Protocol for Fluorescence Recovery After Photobleaching (FRAP) During his Ph.D research in the Laboratory of Professor Alexei Aravin, Dr. Alexandre Webster wrote a detailed protocol for doing FRAP experiments using our Zeiss LSM 710 confocal. This protocol describes how a beginner at microscopy can acquire and analyze Fluorescence Recovery After Photobleaching data using a simplified approach.  Although simplified, this protocol was able to provide a consistent estimate of the mobile fraction of tagged protein expressed in fly ovaries. Professor Wachsmuth's paper is also provided.
Link 1: Drs. Webster and Wachsmuth's Easy Protocol for FRAP
Link 2: Professor Malte Wachsmuth's FRAP paper

Using Z-Correction in the Zeiss Zen Black Software Ms. Emily Wyatt, a graduate student in CCE, wrote a quick and easy to follow tutorial on using the Z-Stack Correction function in the Zen Black software when she was a student in the Bi 227 class (Winter 2016). When collecting a large Z-stack you may notice that as you go deeper into your sample, the image intensity fades. The correction function in the Zen software allows one to increase the laser intensity, the detector gain or both as you go deeper into the sample.
Link 3: Ms. Emily Wyatt's quick guide to Z-correction

Protocol for Image Quantification The Director of the BIF, Andres Collazo, is often asked; how do I compare fluorescent intensities across different images? The following document provides 6 tips for accurate quantification of multiple images. It also mentions two of the more common problems users could encounter. This is geared towards fluorescent samples that fade, such as antibody labeling and fluorescent proteins. The protocol is biased to images taken with a laser scanning confocal microscope but similar tips apply to digital cameras.
Link 4: Andres' tips for quantifying images


Papers published using the resources of the BIF Bibliography of papers published since 2013. This is an incomplete list of the publications that have used the microscopes, software and other resources provided by the BIF. These publications are in high impact journals. Most of the publications listed include a link to the online journal article.

Hochgreb-Hagele, T. and M. E. Bronner (2013). "A novel FoxD3 gene trap line reveals neural crest precursor movement and a role for FoxD3 in their specification." Dev Biol 374(1): 1-11.

Hochgreb-Hagele, T., C. Yin, D. E. Koo, M. E. Bronner and D. Y. Stainier (2013). "Laminin beta1a controls distinct steps during the establishment of digestive organ laterality." Development 140(13): 2734-2745.

Kim, D. Y., J. Fingler, R. J. Zawadzki, S. S. Park, L. S. Morse, D. M. Schwartz, S. E. Fraser and J. S. Werner (2013). "Optical imaging of the chorioretinal vasculature in the living human eye." Proc Natl Acad Sci U S A 110(35): 14354-14359.

Rogers, C. D., A. Saxena and M. E. Bronner (2013). "Sip1 mediates an E-cadherin-to-N-cadherin switch during cranial neural crest EMT." J Cell Biol 203(5): 835-847.

Saxena, A., B. N. Peng and M. E. Bronner (2013). "Sox10-dependent neural crest origin of olfactory microvillous neurons in zebrafish." Elife 2: e00336.

Choi, H. M. T., V. A. Beck and N. A. Pierce (2014). "Next-Generation in Situ Hybridization Chain Reaction: Higher Gain, Lower Cost, Greater Durability." ACS Nano 8(5): 4284-4294.

Hochgreb-Hagele, T., D. E. Koo, N. M. Das and M. E. Bronner (2014). "Zebrafish stem/progenitor factor msi2b exhibits two phases of activity mediated by different splice variants." Stem Cells 32(2): 558-571.

Maier, E. C., A. Saxena, B. Alsina, M. E. Bronner and T. T. Whitfield (2014). "Sensational placodes: neurogenesis in the otic and olfactory systems." Dev Biol 389(1): 50-67.

Martinez, T. F., J. W. Phillips, K. K. Karanja, P. Polaczek, C. M. Wang, B. C. Li, J. L. Campbell and P. B. Dervan (2014). "Replication stress by Py-Im polyamides induces a non-canonical ATR-dependent checkpoint response." Nucleic Acids Res 42(18): 11546-11559.

Sapir, A., A. R. Dillman, S. A. Connon, B. M. Grupe, J. Ingels, M. Mundo-Ocampo, L. A. Levin, J. G. Baldwin, V. J. Orphan and P. W. Sternberg (2014). "Microsporidia-nematode associations in methane seeps reveal basal fungal parasitism in the deep sea." Front Microbiol 5: 43.

Schwartz, D. M., J. Fingler, D. Y. Kim, R. J. Zawadzki, L. S. Morse, S. S. Park, S. E. Fraser and J. S. Werner (2014). "Phase-variance optical coherence tomography: a technique for noninvasive angiography." Ophthalmology 121(1): 180-187.

Shinya, R., K. Hasegawa, A. Chen, N. Kanzaki and P. W. Sternberg (2014). "Evidence of hermaphroditism and sex ratio distortion in the fungal feeding nematode Bursaphelenchus okinawaensis." G3 (Bethesda) 4(10): 1907-1917.

Simoes-Costa, M., J. Tan-Cabugao, I. Antoshechkin, T. Sauka-Spengler and M. E. Bronner (2014). "Transcriptome analysis reveals novel players in the cranial neural crest gene regulatory network." Genome Res 24(2): 281-290.

Ahmed, M., D. W. Pan and M. E. Davis (2015). "Lack of in vivo antibody dependent cellular cytotoxicity with antibody containing gold nanoparticles." Bioconjug Chem 26(5): 812-816.

DeForest, C. A. and D. A. Tirrell (2015). "A photoreversible protein-patterning approach for guiding stem cell fate in three-dimensional gels." Nat Mater 14(5): 523-531.

Deyle, K. M., B. Farrow, Y. Qiao Hee, J. Work, M. Wong, B. Lai, A. Umeda, S. W. Millward, A. Nag, S. Das and J. R. Heath (2015). "A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1." Nat Chem 7(5): 455-462.

Hargrove, A. E., T. F. Martinez, A. A. Hare, A. A. Kurmis, J. W. Phillips, S. Sud, K. J. Pienta and P. B. Dervan (2015). "Tumor Repression of VCaP Xenografts by a Pyrrole-Imidazole Polyamide." PLoS One 10(11): e0143161.

Kang, J. S. and P. B. Dervan (2015). "A sequence-specific DNA binding small molecule triggers the release of immunogenic signals and phagocytosis in a model of B-cell lymphoma." Q Rev Biophys 48(4): 453-464.

McHugh, C. A., C. K. Chen, A. Chow, C. F. Surka, C. Tran, P. McDonel, A. Pandya-Jones, M. Blanco, C. Burghard, A. Moradian, M. J. Sweredoski, A. A. Shishkin, J. Su, E. S. Lander, S. Hess, K. Plath and M. Guttman (2015). "The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3." Nature 521(7551): 232-236.

Singh, C., G. Oikonomou and D. A. Prober (2015). "Norepinephrine is required to promote wakefulness and for hypocretin-induced arousal in zebrafish." Elife 4: e07000.

Uribe, R. A. and M. E. Bronner (2015). "Meis3 is required for neural crest invasion of the gut during zebrafish enteric nervous system development." Mol Biol Cell 26(21): 3728-3740.

Uribe, R. A., A. L. Buzzi, M. E. Bronner and P. H. Strobl-Mazzulla (2015). "Histone demethylase KDM4B regulates otic vesicle invagination via epigenetic control of Dlx3 expression." J Cell Biol 211(4): 815-827.

Yuet, K. P., M. K. Doma, J. T. Ngo, M. J. Sweredoski, R. L. J. Graham, A. Moradian, S. Hess, E. M. Schuman, P. W. Sternberg and D. A. Tirrell (2015). "Cell-specific proteomic analysis in Caenorhabditis elegans." Proceedings of the National Academy of Sciences 112(9): 2705-2710.

Datta, S. S., A. Preska Steinberg and R. F. Ismagilov (2016). "Polymers in the gut compress the colonic mucus hydrogel." Proceedings of the National Academy of Sciences.

DePas, W. H., R. Starwalt-Lee, L. Van Sambeek, S. Ravindra Kumar, V. Gradinaru and D. K. Newman (2016). "Exposing the Three-Dimensional Biogeography and Metabolic States of Pathogens in Cystic Fibrosis Sputum via Hydrogel Embedding, Clearing, and rRNA Labeling." MBio 7(5).

Deverman, B. E., P. L. Pravdo, B. P. Simpson, S. R. Kumar, K. Y. Chan, A. Banerjee, W. L. Wu, B. Yang, N. Huber, S. P. Pasca and V. Gradinaru (2016). "Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain." Nat Biotechnol 34(2): 204-209.

Ho, S. H. and D. A. Tirrell (2016). "Chemoenzymatic Labeling of Proteins for Imaging in Bacterial Cells." Journal of the American Chemical Society 138(46): 15098-15101.

Hulse, B. K., L. C. Moreaux, E. V. Lubenov and A. G. Siapas (2016). "Membrane Potential Dynamics of CA1 Pyramidal Neurons during Hippocampal Ripples in Awake Mice." Neuron 89(4): 800-813.

Mysore, V. S., J. Szablowski, P. B. Dervan and P. J. Frost (2016). "A DNA-binding Molecule Targeting the Adaptive Hypoxic Response in Multiple Myeloma Has Potent Antitumor Activity." Mol Cancer Res 14(3): 253-266.

Ndjamen, B., D. S. Joshi, S. E. Fraser and P. J. Bjorkman (2016). "Characterization of Antibody Bipolar Bridging Mediated by the Human Cytomegalovirus Fc Receptor gp68." J Virol 90(6): 3262-3267.

Sampson, T. R., J. W. Debelius, T. Thron, S. Janssen, G. G. Shastri, Z. E. Ilhan, C. Challis, C. E. Schretter, S. Rocha, V. Gradinaru, M.-F. Chesselet, A. Keshavarzian, K. M. Shannon, R. Krajmalnik-Brown, P. Wittung-Stafshede, R. Knight and S. K. Mazmanian (2016). "Gut Microbiota Regulate Motor Deficits and Neuroinflammation in a Model of Parkinson’s Disease." Cell 167(6): 1469-1480.e1412.

Shah, S., E. Lubeck, M. Schwarzkopf, T.-F. He, A. Greenbaum, C. H. Sohn, A. Lignell, H. M. T. Choi, V. Gradinaru, N. A. Pierce and L. Cai (2016). "Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing." Development 143(15): 2862-2867.

Szablowski, J. O., J. A. Raskatov and P. B. Dervan (2016). "An HRE-Binding Py-Im Polyamide Impairs Hypoxic Signaling in Tumors." Mol Cancer Ther 15(4): 608-617.

Uribe, R. A., T. Gu and M. E. Bronner (2016). "A novel subset of enteric neurons revealed by ptf1a:GFP in the developing zebrafish enteric nervous system." Genesis 54(3): 123-128.

Bedbrook, C. N., A. J. Rice, K. K. Yang, X. Ding, S. Chen, E. M. LeProust, V. Gradinaru and F. H. Arnold (2017). "Structure-guided SCHEMA recombination generates diverse chimeric channelrhodopsins." Proc Natl Acad Sci U S A 114(13): E2624-E2633.

Burke, K. S., K. A. Antilla and D. A. Tirrell (2017). "A Fluorescence in Situ Hybridization Method To Quantify mRNA Translation by Visualizing Ribosome–mRNA Interactions in Single Cells." ACS Central Science.

Kieffer, C., M. S. Ladinsky, A. Ninh, R. P. Galimidi and P. J. Bjorkman (2017). "Longitudinal imaging of HIV-1 spread in humanized mice with parallel 3D immunofluorescence and electron tomography." eLife 6: e23282.

Lee, J. O., V. Narasimhan, J. Du, B. Ndjamen, D. Sretavan and H. Choo (2017). "Biocompatible Multifunctional Black-Silicon for Implantable Intraocular Sensor." Adv Healthc Mater 6(4).

Rapp, P. B., A. K. Omar, J. J. Shen, M. E. Buck, Z. G. Wang and D. A. Tirrell (2017). "Analysis and Control of Chain Mobility in Protein Hydrogels." J Am Chem Soc 139(10): 3796-3804.

Roellig, D., J. Tan-Cabugao, S. Esaian and M. E. Bronner (2017). "Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells." eLife 6: e21620.

Wang, H., J. Liu, S. Gharib, C. M. Chai, E. M. Schwarz, N. Pokala and P. W. Sternberg (2017). "cGAL, a temperature-robust GAL4-UAS system for Caenorhabditis elegans." Nat Meth 14(2): 145-148.

Wu, W. L., E. Y. Hsiao, Z. Yan, S. K. Mazmanian and P. H. Patterson (2017). "The placental interleukin-6 signaling controls fetal brain development and behavior." Brain Behav Immun 62: 11-23.

Yee, D. W., M. D. Schulz, R. H. Grubbs and J. R. Greer (2017). "Functionalized 3D Architected Materials via Thiol-Michael Addition and Two-Photon Lithography." Advanced Materials 29(16): 1605293-n/a.