The low-cost Shifter microscope stage transforms the speed and robustness of protein crystal harvesting

Despite the tremendous success of X-ray cryo-crystallography in recent decades, the transfer of crystals from the drops in which they are grown to diffractometer sample mounts remains a manual process in almost all laboratories. Here, the Shifter, a motorized, interactive microscope stage that transforms the entire crystal-mounting workflow from a rate-limiting manual activity to a controllable, high-throughput semi-automated process, is described. By combining the visual acuity and fine motor skills of humans with targeted hardware and software automation, it was possible to transform the speed and robustness of crystal mounting. Control software, triggered by the operator, manoeuvres crystallization plates beneath a clear protective cover, allowing the complete removal of film seals and thereby eliminating the tedium of repetitive seal cutting. The software, either upon request or working from an imported list, controls motors to position crystal drops under a hole in the cover for human mounting at a microscope. The software automatically captures experimental annotations for uploading to the user's data repository, removing the need for manual documentation. The Shifter facilitates mounting rates of 100–240 crystals per hour in a more controlled process than manual mounting, which greatly extends the lifetime of the drops and thus allows a dramatic increase in the number of crystals retrievable from any given drop without loss of X-ray diffraction quality. In 2015, the first in a series of three Shifter devices was deployed as part of the XChem fragment-screening facility at Diamond Light Source, where they have since facilitated the mounting of over 120 000 crystals. The Shifter was engineered to have a simple design, providing a device that could be readily commercialized and widely adopted owing to its low cost. The versatile hardware design allows use beyond fragment screening and protein crystallography.

Automated calibration of optoPlate LEDs to reduce light dose variation in optogenetic experiments

Optogenetic systems use light to precisely control and investigate cellular processes. Until recently, there had been few instruments available for applying controlled light doses to cultures of cells. The optoPlate, a programmable array of 192 LEDs, was developed to meet this need. However, LED performance varies, and without calibration there are substantial brightness differences between LEDs on an optoPlate. Here we present a method for calibrating an optoPlate that uses a programmable microscope stage and optical power meter to automatically measure all 192 LEDs of an optoPlate. The resulting brightness measurements are used to calculate calibration values that tune the electrical current supplied to each optoPlate LED to reduce brightness variation in optogenetic experiments.

Correlating cell function and morphology by performing fluorescent immunocytochemical staining on the light-microscope stage

ABSTRACTBackgroundCorrelation of fluorescence signals from functional changes in live cells with those from immunocytochemical indicators of their morphology following chemical fixation can be highly informative with regard to function-structure relationship. Such analyses can be technically challenging because they need consistently aligning the images between imaging sessions. Existing solutions include introducing artificial spatial landmarks and modifying the microscopes. However, these methods can require extensive changes to the experimental systems.New methodHere we introduce a simple approach for aligning images. It is based on two procedures: performing immunocytochemistry while a specimen stays on a microscope stage (on-stage), and aligning images using biological structures as landmarks after they are observed with transmitted-light optics in combination with fluorescence-filter sets.ResultsWe imaged a transient functional signal from a fluorescent Ca2+ indicator, and mapped it to neurites based on immunocytochemical staining of a structural marker. In the same preparation, we could identify presynaptically silent synapses, based on a lack of labeling with an indicator for synaptic vesicle recycling and on positive immunocytochemical staining for a structural marker of nerve terminals. On-stage immunocytochemistry minimized lateral translations and eliminated rotations, and transmitted-light images of neurites were sufficiently clear to enable spatial registration, effective at a single-pixel level.Comparison with existing methodsThis method aligned images with minimal change or investment in the experimental systems.ConclusionsThis method facilitates information retrieval across multiple imaging sessions, even when functional signals are transient or local, and when fluorescent signals in multiple imaging sessions do not match spatially.

Instantaneous tension measurements in droplet interface bilayers using an inexpensive, integrated pendant drop camera

Adding an inexpensive horizontal camera to a microscope stage yields faster, simpler, and more accurate measurements of droplet interface bilayers. Measurements of monolayer tension, bilayer tension, and specific capacitance are all improved.

Sufficient condition for reliability measurement of sample geometry by atomic force microscopy

A sufficient condition for determining the reliability of geometry measurements using atomic force microscopy for relatively small cantilever tilt angles is proposed. A relationship between the basic geometric parameters of surface roughness, geometric deviations of the probe, the angles of the cantilever and the inclination of the side faces of the probe, as well as the dimensions of the nonlocal point of the probable contact of its side faces with protrusions of roughness has been established. As a sufficient condition for the reliability of geometry measurements using atomic force microscopy, an obvious requirement is accepted. It determines the smallness of the ratio of the sizes of a nonlocal point to the distance between neighboring nonlocal points. Publications in which the measurement of surface nano-geometry of the samples does not indicate the roughness of the sample surface and the probe, the angles at the tip of the probe and the tilt of the cantilever, as well as the best resolution (smallest step) at which the study is carried out, cannot be accepted as reliable, because the results obtained in them are probabilistic in nature. The surface images obtained using atomic force microscopy without proper justification for the resolution (value of the measurement step) represent only a qualitative picture, on the basis of which it makes no sense to carry out any computational manipulations. In order to increase the reliability of measurements of surface geometry using atomic force microscopy, it is necessary to radically increase the accuracy of the manufacture of probes, as well as use probes with the smallest possible angle at the apex. In addition, it is necessary to make changes in the design of the atomic force microscopy. In particular, the automatic rotation of the microscope stage should be designed. It should provide closeness the probe axis direction to the normal to the average plane of the sample. This “integral” angle of rotation of the microscope stage is easily iteratively determined at the stage of preliminary investigation of the geometry of the surface of the sample. In this case, it will be necessary to geometrically increase the length of the cantilever so that the base extends beyond the limits of the sample.

The Low-Cost, Semi-Automated Shifter Microscope Stage Transforms Speed and Robustness of Manual Protein Crystal Harvesting

Despite the tremendous success of x-ray cryocrystallography over recent decades, the transfer of crystals from the drops where they grow to diffractometer sample mounts, remains a manual process in almost all laboratories. Here we describe the Shifter, a semi-automated microscope stage that offers an accessible and scalable approach to crystal mounting that exploits on the strengths of both humans and machines. The Shifter control software manoeuvres sample drops beneath a hole in a clear protective cover, for human mounting under a microscope. By allowing complete removal of film seals the tedium of cutting or removing the seal is eliminated. The control software also automatically captures experimental annotations for uploading to the user’s data repository, removing the overhead of manual documentation. The Shifter facilitates mounting rates of 100-240 crystals per hour, in a more controlled process than manual mounting, which greatly extends the lifetime of drops and thus allows for a dramatic increase in the number of crystals retrievable from any given drop, without loss of X-ray diffraction quality. In 2015 the first in a series of three Shifter devices was deployed as part of the XChem fragment screening facility at Diamond Light Source (DLS), where they have since facilitated the mounting of over 100,000 crystals. The Shifter was engineered to be simple, allowing for a low-cost device to be commercialised and thus potentially transformative as many research initiatives as possible.SynopsisA motorised X/Y microscope stage is presented that combines human fine motor control with machine automation and automated experiment documentation, to transform productivity in protein crystal harvesting.

Long-term in toto cell tracking using lightsheet microscopy of the zebrafish tailbud

In toto light-sheet imaging allows the tracking of entire growing tissues with high spatial and temporal resolution for many hours. However, this technology requires a sample to be immobilised to ensure that the tissue of interest remains within the field of view throughout the image acquisition period. We have developed a method of mounting and image capture for long-term light-sheet imaging of a growing zebrafish tailbud from the 18 somite stage through to the end of somitogenesis. By tracking the global movement of the tailbud during image acquisition and feeding this back to the microscope stage, we are able to ensure that the growing tissue remains within the field of view throughout image acquisition. Here, we present three representative datasets of embryos in which all nuclei are labelled and tracked until the completion of somitogenesis.