Cell Biology Commons^™

Mcnamara 201412 Nih Scap Innocentive Challenge Solution - T-Bow Rainbow T-Cells And Tumor Cells Spatial Multiplexing Gene Expression Reporter System – Plus Supplement Plus Posters - 20151027 - Please Download "75" Instead, George Mcnamara

George McNamara

McNamara 201412 NIH SCAP InnoCentive Challenge Solution - T-Bow Rainbow T-cells and Tumor Cells Spatial Multiplexing Gene Expression Reporter System – plus supplement plus posters - 20151027.

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Please download the current 20151027 (October 27, 2015) Tattletales and T-Bow update from

http://works.bepress.com/gmcnamara/75/

The bepress web site is not letting me replace the old pdf here at "65" with the additional 10 pages update.

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The download is my/Cooper lab solution (submission) to the 2014 NIH Single Cell Analysis Program (SCAP) InnoCentive Challenge, "Follow That Cell". I submitted the Solution on 20141215Mon (with 20 minutes to spare). The Challenge web page …

Tattletales And T-Bow Update 20151027tue, George Mcnamara

George McNamara

20151027Tue this "75"

http://works.bepress.com/gmcnamara/75

is my update of "65" posting

See text at

http://works.bepress.com/gmcnamara/65/

for text summary. The PDf here in "75" supersedes "65".

The PDF here has 10 pages added to the end from the "65" version (pages 40-49 of PDF when including the bepress cover page)..

here is the text in my cover page (bepress may add its own cover):

20151027Tue: added 10 page e-poster at bottom explaining Binary Tattletales and T-Bow. That is, binary with respect to protein components. For one color (number of repeats, epitope tags, FPs are examples, here rounded to convenient numbers):

1. 100 …

Gpu Deconvolution Wow Result On 2048x2048x32 Plane Z-Series, George Mcnamara

George McNamara

GPU Deconvolution WOW result on 2048x2048x32 plane Z-series ... formerly bad academic code ("you get what you pay for") now impressive

Alternative title: "instant gratification quantitative deconvolution fluorescence microscopy".

http://works.bepress.com/gmcnamara/55/

Please see "74"

http://works.bepress.com/gmcnamara/74/

for 32-bit images from this project (bepress file size limitation prevented me from including them in this ZIP archive).

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Summary: Deconvolution microscopy has historically been painfully slow. The early vendors were:

- Scanalytics (Carrington and Fay), commercialized to try to sell expensive, specialized array processors made by CSPI (the CSPI box likely had less computing power than a first gen smartphone).

- Applied Precision (Sedat …

32-Bit Deconvolution Files Accompanying 55 Page, George Mcnamara

George McNamara

ZIP file with 32-bit images accompanying

http://works.bepress.com/gmcnamara/55

Bepress apparently has an ~1 Gigabyte file upload limit so I have placed the 32-bit images of greatest interest here.

See Word document inside for details.

Light Microscopy Reflection Focusing Cells And Coverglass, George Mcnamara

George McNamara

Light Microscopy Reflection Focusing Cells and Coverglass 20150520Wed

Many light microscopes have the capability of using camera based brightfield, phase contrast, or fluorescence to find the most contrasty specimen focus. When I worked for UIC (1992-1997) we offered this with a combination of Ludl MAC2000 controller, Z-drive, video card thingy, and video camera (most customers at the time used analog video cameras).

Many automated light microscopes now have clever -- and pricey -- autofocus systems based on an NIR LED structured illumination reflecting light off the bottom of the coverglass.

I encourage a simpler approach - my hardware details are …

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/ …

Stellaris Fish Workshop Gadph And Dapi Z-Series, George Mcnamara

George McNamara

Stellaris FISH workshop GADPH and DAPI Z-series at MD Anderson Cancer Center, Houston, TX.

The zip file contains raw and GPU deconvolved image data from a workshop Biosearch Technologies conducted for MDACC researchers in December 2013. Image data was acquired on a Leica DMI6000 microscope with Lumencor SOLA light engine, DAPI and Cy5 filter cubes, Hamamatsu ORCA FLASH4.0 sCMOS camera (500 ms exposure time per plane for Quasar 670).

Pixel size 100 nm XY.

Z-step size 200 nm.

32 planes (power of 2 is optimal for GPU deconvolution). With 500 ms exposure time, the Quasar 670 GADPH FISH probes images …

Stellaris Fishing 20131125mon Part 2 Of 2, George Mcnamara

George McNamara

Stellaris FISH dataset using three FISH probe sets.

Slides courtesy of Biosearch Technologies,

https://www.biosearchtech.com/store/product.aspx?catid=224,318,324

see http://stellarisfish.smugmug.com/ for online gallery by Biosearch.

This experiment was to evaluate the crosstalk between the Biosearch fluorophores:

Quasar 570

CAL Fluor Red 610 (CFR 610)

Quasar 670

DAPI (DNA counterstain)

Autofluorescence (green, but sometimes showing up in other channels).

and our lab's Leica DMI6000 fluorescence microscope with Leica filter sets:

DAPI

GFP (L5)

Cy3 (N3)

Texas Red (TxRed2)

Cy5 (Y5)

I also acquired green channel and red channel with exciter filters in our ASI excitation wheel:

GFP + 492 exciter

Texas Red (TxRed2) + 572 …

Leica Microscope Gpu Deconvolution Stellaris Fish Dataset #1, George Mcnamara

George McNamara

McNamara 20131101Fri Leica widefield microscope CUDA Deconvolution Stellaris FISH probe cultured cells dataset #1.zip

A text file in the zip archive has experiment details. I am posting this online so that researchers - whether academic or commercial - can evaluate the acquired data, the Bruce and Butte 2013 Optics Express ( http://www.opticsinfobase.org/oe/fulltext.cfm?uri=oe-21-4-4766 ) deconvolution result (note: I may not have used optimal settings), and to compare these deconvolution results to other methods. If anyone generates alternative spatial deconvolution output, such as from: * SVI Huygens * Media Cy AutoQuant * Agard's ER-Decon (Arigovindan 2013 PNAS) * Vicidomini SGP GPU Deconv …

Hamamatsu Flash4.0 Scmos Exposure Time Series, George Mcnamara

George McNamara

Hamamatsu FLASH4.0 scientific cMOS camera exposure time series are pairs of images of:

1 millisecond (00,001ms series)

10 millisecond (00,010ms series)

100 millisecond (00,100ms series)

1,000 millisecond (01,000ms series)

4,000 millisecond (04,000ms series)

10,000 millisecond (10,000ms series)

I also included:

* difference images (exposure 2 minus exposure 1 plus 100 intensity values).

* a series of eleven 1 second (1,000 ms) exposure time images in a multi-plane TIFF file (different images than the pair of 1,000ms images above).

* Stack Arithmetic: Median, Average, Minimum, Maximum, of the eleven plane series (Stack Arithmetic is a MetaMorph command).

These images were acquired …

Fluorescence Microscopy Digital Deconvolution Comparison, George Mcnamara, Vinita Popat

George McNamara

Presentation by Ms. Vinita Popat, Cornell University, 2013 summer student in Prof. Laurence J.N. Cooper lab, MD Anderson Cancer Center, Houston, TX. Project was evaluation of several deconvolution software for improving (or making worse!) fluorescence microscopy Z-series.

Flash4 Dark Reference Images, George Mcnamara

George McNamara

Hamamatsu FLASH4.0 dark reference images, acquired with 10 second exposure times, no light to camera. Camera offset (set by Hamamatsu( is ~100 (the average intensity of the first image is always ~1 intensity level higher - an odd feature, but trivial in practice for a 16-bit camera).

George McNamara, Ph.D.

Single Cells Analyst at L.J.N. Cooper Lab

University of Texas M.D. Anderson Cancer Center

Video Codec Performance (Excel Spreadsheet), George Mcnamara

George McNamara

Video codec performance (Excel spreadsheet). Movie was made in 2005-2006 when I worked at City of Hope National Medical Center. VTLF refers to Video Timelapse Light Facility. Videos were outputted from MetaMorph as AVI files. Personally, I always recommend uncompressed video files fro scientific uses. I also encourage posting the original scientific data format (ex. .lsm, .zvi, .lif, .stk).

Pubspectra Tattletales, George Mcnamara

George McNamara

Tattletales for Multiplex Fluorescent Reporters in Single Cells for Metabolomics

George McNamara

As of April 2013: L.J.N. Cooper & D.A. Lee Cellular Immunotherapy Lab, University of Texas M.D. Anderson Cancer Center, Houston, TX

Email: gtmcnamara@mdanderson.org, geomcnamara@earthlink.net

Tattletales is my concept for spatial multiplexing many fluorescent protein (FP) biosensors in the same live cell. For example, there are excellent FP biosensors to Ca++ ions, pH, glucose, ribose, glutamine, glutamate, ATP, redox, ROS, pyruvate, cAMP, cGMP, IP3, PI(3,4,5)P3, cell cycle indicators (Fucci2), PKA, PKC, photsphatases, caspase(s) [1, 2]. However, these are typically used one biosensor per experiment, due in part to flooding …

Halloween 2012 Jack O'Lanterns Trick Or Treat, George Mcnamara

George McNamara

Halloween 2012 makes trick or treating more visual and interactive than in past years.

the download is a ZIP file containing three files.

Print out the (unnumbered) image on as large and nice printer paper as possible - I used glossy 44" wide here in Miami (University of Miami, MillerSchool of Medicine, Calder Library, Biomedical Communications dept - I also made another print on "fabric", also 44" wide to take with me to an HHMI Janelia Farm conference on 'turning images into knowledge' that ends on Oct 31 - might stay up for a second conference, "GFP..." that start Nov …

Mcnamara 20120831fri-20120904tue Cosmic Ray Particles By Ccd Imaging, George Mcnamara

George McNamara

McNamara 20120831Fri-20120904Tue Cosmic Ray Particles by CCD imaging.zip contains image files in support of a Microscopy Today article - please see

http://www.microscopy-today.com/

Cosmic Ray Particles Images With Orca-Ii Erg, George Mcnamara

George McNamara

Cosmic ray particles image series acquired using a Hamamatsu ORCA-II ERG scientific grade CCD camera, cooled to -60 C. Each image is a consecutive 600 second (10 minute) exposure time with no light to the camera.

While processing the data, I discoverd that the background changed around planes 25 and 227 (see Excel file and jpeg screenshots), so I also processed only planes 025-227 (203 planes total, 2030 minutes, 33.83 hours). the CCD industry "rule of thumb" for a "typical" CCD sensor (i.e. 1/3" CCD) is that one cosmic ray particle strikes a sensor approximately every 30 seconds (assuming not …

Flatbed Scanner Report - Optical Density Dynamic Range, George Mcnamara

George McNamara

George McNamara (now at University of Miami) report for Hua Yu and Richard Jove, City of Hope National Medical Center, on optical density dynamic range of several flatbed scanners.

Introduction To Nanoscopy Nano-Talk, George Mcnamara

George McNamara

T7-1 is the designation for the LMRG Nanoscopy session at ABRF in Orlando, FL, on March 20, 2012. The PDF file here is a draft of my presentation.

May not be very helpful since (1) would probably help to know what is in my head and each slide will [hopefully] prompt me to say, and (2) 10 minute talk so I am going to push the "next slide" button after saying very little.

__________________

Publisher statement:

The T7-1 Introduction to Nanoscopy Nano Talk is copyrighted (c) George McNamara, 2012. Except for (1) screenshots from research articles (which are copyrighted by …

Pubspectra - Open Data Access Fluorescence Spectra, George Mcnamara

George McNamara

The Internet is enabling greater access to spectral imaging publications, spectral graphs, and data than that was available a generation ago. The spectral imaging systems discussed in this issue of Cytometry work because reagent and hardware spectra are reproducible, reusable, and provide input to spectral unmixing and spectral components recognition algorithms. These spectra need to be readily available in order to determine what to purchase, how to use it, and what the output means. We refer to several commercially sponsored and academic spectral web sites and discuss our spectral graphing and data sites. Sites include fluorescent dye graph servers from …

Mcnamara 2011 Feature Extraction (Image Analysis), George Mcnamara

George McNamara

Feature Extraction presentation and movies in a ZIP file from a presentation I gave at ISAC 2011 in Baltomore, Md.

Feature extraction is one phrase for image analysis.