Cell Biology Commons^™

Tattletales And T-Bow Update 20141018, George Mcnamara

George McNamara

Tattletales and T-Bow Update 20141018

http://works.bepress.com/gmcnamara/63

Tattletales is my concept to multiplex fluorescent protein biosensors ("Tattletales", which is also the overall concept name) and multicolor FP reporters ("T-Bow" for Rainbow T-cells and tumor cells) to study the physiology of live cells.

These are based on:

1. LacI-GFP :: LacO operator synthetic tandem repeat array (Robinett et al 1996 JCB, Fig 4A, http://jcb.rupress.org/content/135/6/1685.full.pdf )

2. the plethora of fluorescent protein biosensors - see page 1 of this "63" download site.

3. the many colors, and combinations of colors (CY11.5, CyPet-YPet 90% FRET, simple color combination fusions, Steven Vogel's "V6" and "V8" …

Timelapse Data - Gm Imaging Cytometer Needs Stitching Part 1 Of 2, George Mcnamara

George McNamara

Timelapse data - GM imaging cytometer needs stitching part 1 of 2

Imaging challenge: align multiple time series over time to overcome "stage slop" in the imaging cytometer used in this experiment.

http://works.bepress.com/gmcnamara/53 (this)

and

http://works.bepress.com/gmcnamara/54 (next)

are zip files containing 8-bit stacks (MetaMorph multiplane TIFF files) acquired on an imaging cytometer.

"53" is the brightfield data, raw and automatic aligned stacks in MetaMorph (Apps menu, MM7.8.6).

"54" is GFP fluorescence, raw and automatic aligned stacks in MetaMorph (Apps menu, MM7.8.6). The GFP fluorescence decreases over time due to photobleaching. GFP disappears from cells when they die (soluble GFP diffusing …

Timelapse Data - Gm Imaging Cytometer Needs Stitching Part 2 Of 2, George Mcnamara

George McNamara

Timelapse data - GM imaging cytometer needs stitching part 2 of 2

Imaging challenge: align multiple time series over time to overcome "stage slop" in the imaging cytometer used in this experiment.

http://works.bepress.com/gmcnamara/53 (previous) and http://works.bepress.com/gmcnamara/54 (this)

are zip files containing 8-bit stacks (MetaMorph multiplane TIFF files) acquired on an imaging cytometer.

"53" is the brightfield data, raw and automatic aligned stacks in MetaMorph (Apps menu, MM7.8.6). "54" is GFP fluorescence, raw and automatic aligned stacks in MetaMorph (Apps menu, MM7.8.6). The GFP fluorescence decreases over time due to photobleaching. GFP disappears from cells when they die (soluble GFP diffusing …

Tattletales And T-Bow Update 20140602mon, George Mcnamara

George McNamara

Tattletales and T-Bow Update 20140602Mon

http://works.bepress.com/gmcnamara/42

Please see also http://works.bepress.com/gmcnamara/26

Tattletales: multiplex fluorescent protein biosensors by spatial localization with TALE-FPs, Cas9-FPs, ZF-FPs, LacI-FPs, TetR-FPs, etc.

T-Bow: Rainbow T-cells and Tumor cells (and ES cells, iPS cells, other cells and organisms). You can think of this as "Brainbow meets TALENs/Cas9/ZFNs/other DNA sequence specific binding proteins".

If not familiar with Brainbow, see

http://en.wikipedia.org/wiki/Brainbow

If not familiar with TALENs, Cas9, etc, see

http://www.addgene.org/genome_engineering/

Big idea: localizing fluorescent proteins - and/or Nano-Lanterns (Take Nagai) - to tandem repeat arrays - is a great way to improve signal to noise ratio compared to the usual …

Timelapse Data - Gm Vandana 20140502fri Part 2 Of 2, George Mcnamara

George McNamara

Timelapse data - GM Vandana 20140502Fri part 2 of 2

(positions 4 and 5 of 5).

Data from a project with Vandana Kaul, Univ Houston. Vandana and GM know the details of this experiment. Nikon BioStation IM, 20x effective magnification (Nikon 40x lens with de-zoom), 800x600 pixels (by 2x2 binning). Five positions (P1 ... P5), acquired at 1 frame per minute, playback 30 fps.

Videos 1, 2 and 3 are in "part 1" at

http://works.bepress.com/gmcnamara/48/

Timelapse Data - Gm Vandana 20140502fri Part 1 Of 2, George Mcnamara

George McNamara

Timelapse data - GM Vandana 20140502Fri part 1 of 2

(positions 1, 2 and 3).

Data from a project with Vandana Kaul, Univ Houston. Vandana and GM know the details of this experiment. Nikon BioStation IM, 20x effective magnification (Nikon 40x lens with de-zoom), 800x600 pixels (by 2x2 binning). Five positions (P1 ... P5), acquired at 1 frame per minute, playback 30 fps.

Image Z-Series Wrt Nvidia Titan 6 Gb Gpu Cuda Deconvolution, George Mcnamara

George McNamara

Image Z-series wrt NVidia Titan 6 Gb GPU CUDA Deconvolution

This ZIP file contains raw and deconvolved data series from George McNamara, Laurence J.N. Cooper lab, M.D. Anderson Cancer Center. I have also included our lab instructions, and a screenshot of the current CUDA deconvolution settings (for dry objective lenses, Manish Butte wrote me to use the immersion medium refractive index, air = 1.0).

Instrument:

Leica DMI6000 inverted fluorescence microscope, Lumencor SOLA LED light source, Leica "L5" GFP filter cube (480/40ex, 505dm, 527/30em).

Leica 20x/0.75 NA objective lens, 1.6x optovar, for 325 nm XY piel size. For this dataset, I …

Cell Morphometry, George Mcnamara

George McNamara

Cell Morphometry ZIP content by George McNamara

http://works.bepress.com/gmcnamara/41

Robert Murphy's TypIC (typical cell Chooser,

http://murphylab.web.cmu.edu/services/TypIC/

now superseded by PSLID and SLIF

http://murphylab.web.cmu.edu/services/ ), was a "game changer" for me with respect to cell shape analysis. Rather than trying to compute the average of (say) a triangle and a pentagon ... which might result in a square, or a rectange, or some bizarre quadrilateral ... R.M. advocated using the median. OK, in a 2 member dataset this would result in averaging the two shapes (if use the standard way to calculate median of even number datasets), but this could be avoided …

3b Project Data - Raw Tiff Series And Cropped Series, George Mcnamara

George McNamara

Fluorescence microscopy data acquired for 3B Bayesian localization microscopy.

Settings: 100 nm XY pixel size. 50 millisecond exposures, MetaMorph stream acquisition mode (as fast as possible). Stellaris FISH mammalian cells labeled with Quasar 670 fluorophores, one molecule per 20mer FISH probe, ~48 probes to TOP1 mRNA (Topoisomerase 1).

More on the 3B project and Stellaris FISH data at http://works.bepress.com/gmcnamara/38/

and at Susan Cox's web sites http://www.coxphysics.com/3b/ http://superresolved.com/

Blood Vessel Z-Series Image Data Set From Confocal Microscopy 2013 Book, George Mcnamara

George McNamara

Blood Vessel Z-series image data set from Confocal Microscopy 2013 book chapter:

McNamara G, Yanai A, Khankaldyyan V, Laug WE, Boden J, Webster K, Li Y, Wen R. Low magnification confocal microscopy of tumor angiogenesis. Methods Mol Biol. 2014; 1075: 149-75.

doi: 10.1007/978-1-60761-847-8_6. PubMed PMID: 24052350.

See also:

Jeffrey Boden, Jianqin Wei, George McNamara, Hans Layman, Midhat Abdulreda, Fotios Andreopoulos, and Keith A. Webster. Whole-mount imaging of the mouse hindlimb vasculature using the lipophilic carbocyanine dye DiI. Biotechniques Rapid Dispatches, July 2012, pp. 1–4

http://www.biotechniques.com/rapiddispatches/Whole-mount-imaging-of-the-mouse-hindlimb-vasculature-using-the-lipophilic-carbocyanine-dye-DiI./biotechniques-333144.html

Supplemental file:

http://www.biotechniques.com/multimedia/archive/00182/BTNRD-BM-WebsterSUP_182355a.pdf

You may find useful to download Leica LAS AF Lite from

ftp://ftp.llt.de/softlib/LAS_AF_Lite/ …