Label-free proteomics identifies Calreticulin and GRP75/Mortalin as peripherally accessible protein biomarkers for spinal muscular atrophy

BACKGROUND: Spinal muscular atrophy (SMA) is a neuromuscular disease resulting from mutations in the survival motor neuron 1 (SMN1) gene. Recent breakthroughs in preclinical research have highlighted several potential novel therapies for SMA, increasing the need for robust and sensitive clinical trial platforms for evaluating their effectiveness in human patient cohorts. Given that most clinical trials for SMA are likely to involve young children, there is a need for validated molecular biomarkers to assist with monitoring disease progression and establishing the effectiveness of therapies being tested. Proteomics technologies have recently been highlighted as a potentially powerful tool for such biomarker discovery. METHODS: We utilized label-free proteomics to identify individual proteins in pathologically-affected skeletal muscle from SMA mice that report directly on disease status. Quantitative fluorescent western blotting was then used to assess whether protein biomarkers were robustly changed in muscle, skin and blood from another mouse model of SMA, as well as in a small cohort of human SMA patient muscle biopsies. RESULTS: By comparing the protein composition of skeletal muscle in SMA mice at a pre-symptomatic time-point with the muscle proteome at a late-symptomatic time-point we identified increased expression of both Calreticulin and GRP75/Mortalin as robust indicators of disease progression in SMA mice. We report that these protein biomarkers were consistently modified in different mouse models of SMA, as well as across multiple skeletal muscles, and were also measurable in skin biopsies. Furthermore, Calreticulin and GRP75/Mortalin were measurable in muscle biopsy samples from human SMA patients. CONCLUSIONS: We conclude that label-free proteomics technology provides a powerful platform for biomarker identification in SMA, revealing Calreticulin and GRP75/Mortalin as peripherally accessible protein biomarkers capable of reporting on disease progression in samples of muscle and skin

Cavity-enhanced photoionization of an ultracold rubidium beam for application in focused ion beams

A two-step photoionization strategy of an ultracold rubidium beam for application in a focused ion beam instrument is analyzed and implemented. In this strategy the atomic beam is partly selected with an aperture after which the transmitted atoms are ionized in the overlap of a tightly cylindrically focused excitation laser beam and an ionization laser beam whose power is enhanced in a build-up cavity. The advantage of this strategy, as compared to without the use of a build-up cavity, is that higher ionization degrees can be reached at higher currents. Optical Bloch equations including the photoionization process are used to calculate what ionization degree and ionization position distribution can be reached. Furthermore, the ionization strategy is tested on an ultracold beam of $^{85}$Rb atoms. The beam current is measured as a function of the excitation and ionization laser beam intensity and the selection aperture size. Although details are different, the global trends of the measurements agree well with the calculation. With a selection aperture diameter of 52 $\mu$m, a current of $\left(170\pm4\right)$ pA is measured, which according to calculations is 63% of the current equivalent of the transmitted atomic flux. Taking into account the ionization degree the ion beam peak reduced brightness is estimated at $1\times10^7$ A/(m$^2\,$sr$\,$eV).Comment: 13 pages, 9 figure

High quality ultrafast transmission electron microscopy using resonant microwave cavities

Ultrashort, low-emittance electron pulses can be created at a high repetition rate by using a TM$_{110}$ deflection cavity to sweep a continuous beam across an aperture. These pulses can be used for time-resolved electron microscopy with atomic spatial and temporal resolution at relatively large average currents. In order to demonstrate this, a cavity has been inserted in a transmission electron microscope, and picosecond pulses have been created. No significant increase of either emittance or energy spread has been measured for these pulses. At a peak current of $814\pm2$ pA, the root-mean-square transverse normalized emittance of the electron pulses is $\varepsilon_{n,x}=(2.7\pm0.1)\cdot 10^{-12}$ m rad in the direction parallel to the streak of the cavity, and $\varepsilon_{n,y}=(2.5\pm0.1)\cdot 10^{-12}$ m rad in the perpendicular direction for pulses with a pulse length of 1.1-1.3 ps. Under the same conditions, the emittance of the continuous beam is $\varepsilon_{n,x}=\varepsilon_{n,y}=(2.5\pm0.1)\cdot 10^{-12}$ m rad. Furthermore, for both the pulsed and the continuous beam a full width at half maximum energy spread of $0.95\pm0.05$ eV has been measured

Gigahertz repetition rate thermionic electron gun concept

We present a novel concept for the generation of gigahertz repetition rate high brightness electron bunches. A custom design 100 kV thermionic gun provides a continuous electron beam, with the current determined by the filament size and temperature. A 1 GHz rectangular RF cavity deflects the beam across a knife-edge, creating a pulsed beam. Adding a higher harmonic mode to this cavity results in a flattened magnetic field profile which increases the duty cycle to 30%. Finally, a compression cavity induces a negative longitudinal velocity-time chirp in a bunch, initiating ballistic compression. Adding a higher harmonic mode to this cavity increases the linearity of this chirp and thus decreases the final bunch length. Charged particle simulations show that with a 0.15 mm radius LaB6 filament held at 1760 K, this method can create 279 fs, 3.0 pC electron bunches with a radial rms core emittance of 0.089 mm mrad at a repetition rate of 1 GHz.Comment: 12 pages, 12 figure

Design and experimental validation of a compact collimated Knudsen source

In this paper we discuss the design and performance of a collimated Knudsen source which has the benefit of a simple design over recirculating sources. Measurements of the flux, transverse velocity distribution and brightness at different temperatures were conducted to evaluate the performance. The scaling of the flux and brightness with the source temperature follow the theoretical predictions. The transverse velocity distribution in the transparent operation regime also agrees with the simulated data. The source was found able to produce a flux of $10^{14}$ s$^{-1}$ at a temperature of 433 K. Furthermore the transverse reduced brightness of an ion beam with equal properties as the atomic beam reads $1.7 \times 10^2$ A/(m${}^2$ sr eV) which is sufficient for our goal: the creation of an ultra-cold ion beam by ionization of a laser-cooled and compressed atomic rubidium beam

Theory and particle tracking simulations of a resonant radiofrequency deflection cavity in TM110_{110} mode for ultrafast electron microscopy

We present a theoretical description of resonant radiofrequency (RF) deflecting cavities in TM$_{110}$ mode as dynamic optical elements for ultrafast electron microscopy. We first derive the optical transfer matrix of an ideal pillbox cavity and use a Courant-Snyder formalism to calculate the 6D phase space propagation of a Gaussian electron distribution through the cavity. We derive closed, analytic expressions for the increase in transverse emittance and energy spread of the electron distribution. We demonstrate that for the special case of a beam focused in the center of the cavity, the low emittance and low energy spread of a high quality beam can be maintained, which allows high-repetition rate, ultrafast electron microscopy with 100 fs temporal resolution combined with the atomic resolution of a high-end TEM. This is confirmed by charged particle tracking simulations using a realistic cavity geometry, including fringe fields at the cavity entrance and exit apertures

Direct magneto-optical compression of an effusive atomic beam for high-resolution focused ion beam application

An atomic rubidium beam formed in a 70 mm long two-dimensional magneto-optical trap (2D MOT), directly loaded from a collimated Knudsen source, is analyzed using laser-induced fluorescence. The longitudinal velocity distribution, the transverse temperature and the flux of the atomic beam are reported. The equivalent transverse reduced brightness of an ion beam with similar properties as the atomic beam is calculated because the beam is developed to be photoionized and applied in a focused ion beam. In a single two-dimensional magneto-optical trapping step an equivalent transverse reduced brightness of $(1.0\substack{+0.8-0.4})$ $\times 10^6$ A/(m$^2$ sr eV) was achieved with a beam flux equivalent to $(0.6\substack{+0.3-0.2})$ nA. The temperature of the beam is further reduced with an optical molasses after the 2D MOT. This increased the equivalent brightness to $(6\substack{+5-2})$$\times 10^6$ A/(m$^2$ sr eV). For currents below 10 pA, for which disorder-induced heating can be suppressed, this number is also a good estimate of the ion beam brightness that can be expected. Such an ion beam brightness would be a six times improvement over the liquid metal ion source and could improve the resolution in focused ion beam nanofabrication.Comment: 10 pages, 8 figures, 1 tabl

Measurement of the temperature of an ultracold ion source using time-dependent electric fields

We report on a measurement of the characteristic temperature of an ultracold rubidium ion source, in which a cloud of laser-cooled atoms is converted to ions by photo-ionization. Extracted ion pulses are focused on a detector with a pulsed-field technique. The resulting experimental spot sizes are compared to particle-tracking simulations, from which a source temperature $T = (1 \pm 2)$ mK and the corresponding transversal reduced emittance $\epsilon_r = 7.9 X 10^{-9}$ m rad $\sqrt{\rm{eV}}$ are determined. We find that this result is likely limited by space charge forces even though the average number of ions per bunch is 0.022.Comment: 8 pages, 11 figure