PMID- 28370211
OWN - NLM
STAT- MEDLINE
DCOM- 20190627
LR  - 20190627
IS  - 1365-2818 (Electronic)
IS  - 0022-2720 (Linking)
VI  - 267
IP  - 2
DP  - 2017 Aug
TI  - A protocol for registration and correction of multicolour STED superresolution 
      images.
PG  - 160-175
LID - 10.1111/jmi.12556 [doi]
AB  - Multicolour fluorescence imaging by STimulated Emission Depletion (STED) 
      superresolution microscopy with doughnut-shaped STED laser beams based on 
      different wavelengths for each colour channel requires precise image 
      registration. This is especially important when STED imaging is used for 
      co-localisation studies of two or more native proteins in biological specimens to 
      analyse nanometric subcellular spatial arrangements. We developed a robust 
      postprocessing image registration protocol, with the aim to verify and ultimately 
      optimise multicolour STED image quality. Importantly, this protocol will support 
      any subsequent quantitative localisation analysis at nanometric scales. 
      Henceforth, using an approach that registers each colour channel present during 
      STED imaging individually, this protocol reliably corrects for optical 
      aberrations and inadvertent sample drift. To achieve the latter goal, the 
      protocol combines the experimental sample information, from corresponding STED 
      and confocal images using the same optical beam path and setup, with that of an 
      independent calibration sample. As a result, image registration is based on a 
      strategy that maximises the cross-correlation between sequentially acquired 
      images of the experimental sample, which are strategically combined by the 
      protocol. We demonstrate the general applicability of the image registration 
      protocol by co-staining of the ryanodine receptor calcium release channel in 
      primary mouse cardiomyocytes. To validate this new approach, we identify 
      user-friendly criteria, which - if fulfilled - support optimal image 
      registration. In summary, we introduce a new method for image registration and 
      rationally based postprocessing steps through a highly standardised protocol for 
      multicolour STED imaging, which directly supports the reproducibility of protein 
      co-localisation analyses. Although the reference protocol is discussed 
      exemplarily for two-colour STED imaging, it can be readily expanded to three or 
      more colours and STED channels.
CI  - (c) 2017 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on 
      behalf of Royal Microscopical Society.
FAU - Hebisch, E
AU  - Hebisch E
AD  - Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 
      Gottingen, Germany.
FAU - Wagner, E
AU  - Wagner E
AD  - Heart Research Center Gottingen, Department of Cardiology & Pulmonology, 
      University Medical Center Gottingen, Gottingen, Germany.
AD  - German Center for Cardiovascular Research (DZHK) site Gottingen, Gottingen, 
      Germany.
FAU - Westphal, V
AU  - Westphal V
AD  - Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 
      Gottingen, Germany.
FAU - Sieber, J J
AU  - Sieber JJ
AD  - Leica Microsystems CMS GmbH, Mannheim, Germany.
FAU - Lehnart, S E
AU  - Lehnart SE
AD  - Heart Research Center Gottingen, Department of Cardiology & Pulmonology, 
      University Medical Center Gottingen, Gottingen, Germany.
AD  - German Center for Cardiovascular Research (DZHK) site Gottingen, Gottingen, 
      Germany.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20170403
PL  - England
TA  - J Microsc
JT  - Journal of microscopy
JID - 0204522
RN  - 0 (Ryanodine Receptor Calcium Release Channel)
SB  - IM
MH  - Animals
MH  - Cells, Cultured
MH  - Image Processing, Computer-Assisted/*methods
MH  - Mice
MH  - Myocytes, Cardiac/*enzymology
MH  - Optical Imaging/*methods
MH  - Ryanodine Receptor Calcium Release Channel/*analysis
OTO - NOTNLM
OT  - Image registration
OT  - STED microscopy
OT  - multicolour imaging
OT  - ryanodine receptor
OT  - targeted superresolution microscopy
EDAT- 2017/04/04 06:00
MHDA- 2019/06/30 06:00
CRDT- 2017/04/04 06:00
PHST- 2016/08/09 00:00 [received]
PHST- 2017/02/01 00:00 [revised]
PHST- 2017/02/21 00:00 [accepted]
PHST- 2017/04/04 06:00 [pubmed]
PHST- 2019/06/30 06:00 [medline]
PHST- 2017/04/04 06:00 [entrez]
AID - 10.1111/jmi.12556 [doi]
PST - ppublish
SO  - J Microsc. 2017 Aug;267(2):160-175. doi: 10.1111/jmi.12556. Epub 2017 Apr 3.