Cell and Developmental 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.

///

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.

///

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).

//

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.

Mcnamara 2010 Metamatters Newletter Series - Rogers Pmn The Chase And Temporal Area Maps, George Mcnamara

George McNamara

My four 2010 MetaMorph MetaMatters newsletter articles on "The Chase" (video available for download here on my bepress site) and history / use of temporal area maps.

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 …

Iron Alters Cell Survival In A Mitochondria-Dependent Pathway In Ovarian Cancer Cells., Edward Haller

Edward Haller

ABSTRACT The role of iron in the development of cancer remains unclear. We previously reported that iron reduces cell survival in a Ras/mitogen-activated protein kinase (MAPK)-dependent manner in ovarian cells; however, the underlying downstream pathway leading to reduced survival was unclear. Although levels of intracellular iron, ferritin/CD71 protein and reactive oxygen species did not correlate with iron-induced cell survival changes, we identified mitochondrial damage (via TEM) and reduced expression of outer mitochondrial membrane proteins (translocase of outer membrane: TOM20 and TOM70) in cell lines sensitive to iron. Interestingly, Ru360 (an inhibitor of the mitochondrial calcium uniporter) reversed mitochondrial changes and …

Gadph Single Molecule Mrna Fish 20141104tue, George Mcnamara

George McNamara

GADPH single molecule mRNA FISH 20141104Tue

Raw data on NVidia TITAN GPU card deconvolution using original Bruce and Butte 2013 Optics Express software running inside Fiji ImageJ.

B&B GPU Deconvolution Reference:

http://www.opticsinfobase.org/oe/fulltext.cfm?uri=oe-21-4-4766&id=249375

Acquired at 63x1x using leica plan apo 1.4 NA objective lens on Cooper lab Leica DMI6000 microscope, SOLA (initial release) LED lamp using only "green" LED, 554x Semrock exciter filter (in ASI ex wheel), Leica N3 (Cy3) filter cube, center 600 nm emission wavelength.

Hamamatsu FLASH4.0 sCMOS, 100 ms exposure time so 6.4 seconds acquisition time (64 planes X 4 seconds). Acquired with MetaMorph 7.8.8.

XY 100 nm …

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" …

Gpu Deconvolution Good Result On 2048x2048x32 Plane Z-Series (Update From Dataset 56), George Mcnamara

George McNamara

GPU Deconvolution Good result on 2048x2048x32 plane Z-series (update from dataset 56)

This web page and download is

http://works.bepress.com/gmcnamara/62

Marc Bruce sent me the file here (web page 62) that is a preliminary bug fix (apparently of NVidia CUDA FFT code!!!) from the original 'raw' Z-series that is part of the

http://works.bepress.com/gmcnamara/56

web page download. Please see that web page for details on the project (GADPH single molecule mRNA detection with Stellaris FISH probe sets).

Marc is:

MARC BRUCE, PHD

CTO and Co-Founder

MICROVOLUTION, LLC

http://www.microvolution.com/

//

The 730 Mb ZIP download includes 3 files:

* 32-bit TIFF series sent …

Enhancement Of Chronically Induced Breast Carcinogenesis By Combined Environmental And Dietary Carcinogens And Suppression By Dietary Agents, Lenora A. Pluchino Ph.D.

Lenora A. Pluchino, Ph.D.

Most breast cancers occur sporadically due to long-term exposure to low-dose carcinogens present in our environment and diet. American lifestyles involve frequent exposures to smoke, polluted air, and high temperature-cooked meats comprising multiple carcinogens, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), benzo[α[alpha]]pyrene (B[α[alpha]]P), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). To investigate whether these carcinogens may act together to enhance breast cell carcinogenesis, we used our chronically-induced breast cell carcinogenesis model wherein we repeatedly expose non-cancerous human breast epithelial MCF10A cells to physiologically-achievable doses of carcinogens to progressively induce cellular acquisition of cancer-associated properties including reduced dependence on growth factors, anchorage-independent growth, increased cell proliferation, migration and …

Experimental Demonstration Of Bindingless Signal Delivery In Human Cells Via Microfluidics, Fang-Tzu Chuang

Fang-Tzu Chuang

The cellular signal transduction is commonly believed to rely on the direct “contact” or “binding” of the participating molecule reaction that depends positively on the corresponding molecule concentrations. In living systems, however, it is somewhat difficult to precisely match the corresponding rapid “binding,” depending on the probability of molecular collision, existing in the cellular receptor-ligand interactions. Thus, a question arises that if there is another mechanism (i.e., bindingless) that could promote this signal communication. According to this hypothesis, we report a cellular model based on the examination of intracellular calcium concentration to explore whether the unidentified signal delivery in cells …

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 …

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 …

More Fish (1 Of 4), George Mcnamara

George McNamara

More Stellaris FRISH and GPU Deconvolution, 20140313Thur - part 1 of 4

Please see other content in the FISH Imaging section of

http://works.bepress.com/gmcnamara/subject_areas.html

for details on the microscope. Briefly: Leica DMI6000 inverted microscope, SOLA lamp, Hamamatsu FLASH4.0 sCMOS camera, 100 nm XY, 200 nm Z pixels, 32 planes (16 um Z), MetaMorph 7.8 acquisition software.

Four zip files:

http://works.bepress.com/gmcnamara/43

http://works.bepress.com/gmcnamara/44

http://works.bepress.com/gmcnamara/45

http://works.bepress.com/gmcnamara/46

with two dataset (001, 002), raw and deconvolved using Bruce & Butte GPU deconvolution in Fiji ImageJ,

http://www.ncbi.nlm.nih.gov/pubmed/23482010

http://www.opticsinfobase.org/vjbo/fulltext.cfm?uri=oe-21-4-4766&id=249375 (free download, as are all Optics Express papers).

http://tcell.stanford.edu/software.html

http://lists.umn.edu/cgi-bin/wa?A2=ind1312&L=confocalmicroscopy&P=5534

Note: "001 raw" (bepress 43) contains my "imaging SOP" …

More Fish (2 Of 4), George Mcnamara

George McNamara

More Stellaris FISH and GPU Deconvolution, 20140313Thur - part 2 of 4

Please see other content in the FISH Imaging section of

http://works.bepress.com/gmcnamara/subject_areas.html

for details on the microscope. Briefly: Leica DMI6000 inverted microscope, SOLA lamp, Hamamatsu FLASH4.0 sCMOS camera, 100 nm XY, 200 nm Z pixels, 32 planes (16 um Z), MetaMorph 7.8 acquisition software.

Four zip files:

http://works.bepress.com/gmcnamara/43

http://works.bepress.com/gmcnamara/44

http://works.bepress.com/gmcnamara/45

http://works.bepress.com/gmcnamara/46

with two dataset (001, 002), raw and deconvolved using Bruce & Butte GPU deconvolution in Fiji ImageJ,

http://www.ncbi.nlm.nih.gov/pubmed/23482010

http://www.opticsinfobase.org/vjbo/fulltext.cfm?uri=oe-21-4-4766&id=249375 (free download, as are all Optics Express papers).

http://tcell.stanford.edu/software.html

http://lists.umn.edu/cgi-bin/wa?A2=ind1312&L=confocalmicroscopy&P=5534

Note: "001 raw" (bepress 43) contains my "imaging SOP" …

More Fish (3 Of 4), George Mcnamara

George McNamara

More Stellaris FISH and GPU Deconvolution, 20140313Thur - part 3 of 4

Please see other content in the FISH Imaging section of

http://works.bepress.com/gmcnamara/subject_areas.html

for details on the microscope. Briefly: Leica DMI6000 inverted microscope, SOLA lamp, Hamamatsu FLASH4.0 sCMOS camera, 100 nm XY, 200 nm Z pixels, 32 planes (16 um Z), MetaMorph 7.8 acquisition software.

Four zip files:

http://works.bepress.com/gmcnamara/43

http://works.bepress.com/gmcnamara/44

http://works.bepress.com/gmcnamara/45

http://works.bepress.com/gmcnamara/46

with two dataset (001, 002), raw and deconvolved using Bruce & Butte GPU deconvolution in Fiji ImageJ,

http://www.ncbi.nlm.nih.gov/pubmed/23482010

http://www.opticsinfobase.org/vjbo/fulltext.cfm?uri=oe-21-4-4766&id=249375 (free download, as are all Optics Express papers).

http://tcell.stanford.edu/software.html

http://lists.umn.edu/cgi-bin/wa?A2=ind1312&L=confocalmicroscopy&P=5534

Note: "001 raw" (bepress 43) contains my "imaging SOP" …

More Fish (4 Of 4), George Mcnamara

George McNamara

More Stellaris FISH and GPU Deconvolution, 20140313Thur - part 4 of 4

Please see other content in the FISH Imaging section of

http://works.bepress.com/gmcnamara/subject_areas.html

for details on the microscope. Briefly: Leica DMI6000 inverted microscope, SOLA lamp, Hamamatsu FLASH4.0 sCMOS camera, 100 nm XY, 200 nm Z pixels, 32 planes (16 um Z), MetaMorph 7.8 acquisition software.

Four zip files:

http://works.bepress.com/gmcnamara/43

http://works.bepress.com/gmcnamara/44

http://works.bepress.com/gmcnamara/45

http://works.bepress.com/gmcnamara/46

with two dataset (001, 002), raw and deconvolved using Bruce & Butte GPU deconvolution in Fiji ImageJ,

http://www.ncbi.nlm.nih.gov/pubmed/23482010

http://www.opticsinfobase.org/vjbo/fulltext.cfm?uri=oe-21-4-4766&id=249375 (free download, as are all Optics Express papers).

http://tcell.stanford.edu/software.html

http://lists.umn.edu/cgi-bin/wa?A2=ind1312&L=confocalmicroscopy&P=5534

Note: "001 raw" (bepress 43) contains my "imaging SOP" …

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 …

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).