Postdoctoral positions in my lab

I recently got an R01 funded and so I am looking to hire two postdoctoral fellows to work on a project to develop novel bead-based biochemical assays. This project seeks to apply our recently developed technology for making spectrally encoded microspheres (see Gerver et al. Lab Chip 2012) as a substrate for developing novel assays for probing antibody reactivity in patient samples.
One postdoctoral fellow will head the assay development part of the project, and must have substantial experience in biology and or chemistry. Experience with peptide synthesis or antibody binding assays is a plus. The second postdoc will develop new microfluidic devices to automate these assays and must have substantial expertise in microfluidic device design and fabrication. For both positions, programming experience is desirable. Applicants must have a PhD or expect to receive their PhD by the end of the year.
This project is a close collaboration with Dr. Joseph DeRisi at UCSF and Dr. Polly Fordyce at Stanford University, and the individuals hired for these positions will work closely with both of these laboratories. In particular, the postdoc working on the microfluidic aspects of this project will be expect to spend part of his or her time at Stanford in the Fordyce lab.
To apply, please send your CV and names of three references to me: kurt.thorn@ucsf.edu.

Spinning disk upgrades: Evolve Delta and directly modulated lasers

We’ve recently undergone a round of upgrades to our spinning disk microscope that may be of interest to readers of this blog. For several years, we’ve had a CSU-22 spinning disk confocal with a four-line laser launch, an EMCCD, and a motorized XY stage with Piezo Z insert, all run through Micro-manager. This system has been a real workhorse – it’s routinely used 12-15 hours a day, and the time had come to make a few improvements to it.

First, we replaced the Evolve EMCCD we had with the new Evolve Delta (thanks to a generous loan from Photometrics). The Evolve Delta is a faster version of the Evolve, and is capable of running at 67 fps for the full 512 x 512 pixel field of view.

Second, we replaced our 488 and 561 nm lasers with new OBIS lasers from Coherent, which can be directly modulated at high speed. This enabled us to eliminate the AOTF from our laser launch and control the lasers directly, increasing brightness and maximizing laser lifetime (because the lasers are only on when we are acquiring data). The lasers are driven with a driver board from ES|Imaging which converts the AOTF connector from our controller to low impedance digital signals that can be directly connected to the lasers. I had fun with some scrap plastic and laser cut a rainbow enclosure for it.

DriverBox

Laser cut enclosure for the laser driver board.

The resulting system allows both the lasers and the piezo Z-stage to be triggered from the camera so that the entire system can run at the full frame rate of the camera. This means that with our new Evolve Delta, we can acquire multicolor Z-stacks at 67 fps. When doing this, we don’t switch emission filters; we just use a single multipass emission filter and choose the channel being imaged by the laser line exciting the sample. A schematic of the trigger cabling is shown below.

SD-triggering

Schematic of the trigger cabling. The laser and piezo controller is a custom card but could be replaced with ESio controllers. There are also analog connections to the lasers to set power, but these are omitted for clarity.

One of the beautiful things about Micro-manager is that it will run acquisitions in triggered mode whenever possible, without any action on the part of the user. On this system, the only requirements for triggered acquisition is to use the correct channel setting (so that the multipass filter is used and no filter wheels have to move) and to have the same exposure time in all channels. Photometrics is working to relax this last requirement with a feature they call SMART streaming; once this is implemented in the Micro-manager driver we should be able to run triggered acquisitions with different exposure times in each channel.

Overall, I’m very pleased with the system. The Evolve Delta performs great and the system is really fast – we were able to acquire a 3 channel Z stack (64 slices) in just under 3 seconds.  Eliminating the AOTF improved our light throughput by about 30% and eliminated one component to align. Finally, with the lasers now only on when the sample is being illuminated, we expect the lasers to last much longer.

A movie about Klaus Kemp, diatom slide maker

We have long used diatom slides as nice test samples and resolution targets for brightfield microscopy. The diatoms have silica shells with regularly spaced small holes in them. The hole spacing varies from species to species and ranges from a few hundred nanometers to a little over a micron, making them perfect for showing the affect of NA on microscope resolution. For a long time, we have gotten slides from Klaus Kemp, who is apparently the last producer of diatom slides.

The New York Times is now hosting a very nice short movie on Klaus’s work, called The Diatomist. It’s worth checking out if you’ve ever used a diatom slide or wondered what went into making them.

Paper Roundup – August 2014

  • A new clearing method for whole mice; similar to CLARITY, it uses hydrogel cross-linking by perfusion followed by detergent to remove lipids. [1]
  • A simple lens-less imaging system for time lapse imaging of large fields of cells [2]
  • A tomographic imaging system using flow of objects past a slit aperture in a microfluidic device to reconstruct 3D images [3]
  • Intravital imaging of single cells in a beating heart by timing the acquisition based on the EKG [4]
  • Single molecule localization super-resolution imaging of mCherry [5]
  • A new, more accurate method for aligning serial sections to each other [6]. A plugin is available for ImageJ.
  • Fluorescent voltage reporters by voltage dependent quenching of fluorescent proteins [7]
  • A comparison of the performance of different SNAP-tag conjugates for single molecule tracking [8]
  • Imaging through the skull in mice, using infrared fluorescent carbon nanotubes [9]
  • A micro-manager script for microscope benchmarking.  We have been using it routinely for checking our microscopes in the NIC and it works well. [10]
  • A clever screen for receptor internalization using a fluorophore activated at low pH [11]
  • Combined SIM/STORM imaging of biological samples [12]
  • Using a single dichroic mirror and independent component analysis to separate two different dyes with different spatial localizations [13]

References

  1. B. Yang, J. Treweek, R. Kulkarni, B. Deverman, C. Chen, E. Lubeck, S. Shah, L. Cai, and V. Gradinaru, "Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing", Cell, vol. 158, pp. 945-958, 2014. http://dx.doi.org/10.1016/j.cell.2014.07.017
  2. S.V. Kesavan, F. Momey, O. Cioni, B. David-Watine, N. Dubrulle, S. Shorte, E. Sulpice, D. Freida, B. Chalmond, J.M. Dinten, X. Gidrol, and C. Allier, "High-throughput monitoring of major cell functions by means of lensfree video microscopy", Scientific Reports, vol. 4, 2014. http://dx.doi.org/10.1038/srep05942
  3. N.C. Pégard, M.L. Toth, M. Driscoll, and J.W. Fleischer, "Flow-scanning optical tomography", Lab Chip, vol. 14, pp. 4447-4450, 2014. http://dx.doi.org/10.1039/C4LC00701H
  4. A.D. Aguirre, C. Vinegoni, M. Sebas, and R. Weissleder, "Intravital imaging of cardiac function at the single-cell level", Proceedings of the National Academy of Sciences, vol. 111, pp. 11257-11262, 2014. http://dx.doi.org/10.1073/pnas.1401316111
  5. C.M. Winterflood, and H. Ewers, "Single-Molecule Localization Microscopy using mCherry", ChemPhysChem, vol. 15, pp. 3447-3451, 2014. http://dx.doi.org/10.1002/cphc.201402423
  6. C. Wang, S. Ka, and A. Chen, "Robust image registration of biological microscopic images", Scientific Reports, vol. 4, 2014. http://dx.doi.org/10.1038/srep06050
  7. P. Zou, Y. Zhao, A.D. Douglass, D.R. Hochbaum, D. Brinks, C.A. Werley, D.J. Harrison, R.E. Campbell, and A.E. Cohen, "Bright and fast multicoloured voltage reporters via electrochromic FRET", Nature Communications, vol. 5, 2014. http://dx.doi.org/10.1038/ncomms5625
  8. P. Bosch, I. Corrêa, M. Sonntag, J. Ibach, L. Brunsveld, J. Kanger, and V. Subramaniam, "Evaluation of Fluorophores to Label SNAP-Tag Fused Proteins for Multicolor Single-Molecule Tracking Microscopy in Live Cells", Biophysical Journal, vol. 107, pp. 803-814, 2014. http://dx.doi.org/10.1016/j.bpj.2014.06.040
  9. G. Hong, S. Diao, J. Chang, A.L. Antaris, C. Chen, B. Zhang, S. Zhao, D.N. Atochin, P.L. Huang, K.I. Andreasson, C.J. Kuo, and H. Dai, "Through-skull fluorescence imaging of the brain in a new near-infrared window", Nature Photonics, vol. 8, pp. 723-730, 2014. http://dx.doi.org/10.1038/nphoton.2014.166
  10. M. Halter, E. Bier, P.C. DeRose, G.A. Cooksey, S.J. Choquette, A.L. Plant, and J.T. Elliott, "An automated protocol for performance benchmarking a widefield fluorescence microscope", Cytometry Part A, vol. 85, pp. 978-985, 2014. http://dx.doi.org/10.1002/cyto.a.22519
  11. M. Isa, D. Asanuma, S. Namiki, K. Kumagai, H. Kojima, T. Okabe, T. Nagano, and K. Hirose, "High-Throughput Screening System To Identify Small Molecules That Induce Internalization and Degradation of HER2", ACS Chemical Biology, vol. 9, pp. 2237-2241, 2014. http://dx.doi.org/10.1021/cb500654q
  12. V. Hamel, P. Guichard, M. Fournier, R. Guiet, I. Flückiger, A. Seitz, and P. Gönczy, "Correlative multicolor 3D SIM and STORM microscopy", Biomedical Optics Express, vol. 5, pp. 3326, 2014. http://dx.doi.org/10.1364/BOE.5.003326
  13. L. DAO, B. LUCOTTE, B. GLANCY, L. CHANG, L. HSU, and R. BALABAN, "Use of independent component analysis to improve signal-to-noise ratio in multi-probe fluorescence microscopy", Journal of Microscopy, vol. 256, pp. 133-144, 2014. http://dx.doi.org/10.1111/jmi.12167