Cell and Developmental Biology Commons^™

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 …