Circularly polarised luminescence laser scanning confocal microscopy to study live cell chiral molecular interactions
Authors: Lewis MacKenzie
Robert Pal
Patrycja Stachelek
David Parker
Journal: Nature Communications (2022)
Discipline: Multidisciplinary
Keywords: chiral building blocks; live cell bio-imaging; Luminescent chiral molecules; circularly polarized light (CPL); CPL Laser Scanning Confocal Microscope (CPL-LSCM)
Affiliations: Department of Chemistry, Durham University, South Road, Durham, DH1 3LE, UK., Department of Chemistry, Durham University, South Road, Durham, DH1 3LE, UK. .

Abstract:  The molecular machinery of life is founded on chiral building blocks, but no experimental technique is currently available to distinguish or monitor chiral systems in live cell bio-imaging studies. Luminescent chiral molecules encode a unique optical fingerprint within emitted circularly polarized light (CPL) carrying information about the molecular environment, conformation, and binding state. Here, we present a CPL Laser Scanning Confocal Microscope (CPL-LSCM) capable of simultaneous chiroptical contrast based live-cell imaging of endogenous and engineered CPL-active cellular probes. Further, we demonstrate that CPL-active probes can be activated using two-photon excitation, with complete CPL spectrum recovery. The combination of these two milestone results empowers the multidisciplinary imaging community, allowing the study of chiral interactions on a sub-cellular level in a new (chiral) light. 

Retracted: No

PubPeer comments:
 Figure s3, which compares the CPL spectra of the right and left enantiomers of a europium complex in three different solvents, is in fact the duplication of a single figure that has been modified by a simple vertical flip. This obvious edition is hilighted in the following figure where A and flipped B side have been superimposed. ![file] , 
 Fig. 1B and Fig. 3B show the excitation power dependency of the two-photon excitation induced photoluminescence intensity that have been performed with two different europium complexes at two different excitation wavelengths. However, surprisingly, the two plots can be perfectly superimposed, suggesting that the two data are identical. ![file] , 
 #2 We thank the anonymous commenter u201cPrionasciadium humileu201d for the eagle eyed observations and close reading of our manuscript. We wish to address their comments in two parts. Firstly, regarding apparent similarities in Fig. 1B and Fig.3B, the excitation power dependency of these two different complexes is different (see tables of data below). However, as the commenter pointed out, when plotted and presented in this format, their power dependencies are very similar to the eye. This similarity is exacerbated because of (a) the log-log nature of the plot and (b) the size of the diamond markers used in each plot. These combine to hide the small differences in the plot. However, the differences are evidenced by the 0.04 difference in the gradient of the slope for the plots given below. We further wish to comment on the methods of the powder dependency measurement. The data for the complex in Fig.1B is recorded at the excitation wavelength of 720 nm and the Fig. in 3B is recorded at the excitation wavelength at 700 nm. At these wavelengths, the maximum power output of the tuneable near-infrared laser used is 1410 mW and 1421 mW respectively, which is nearly identical. Further, the laser power is controlled precisely within the laser via software-controlled variable neutral density filter cross polarisers, providing highly repeatable measurements. Emission for each complex was monitored at similar, but slightly different europium emission wavelengths (615 nm for Fig.1B, and 613.5 nm for Fig. 3B) representing peak of emission for each complex. Measurements were repeated three times and the data plotted is the average of these three measurements. Due to the near-identical and well controlled experimental conditions, the log-log plot produces similar empirical slopes within the measure error range of u00b10.1. The observed similarities in the photophysical properties of the two complexes is coincidental. We have checked and verified this several times. Fig1B. Alternative plot of data also showing the fit outputs https: www.dropbox.com s ylegl03oibog48z Slide1.JPG?dl=0 Fig3B. Alternative plot of data also showing the fit outputs https: www.dropbox.com s m9pr19k1awneyva Slide2.JPG?dl=0 Below: overlay of alternative plots of data in Fig. 1B and Fig. 3B, showing that the difference between the two plots is nearly impossible to see by eye. https: www.dropbox.com s ifc65e3w20wq9rf Slide3.JPG?dl=0 Therefore, we feel that despite the concerns raised and that the plots do appear to be similar to the naked eye as presented in the manuscript we will take no further action on this concern raised , 
 #1 Regarding Fig. S3, we agree that this plot is incorrect. We agree that on this plot the incorrect inverted enantiopure original data of one enantiomer has been plotted when all 3 spectra have been normalised. We are extremely grateful that the reviewer has brought this to our attention because they have identified a mistake in our Origin data file. This mistake arose due to human error. Further, we have noticed that the figure legend for the CPL (mV) axis is also incorrectly formatted and should have ranged from -2 to +2 and at no CPL activity it should always be 0 mW! To our trained eyes this should have immediately drawn our attention to the figure being wrong. To elaborate further we believe these errors originated due to human error not realising an unusual error in the employed normalisation function (OriginPro) being corrupted arising from a hasty attempt to normalise the CPL spectra so as to compare solution-state (i.e. MeOH and NMP) and solid-state PVP results, with the intent of showing that the sign and nature of CPL emission is conserved. We should have noticed these errors when producing the manuscript and during the peer-review process. There is no excuse for these mistakes and apologise for these errors wholeheartedly. To rectify these errors in Fig. S3, we have replotted the CPL spectra with the correct raw data for both the lambda and delta enantiomers and not normalised the data. From this new plot (see Corrected Fig. S3 below) it is clear to see that the mirror CPL sign is preserved, but the spectra are different, including the noise in measurement and most importantly with appropriate axis values. We have contacted the Editorial Team at Nature Communications to issue a correction as an erratum. We once again wish to thank the anonymous commentor, who woe could like to credit in the erratum. If the anonymous commentor would like to take credit for their assistance, then please contact the authors of the manuscript. https: www.dropbox.com s 8u0moqeaclmww1w Slide4.JPG?dl=0 SI Fig. 3. Key spectra of u039b- and u0394 Eu:L1 in relevant solvents and PVP. (A) One photon CPL spectra (u03bbex.= 355 nm) of u0394 Eu:L1 in MeOH (solid black line) and N-Methyl-2-pyrrolidone (NMP) (blue) and Poly(vinyl-pyrrolidine) (PVP) matrix (solid red line). (B) One photon CPL spectra (u03bbex.= 355 nm) of u039b Eu:L1 in MeOH (black) and N-Methyl-2-pyrrolidone (NMP) (solid blue line) and Poly(vinyl-pyrrolidine) (PVP) matrix (red). ,