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 exciter

(these helped a bit in improving specificity).

DAPI acquired 10 ms.

All other channels were acquired at 500 ms.

Leica 63x/1.4 NA objective lens, 1x optovar setting, Hamamatsu FLASH4.0 sCMOS camera.

100 nm XY pixel size.

Images were acquired and saved with MetaMorph 7.8.2, in MetaMorph TIFF format with most acquisition settings stored in the TIFF file.

Several datasets were further processed to reduce the crosstalk between the Cy3 cube (want Q570) and TxRed2cube+572ex (want CFR610) by doing:

1. unsharp mask Cy3cube

2. unsharp mask TxRedASI572

3. Arithmetic: Cy3cube_USM minus TxRedASI572_USM

4. Arithmetic: TxRedASI572_USM minus Cy3cube_USM

These steps nicely black out the crosstalk between the Q570 and CFR610 in the Leica filter sets. The experiment is single molecule FISH - each bright dot is (very likely) a single mRNA (or pre-mRNA in the nucleus). The probability of two different mRNA molecules "colocalizing" in large flat cells is low, so unsharp masking and "cross" subtraction is a reasonable image processing step.

I am discussing with Biosearch, Leica, Semrock and Lumen Dynamics, upgrades to the microscope to improve wavelength separation. For now, unsharp masking works ok (and we already have all the components.

Some slides also show some cells have autofluorescence -- possibly multiple types of autofluorescence -- that are both green channel and Cy3 and TxRed2 cube channels.

Entire data set would be a 983 Mb zip file. The bepress file size limit is 800 Mb, so I am uploading in two parts (this and adjacent web page numbers). GM thanks bepress for allowing posting datasets.