CryoEM at 100†keV: a demonstration and prospects.

100 kV is investigated as the operating voltage for single-particle electron cryomicroscopy (cryoEM). Reducing the electron energy from the current standard of 300 or 200 keV offers both cost savings and potentially improved imaging. The latter follows from recent measurements of radiation damage to biological specimens by high-energy electrons, which show that at lower energies there is an increased amount of information available per unit damage. For frozen hydrated specimens around 300 Šin thickness, the predicted optimal electron energy for imaging is 100 keV. Currently available electron cryomicroscopes in the 100-120 keV range are not optimized for cryoEM as they lack both the spatially coherent illumination needed for the high defocus used in cryoEM and imaging detectors optimized for 100 keV electrons. To demonstrate the potential of imaging at 100 kV, the voltage of a standard, commercial 200 kV field-emission gun (FEG) microscope was reduced to 100 kV and a side-entry cryoholder was used. As high-efficiency, large-area cameras are not currently available for 100 keV electrons, a commercial hybrid pixel camera designed for X-ray detection was attached to the camera chamber and was used for low-dose data collection. Using this configuration, five single-particle specimens were imaged: hepatitis B virus capsid, bacterial 70S ribosome, catalase, DNA protection during starvation protein and haemoglobin, ranging in size from 4.5 MDa to 64 kDa with corresponding diameters from 320 to 72 Å. These five data sets were used to reconstruct 3D structures with resolutions between 8.4 and 3.4 Å. Based on this work, the practical advantages and current technological limitations to single-particle cryoEM at 100 keV are considered. These results are also discussed in the context of future microscope development towards the goal of rapid, simple and widely available structure determination of any purified biological specimen.

Digitisation of electron microscope films: six useful tests applied to three film scanners.

A series of simple tests have been used to measure the performance of flat-bed film scanners suitable for digitisation of electron micrographs. Two of the film scanners evaluated are commercially available and one has been constructed in the laboratory paying special attention to the needs of the electron microscopist. The tests may be useful for others.

Efficacy of supplementary intracameral lidocaine in routine phacoemulsification under topical anesthesia.

OBJECTIVE: To determine whether the routine use of supplementary intracameral lidocaine has any benefit over topical anesthesia alone when performing phacoemulsification surgery. DESIGN: A prospective single-center, randomized, double-masked, clinical trial. PARTICIPANTS: A total of 204 patients undergoing phacoemulsification surgery with lens implantation under planned topical anesthesia. METHODS: Patients were randomly allocated to receive either topical anesthesia plus 0.5 ml intracameral balanced salt solution or topical anesthesia plus 0.5 ml preservative-free 1% intracameral lidocaine. MAIN OUTCOME MEASURES: On the day after surgery, patients were asked to document the discomfort they had experienced using a visual analog scale. Intraoperative discomfort, postoperative discomfort, and discomfort caused by the microscope light were assessed. RESULTS: Multiple linear regression analysis did not show any significant relationship between the use of intracameral lidocaine and either intraoperative (P = 0.34) or postoperative (P = 0.45) pain scores. There was a small reduction in the discomfort caused by the operating microscope when intracameral lidocaine was used (P = 0.04). CONCLUSIONS: In this study, the routine use of intracameral lidocaine as a supplement to topical anesthesia was shown not to have a clinically useful role.

Can convulsions alone cause retinal haemorrhages in infants?

AIM: To evaluate the likelihood that, in children under the age of 2 years, convulsions alone may cause retinal haemorrhages. METHODS: Children under the age of 2 years admitted to hospital following convulsions, were examined within 48 hours of admission. The convulsions were classified by a paediatric neurologist and detailed ocular examination, including indirect ophthalmoscopy, was performed by an ophthalmologist. Statistical analysis was undertaken using Hanley's rule of three. RESULTS: 32 consecutive children admitted with convulsions were examined; 10 of them were admitted following epileptic seizures and 22 following febrile convulsions. Two of the children with febrile convulsions were admitted in status epilepticus. None of these children had retinal haemorrhages. Therefore, using Hanley's rule of three, the upper limit of 95% confidence interval of retinal haemorrhages following convulsions in children under the age of 2 years, is less than 10/100. CONCLUSIONS: In children under the age of 2 years convulsions alone are unlikely to cause retinal haemorrhages. By combining the results of this study with those previously reported from this unit in older children, the upper limit of 95% confidence interval of retinal haemorrhages, following convulsions in children under the age of 14 years, is less than 5/100. Therefore, the finding of retinal haemorrhages in a child admitted with a history of convulsion should trigger a meticulous search for other causes of these haemorrhages, particularly non-accidental injury.

Inferior rectus recession--an effective procedure?

AIMS: To examine the postoperative stability of inferior rectus recession, with particular reference to the incidence of progressive overcorrection. METHODS: The results of consecutive patients undergoing inferior rectus recession over a 3 year period were reviewed. RESULTS: 21 patients underwent inferior rectus recession, using an adjustable suture technique in all but three cases. In 16 patients additional vertical muscle surgery was performed at the time of the inferior rectus recession. All patients were followed for a minimum of 3 months postoperatively, with a mean follow up of 9.3 months. At the final postoperative visit 11 patients were well aligned, eight were undercorrected, and two were overcorrected. In five of the eight undercorrected cases, the residual deviation was the result of postoperative drift in the direction of the preoperative deviation, following an initially good alignment. Review of the results failed to reveal any factor predictive for this postoperative drift. CONCLUSION: The risk of postoperative overcorrection following inferior rectus recession should be considered, but in this study, undercorrection occurred more frequently than overcorrection. The possible reasons for overcorrection and undercorrection are discussed.

Pulfrich's phenomenon in unilateral cataract.

AIMS: To determine whether unilateral cataract causes a pathological Pulfrich's phenomenon. METHODS: 29 subjects with unilateral cataract and contralateral pseudophakia were assessed on their ability to perceive the Pulfrich phenomenon. Using a computer generated pendulum image, and graded neutral density filters, a series of forced choice trials were performed in which the subject was required to describe the direction of any apparent pendulum rotation. A pathological Pulfrich effect was said to occur when apparent rotation was perceived in the presence of a zero strength neutral density filter. The size of any pathological Pulfrich effect which was present was quantified by neutralising the perceived pendulum rotation with neutral density filters of varying strength placed before the better seeing eye. RESULTS: 20 out of 29 subjects were able to perceive apparent pendulum rotation when uniocular filtering was performed. In the group (n = 12) which was tested both before and after cataract extraction with intraocular lens implantation, a statistically significant pathological Pulfrich effect was demonstrated preoperatively, compared with a group of normal control subjects. This effect was abolished after cataract extraction (p = 0.009). The median size of the effect was equivalent to a 0.25 log unit neutral density filter over the non-cataractous eye. The subjects who were unable to perceive the Pulfrich phenomenon at all had a significantly greater difference in the visual acuity of each eye (p = 0.045) and significantly worse stereoacuity than those who were able to perceive the effect (p = 0.002). CONCLUSIONS: Unilateral cataract can cause a pathological Pulfrich phenomenon. This finding may explain why some patients with unilateral cataract complain of visual symptoms that are not easily accounted for in terms of visual acuity, contrast sensitivity, or stereoacuity.