration of Type II Antipneumococcic Horse Sera

The Neufeld reaction has recently been advocated for the diagnosis of the different types of pneumonia directly from the patient's sputum. For this purpose, he advised a monovalent antipneumococcic rabbit serum in order to avoid the non-specific reactions which he obtained with the use of antipneumococcic horse serum. More recently, Cooper and Walter reported this same “swelling” phenomenon when antipneumococcic serum from rabbits was mixed with the homologous pure culture grown in artificial media. This observation led us to examine different samples of antipneumococcic serum by mixing them with specific pure cultures and observing the degree of capsular swelling which resulted. It was found that a serum high in mouse protective units caused much more swelling than a serum low in mouse protective units, also that a serum of high potency could be diluted many times and still give a typical “swelling” reaction. These experiments suggested a possible quantitative application of the Neufeld reaction. Preliminary work with Type II broth cultures and specific antipneumococcus horse sera showed a definite linear relationship between the number of organisms used and the least amount of antibody required to produce an enlarged capsule. This reaction appeared to be specific. The following method of titration of Type II antibody was then developed: The culture, either live or formalinized, is diluted with 1% peptone to the required density; the serum is diluted with physiological saline solution. Appropriate amounts of a constant culture dilution and of varying serum dilutions are measured with standard platinum loops, placed on a thin cover-slip, mixed well with a loopful of methylene blue solution, and inverted on a microscope slide. These preparations are incubated 30 minutes in a moist chamber at 37°C., and then examined under a fluorite oil-immersion lens.