GM 20240808H Opal TSA Z-series Leica HyVolution #2 - raw and deconvolved using HyVolution2.

1.3 GB Leica LIF image(s) container file.

* See https://works.bepress.com/gmcnamara/97 for modest size jpg image showing raw and deconvolved from Series003 (bepress not good at displaying thumbnails - I am happy it let me upload 1.3 GB file).

Leica SP8 confocal microscope, 488nm laser line, 600 Hz, XY=50nm pixel size, Z=150nm step size, 510-540nm emission range, confocal sweetest spot 0.66 Airy Units (re: Jeff Reece, NIH,confocal sweet range 0.6-0.7 Airy Unit), HyD (2nd generation, linear range up to 60 photons per microsecond).

Dell Z640 workstation, NVidia M6000 GPU (Maxwell generation, 48 GB RAM); GM installed ASUS PCIe gen3 x16 card with four NVMe (M.2) SSDs as fast data drive.

Screen shot is raw on left, deconvolved on rigght from dataset series003, plane 15, upper center.

Series002 used 1 line accumulation, acquired in ~2 minutes (16-bit mode).

Series003 used 10 line accumulation, acquired in 25 minutes (16-bit mode).

Tyramide signal amplification boosts the signal 10x to 100x higher than conventional unlabeled primary + fluorescent dye(s) secondary antibodies. Andrew Belmont (see TSA-Seq publication and earler from their lab) publisheddiffusion radius of activated tyramide radical is ~50 nm in water (aqueous buffer) and can be made less (ex. 20nm) by adding sucrose or other reagent(s) to increase viscosity.

Specimen: Opal 8plex tryramide signal amplification slide from Prof. Janis Taube & lab manager, JHU SOM Dermatopathology - see their AStroPath Science paper (astronomy meets pathology) with Alex Szalay (JHU Astronomy - a key player in Sloan Digital Sky Survey, petabyte, maybe exabyte scale astronomy data online).

The leica LIF file can be viewer with Leica LAS X

https://webshare.leica-microsystems.com/latest/core/confocal  (free download, use "setup" not F-techniques),

MetaMorph (GM was the lead MetaMorph applications and technical support scientist, 1992-1997), Fiji ImageJ and many other programs (the first three all have export capability).

* I have more on tyramide signal amplification at http://confocal.jhu.edu/mctips (search page for TSA or tyramide).