DOI: 10.5281/zenodo.15438495
title: Confocal- and STED-FLIM images of neuronal proteins

description: Confocal- and STED-FLIM images of different protein pairs and fluorophore combinations in live and fixed hippocampal neuronal cultures. Acquired on an Expert Line STED microscope (Abberior Instruments) equipped with a Time Tagger Ultra 8 (Swabian Instruments). Provided both as raw .msr files and .tiff files. Tiff files are structured as (x,y,c=2,t=250) c being Confocal and STED, t being the time bins. 

Pixel size is 20 nm for images of fixed cells and 25 nm for images of live cells. Time bins are 80ps (250 bins in the 0-20 ns interval). STED images acquired with different depletion powers. The acquisition of the images in this dataset was automated using a python script based on the Abberior-STED package.

Details concerning the imaging parameters used are provided in the Methods section below.
description: Folders:





RedDyes_SingleLabel (265 images, 34.2GB )





B2Spectrin_STORANGE




Homer_STORANGE





ShortAcq




MediumAcq




LongAcq






msBassoon_CF594





ShortAcq




MediumAcq




LongAcq






rabBassoon_CF594




PSD95_STORANGE






RedDyes_DualLabel (159 images, 25GB)





Homer_STOrange_msBassoon_CF594





ShortAcq




MediumAcq




LongAcq






PSD95_STOrange_rabBassoon_CF594




Spectrin_STOrange_rabBassoon_CF594









FarRedDyes_SingleLabel (188 images, 44.2GB)





Fixed cells





alphaTubulin_Alexa647




B2Spectrin Alexa647




PSD95 Alexa647




rabBassoon STAR635P






Live cells





SiR_Actin




Tubulin_LIVE610








FarRedDyes_DualLabel (102 images, 33.7GB)





Fixed cells





msB2Spectrin_AF647_rabBassoon_STAR635P




msPSD95_AF647_rabBassoon_STAR635P




Tubulin_Alexa647_Bassoon_STAR635P






Live cells





SiR_Actin_Tubulin_LIVE610
description: Red Dyes - Fixed cells 

Homer-STAR ORANGE and Bassoon-CF594




 
ShortAcq
ShortAcq
MediumAcq 
MediumAcq
LongAcq
LongAcq


 Parameter
Confocal-FLIM
STED-FLIM
Confocal-FLIM
STED-FLIM
Confocal-FLIM
STED-FLIM


Excitation wavelength
561 nm
561 nm
561 nm
561 nm
561 nm
561 nm


Excitation power
2.6 μW
2.6 μW
2.6 μW
2.6 μW
2.5 μW
2.5 μW


Depletion wavelength
 
775 nm
 
775 nm
 
775 nm


Depletion powers
 
44, 88, 132, 176 mW
 
44, 88, 132, 176 mW
 
44, 88, 132, 176 mW


Detection wavelengths
605-625 nm
605-625 nm
605-625 nm
605-625 nm
605-625 nm
605-625 nm


Pixel dwelltime
15 μs
15 μs
15 μs
15 μs
12 μs
12 μs


Line steps
4
8
6
12 
10
38


Detection time bins
250 bins of 80 ps
250 bins of 80 ps
250 bins of 80 ps
250 bins of 80 ps
250 bins of 80 ps
250 bins of 80 ps


Pixel size
20 nm
20 nm
20 nm
20 nm
20 nm
20 nm


Pinhole size
1.0 A.U.
1.0 A.U.
1.0 A.U.
1.0 A.U.
1.0 A.U.
1.0 A.U.




PSD95- STAR ORANGE and Bassoon - CF594




 Parameter
Confocal-FLIM
STED-FLIM


Excitation wavelength
561 nm
561 nm


Excitation power
2.6 μW
2.6 μW


Depletion wavelength
 
775 nm


Depletion powers
 
44, 88, 132, 176 mW


Detection wavelengths
605-625 nm
605-625 nm


Pixel dwelltime
15 μs
15 μs


Line steps
6
25


Detection time bins
250 bins of 80 ps
250 bins of 80 ps


Pixel size
20 nm
20 nm


Pinhole size
1.0 A.U.
1.0 A.U.




β2 Spectrin- STAR ORANGE and Bassoon - CF594




 Parameter
Confocal-FLIM
STED-FLIM


Excitation wavelength
561 nm
561 nm


Excitation power
2.6 μW
2.6 μW


Depletion wavelength
 
775 nm


Depletion powers
 
44, 88, 132, 176 mW


Detection wavelengths
605-625 nm
605-625 nm


Pixel dwelltime
15 μs
15 μs


Line steps
6
12


Detection time bins
250 bins of 80 ps
250 bins of 80 ps


Pixel size
20 nm
20 nm


Pinhole size
1.0 A.U.
1.0 A.U.




Far Red Dyes - Fixed cells




Parameter
Confocal-FLIM
STED-FLIM


Excitation wavelength
640 nm
640 nm


Excitation power
1 μW
2 μW


Depletion wavelength
 
775 nm


Depletion powers
 
22, 44, 66, 88 mW


Detection wavelengths
650-720 nm
650-720 nm


Pixel dwelltime
15 μs
15 μs


Line steps
8
10


Detection time bins
250 bins of 80 ps
250 bins of 80 ps


Pixel size
20 nm
20 nm


Pinhole size
1.0 A.U.
1.0 A.U.




Far Red Dyes - Live cells




Parameter
Confocal-FLIM
STED-FLIM


Excitation wavelength
640 nm
640 nm


Excitation power
2.5 μW
3.6 μW


Depletion wavelength
 
775 nm


Depletion powers
 
 44, 66, 88,132,176 mW


Detection wavelengths
650-720 nm
650-720 nm


Pixel dwelltime
15 μs
15 μs


Line steps
3
16


Detection time bins
250 bins of 80 ps
250 bins of 80 ps


Pixel size
25 nm
25 nm


Pinhole size
1.0 A.U.
1.0 A.U.