Transmitted Light Techniques Research Articles

A Confocal Reflection Super-Resolution Technique to Image Golgi-Cox Stained Neurons.

Metal-based Golgi-Cox (GC) staining is an established method used to visualise neurons with great morphological detail. Although GC stained samples are imaged routinely under transmitted light microscopy, this method is unable to yield information on the three-dimensional structure of dendrites and neurons and thus help reveal the connective properties of the central nervous system. Although a few studies have attempted simultaneous visualisation of GC staining and antigen-specific fluorescent labelling under a confocal reflection technique, the resolution of both confocal reflection and fluorescence modalities used to acquire GC reflection and fluorescently stained antibody signals are still limited by the diffraction limit of light at about 220 nm. Here, we report a confocal reflection super-resolution technique (CRSR) to break this diffraction barrier, which is achieved by minimising the pinhole size from 1 airy unit (AU) to 0.1 AU. This is achieved by minimising or closing the confocal pinhole size and is possible in this reflection modality, unlike fluorescence, because it is not a photon limited technique. Utilising the lowest wavelength of light available in the system (405 nm), the CRSR technique results in ∼30% lateral and axial resolution improvement. We also show that the CRSR technique can be used in conjunction to visualise both GC and immunofluorescence targets to create precise and improved three-dimensional visualisation and analysis. In addition, using these superresolution confocal reflection data sets from GC in CRSR mode significantly reduced the data overestimation, improving the accuracy of statistical analysis of dendritic spine density and average spine dimensions. Combining the 0.1 AU setting with deconvolution routines, the signal-to-noise ratio and resolution could further be improved an additional ∼20-25%, yielding CRSR images with resolutions up to 2-fold over the diffraction limit both laterally and axially. The improved precision of both visualisation and quantification of subdiffraction limited dendritic spines using the CRSR technique may prove to be critical in investigations that concern changes in detailed neuron morphology under central nervous system disease conditions such as multiple sclerosis and Alzheimer's disease. LAY DESCRIPTION: For over a century, Golgi-Cox (GC) has been a leading staining technique in the field of neuroscience, used to visualise neurons with great morphological detail. GC stained brain or spinal cord samples are conventionally visualised under transmitted light techniques. This limits the view of Golgi-staining to a two-dimensional image. A recent report showed that Golgi staining can be visualised in three-dimensions using the reflection modality of the confocal microscope. This visualisation also allows for the simultaneous acquisition of immunofluorescence signals. However, the reported resolution of Golgi staining confocal reflection is limited by the diffraction limit of light, which is around 220 nm. Here, we report a superresolution confocal reflection technique (CRSR) that achieves superresolution by minimising the pinhole size used in confocal microscopy. The CRSR technique results in ∼30% lateral and axial resolution improvement. Adding a deconvolution step in the final processing could improve the SNR and resolution even further up to 2-fold improvement in resolution over the diffraction limit both laterally and axially. We hope that this improved visualisation will help in investigations that concern changes in detailed neuron morphology under central nervous system disease conditions such as multiple sclerosis and Alzheimer's disease.

Experimental study of the onset of downstream motion of adhering droplets in turbulent shear flows

In this study, the dynamics of adhering liquid droplets on various solid surfaces in shear flow, which is driven by a controlled air flow, are investigated experimentally. A series of experiments are carried out to understand the effects of fluid properties, wetting characteristics, droplet sizes and flow velocities. A rectangular Plexiglas-channel is used for the experiments. Droplets are placed on the bottom wall of that channel. The droplet shape and contour position are measured by the transmitted light technique and are further analyzed by using image processing. The shear flow leads to a deformation and oscillation of the droplet and eventually to a movement downstream. Three regimes are identified: regime I corresponds to a deformation and oscillation, whereas in regime II the deformed and still oscillating droplets moves downstream. Regime III characterizes a continuous downstream movement in a gliding manner. The start of regime II, i.e. the onset of downstream motion is defined by a critical air velocity. Results show that the contact angle hysteresis and surface energy have a strong influence on the critical air velocity for the onset of droplet motion, as well as the wetting characteristics and droplet volume. To find a global empirical law for the critical velocity, a dimensionless approach is derived based on the droplet Reynolds number and a modified Laplace number. The empirical law is in good agreement with experimental data and may serve as an estimator of the critical velocity for the onset of droplet motion.

Overview of Live‐Cell Imaging: Requirements and Methods Used

Overview of Live‐Cell Imaging: Requirements and Methods Used

Light transmission from a twisted nematic liquid crystal: accurate methods to measure the azimuthal anchoring energy.

In this paper, we analyze the light transmission from a twisted nematic liquid crystal (NLC) and we propose two accurate and very direct optical methods to measure the azimuthal anchoring energy. In both of them, a monochromatic beam of wavelength lambda with a polarization vector that rotates at an angular frequency omega impinges on a twisted nematic liquid crystal. The intensity of the transmitted beam is modulated at angular frequency 2omega with a phase shift beta, which is related to the surface azimuthal director angle phi(1) at the investigated interface. It is shown that there exists a special geometry where the simple adiabatic relation phi(1)=beta/2 is satisfied up to second order in the small perturbative parameter alpha=lambda/(2piDelta(n)xi), where Delta(n) is the anisotropy of the refractive indices of the NLC and xi is the twist distortion length. Furthermore, the small residual higher order correction terms can be greatly reduced by choosing a proper geometry for the experiment. With this choice, the range of validity of the adiabatic theory is greatly extended. The perturbative theoretical results are fully confirmed by numerical calculations. The azimuthal anchoring energy coefficient can be obtained by measuring phase shift beta versus the intensity of an applied magnetic field. These methods greatly improve the accuracy of the previous transmitted light techniques and also provide accurate measurements of strong azimuthal anchoring energies.

THE USE OF INTERFERENCE REFLECTION CONTRAST IN THE EXAMINATION OF DIATOM VALVES.

DIC, differential interference contrast; IRC, interference reflection contrast; LM, light microscopy.

An investigation into the effects of midazolam and propofol on human respiratory cilia beat frequency in vitro.

Patients in intensive care are known to be prone to both upper and lower respiratory tract infection. Respiratory mucus forms a barrier to infection. Mucus transport rate (MTR) depends upon both the physical properties of mucus and the action of respiratory cilia. Patients undergoing anaesthesia are known to have a reduced MTR that may be related to a depressant effect on cilia beat frequency (CBF) by anaesthetic drugs. The aim of this study was to investigate the effects of two commonly used intensive care sedative agents, midazolam and propofol, on CBF using human nasal turbinate explants in vitro. We exposed ciliated tissue from human nasal turbinate explants to midazolam and propofol in supraclinical concentrations (20 microM midazolam and 70 microM propofol) in a controlled and blinded manner for 90 min and measured CBF by the transmitted light technique. After 90 min, mean (SEM) CBF in the group exposed to midazolam and its control group were 13.0 (0.2) Hz and 12.9 (0.3) Hz, respectively. Mean (SEM) CBF in the group exposed to propofol was 13.6 (0.4) Hz and in the control group the value was 12.0 (0.6) Hz. There was no significant change in CBF (midazolam: p = 0.21, propofol: p = 0.31, MANOVA for repeated measures). We have found no effect of midazolam or propofol in supra-clinical concentrations upon CBF in human turbinate explants after a 90-min exposure. This contrasts with previous work that has shown a depressant effect of inhalational anaesthetic agents on CBF.

Effects of morphine on human nasal cilia beat frequency in vitro

Using human nasal cytological brushings, we have investigated the effects of morphine on ciliary function by measurement of cilia beat frequency in vitro, and we have also determined opioid receptor binding in these specimens. We obtained ciliated samples from seven volunteers, and measured cilia beat frequency using the transmitted light technique during exposure to morphine 10 mumol litre-1 for 4 h. Mean cilia beat frequency of the samples exposed to morphine was 11.1 (95% confidence interval 10.9-11.5) Hz and that of the controls 11.3 (11.1-11.7) Hz. There was no significant effect of morphine on human cilia beat frequency in vitro (MANOVA for repeated measures and nested, F = 0.61, P = 0.66). In a separate study, we obtained nasal brushings from 20 patients and measured the binding of the opioid antagonist tritiated diprenorphine ([3H]DPN). Mean disintegrations per minute (dpm) for total and non-specific binding were 9036 (8105-9967) dpm and 9130 (8054-10206) dpm, respectively. These values did not differ significantly (paired t test, t = 0.22, P = 0.83). We conclude that morphine had no effect on cilia beat frequency in vitro and we were unable to demonstrate any significant numbers of opioid receptors on nasal ciliated epithelium.

Effects of 3 MAC of halothane, enflurane and isoflurane on cilia beat frequency of human nasal epithelium in vitro

We have measured the effects of three times the minimum alveolar concentration (MAC) of halothane, enflurane and isoflurane on cilia beat frequency of human nasal epithelial brushings from 18 healthy adult patients. Using the transmitted light technique and paired perfusion chambers, the cilia were exposed to 2.25% halothane, 5% enflurane or 3.6% isoflurane in air, or air alone, in a controlled and blinded manner. Over a 4-h observation period, cilia beat frequency of the samples exposed to inhalation anaesthetic agents demonstrated a significant reduction in frequency compared with controls exposed to air alone. Mean cilia beat frequency for the samples exposed to halothane was 9.3 (SEM 1.3) compared with its controls of 11.4 (1.0); for the samples exposed to enflurane, 10.9 (1.3) compared with its controls of 11.6 (1.2); and for the samples exposed to isoflurane, 10.8 (1.1) compared with its controls of 11.6 (1.2). There was a statistically significant difference between the samples exposed to all three volatile agents and their associated controls (halothane, P = 0.01; enflurane, P = 0.03; isoflurane, P = 0.01; nested repeated measures analysis of variance utilizing polynomial contrasts).

Effect of halothane on cilia beat frequency of ciliated human respiratory epithelium in vitro

The effect of halothane on human ciliated nasal epithelium was studied in vitro. Samples from 24 healthy adult volunteers were exposed to halothane in varying concentrations and cilia beat frequency was measured using the transmitted light technique. Mean cilia beat frequency was measured at 30-min intervals. There was a significant decrease in cilia beat frequency at 2 h in samples that were exposed to halothane (mean 8.4 (SD 2.5) Hz, 9.18 (2.6) Hz and 6.99 (4.9) Hz) compared with air (10.8 (2.7) Hz, 11.6 (2.1) Hz and 12.1 (2.3) Hz) (P < 0.01). The coefficient of variation of cilia beat frequency measurements increased after exposure to halothane. There was no change in the cilia beat frequency of controls exposed to air over a 3-h period.

Cryopreservation of canine trachea: Functional and histological changes

The purpose of this study was to evaluate tracheal viability and to document histological changes in an autograft implanted in the abdominal wall after a 1-week period of cryopreservation. A 5-cm segment of cervical trachea was resected in 6 dogs. One-half of the segment was cryopreserved and stored in liquid nitrogen at −196 °C for 1 week. The other half was immediately implanted in an abdominal pouch fashioned from the rectus abdominis muscle (control). One week later, the cryopreserved segment was thawed and then implanted in a similar contralateral muscular pouch. Five weeks later, the control and cryopreserved autografts were removed and compared with the in situ trachea. Ciliary beat frequency was assessed by transmitted light technique. Histology was evaluated by light microscopy. Gross anatomy and mucus production were maintained after cryopreservation. Histologically the cryopreserved segment displayed both normal epithelium and smooth muscle cells, but the cartilage was abnormal as characterized by empty lacunae. Mean ciliary beat frequency of in situ, control, and cryopreserved segments was 13.3 ± 1.8, 13.5 ± 1.5, and 13.3 ± 1.1 beats per second (± the standard deviation), respectively. We conclude that smooth muscle, epithelium with mucus production, and ciliary function were retained after cryopreservation and reimplantation. Histological changes, however, were suggestive of early cartilage ischemia. These findings support further evaluation of cryopreserved canine tracheal grafts.