Peer review of: Multiplexed Optical Sensors in Arrayed Islands of Cells for multimodal recordings of cellular (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
I enjoyed reading this manuscript, which describes a smart technical approach to simultaneously 
apply different genetically encoded probes in a microarray assay. The authors used defined 
lentivirus particles that were blotted on a microarray to infect cells and consequently express 
different genetically encoded probes in cells of interest. A special wide-field microscope was used 
to image the grouped fluorescent probes in HEK293 cells or cardiac myocytes simultaneously. The 
novel technology was named MOSAIC, which seems an appropriate name. The sophisticated 
technology was well described and its potential was nicely demonstrated. 
The following corrections are necessary prior to publication of the work: 
On page 4 it should be “Figure 2e” not “Figure 3e” and “Figure 2f” and not “Figure 3f”. 
I have these concerns/suggestions: 
1.) The Ca2+ measurements shown in Figure 5 are less convincing. Why at the beginning of the 
protocol Ca2+ in the ER and cytosol is decreasing while mitochondrial Ca2+ seems to increase 
stepwise? If under these conditions of energy stress, Ca2+ is mobilized from the ER, Ca2+ should 
consequently first increase in the cytosol (which is not the case) and then within mitochondria. It 
is also difficult to understand why Ca2+ increases transiently within the cytosol and ER during 
addition of rotenone and oligomycine. During this phase of the protocol, Ca2+ is restored within 
depolarized mitochondria. The authors should give a plausible explanation for these subcellular 
Ca2+ fluxes. In addition it might help to study and compare well-known Ca2+ signals in response 
to an activation of e.g. the IP3 pathway using ATP. Ca2+ signals between the sub-compartments 
(cytosol, ER, Mitochondria) from the cell population might not correlate well due to cell-to-cell 
variations. Hence, some comparisons with single cell responses might be helpful. 
2.) Also in Figure 5: Why is the glucose level not reduced when glucose is removed from cells? 
Was glucose already removed prior to imaging? 
3.) Measurement of mitochondrial ATP dynamics might represent a more informative response 
compared to cytosolic ATP changes; see Depaoli et. all Cell Reports 2018; DOI: 
10.1016/j.celrep.2018.09.027. Also for ATP changes huge cell-to-cell variations have been shown. 
The authors might better discuss that the low resolution (2 x objective) does not well allow to 
resolve single cell responses. 
4.) The authors should cite Kuchenov et al., 2016, Cell Chemical Biology; 
http://dx.doi.org/10.1016/j.chembiol.2016.11.008. This work describes a similar approach using 
plasmids and FRET-based genetically encoded probes. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
The authors have developed an impressive tool to parallelize a large number of optical 
measurements in a single system. The technique might be very useful for the field and is 
thoroughly characterized. Internal correction of the probe signals for pH effects is elegant, highly 
valuable and makes the setup robust. 
While the technique itself is impressive and suitable for the journal, the biological relevance and 
results are not yet convincing and require some extra work. hPSC-CM experiments seem minimal. 
Furthermore, it should be made clear whether this tool is ‘just’ an all-in-one tool to measure 
multiple parameters or whether there is a benefit in measuring those parameters simultaneously – 
as is claimed. 
 
Major points: 
- Advantage 4 (“For cells that communicate over long distances via electrical signaling such as a 
cardiomyocytes or neurons, behavior can be synchronized throughout the well.”) does not take 
into account propagation times required for signals to travel. While this might not be problematic 
for slow response behaviour this will limit time-correlation between fast-response transients such 

as AP, Ca and contractility. In turn, this might also provide an opportunity: is it possible to assess 
propagation, a key parameter for excitable cells, using this system? 
- Details are required on the efficiency of transfection, especially when using hPSC-CMs since they 
are known to be not easily transfected. The manuscript should include transfection efficiency 
numbers so the number of cells from which the signals originate can be assessed. For example: 
cardiomyocytes are quite large so according to the calculations, islands might contain tens of cells. 
If the transfection efficiency is below 20% - which might not be an unrealistic estimate based on 
sensor 20 in figure 7b for example – this would result in maybe 30 cells * 20 percent = 6 cells. 
Since hPSC-CM populations are not entirely pure, supporting cell types such as fibroblasts might 
be the ones transfected instead of cardiomyocytes. More quantitative details on the transfection 
efficiency and identity of the target cells are required. 
- Importantly, the results from the measurements with cardiomyocytes seem to be a single 
experiment and results are not quantitatively analysed. As standard in the field, this experiment 
should be repeated at least three times and claims such as changes in triangulation, oscillations, 
amplitudes, etc. should be made based on quantitative data from those repeated experiments. In 
addition, it should be corrected for baseline/vehicle measurements (wash in, wash out of buffers, 
etc.). 
- In line with the previous point, the authors mention “The increase in cytosolic calcium transient 
amplitude, expected for positive inotropic β2-adrenergic stimulation72, did not occur immediately 
after perfusion but was observed in the subsequent high-speed recording 2 min later, implying 
that a signaling cascade was required to activate the inotropic effect.” but also such an increase in 
AP amplitude is seen which is unexpected. How can the reader be sure this increase in amplitude is 
not a washing artefact? Again, statistical comparison to baseline changes from multiple 
experiments are needed before making such claims. 
- In the discussion, the authors need to be more critically reflective on how their system compares 
to others. For example, how does it compare to other integrated optical solutions (For example 
Clyde Bioscience System and https://www.nature.com/articles/s41467-019-12354-8)? What 
conclusions can be drawn that cannot be drawn if the data are not measured simultaneously? Or is 
it merely increased throughput? What are the limitations of the system? Also, the analysis seems 
to require a lot of manual tweaking because of cross contamination. How does this affect the 
throughput? 
- Figure 7b, in 11 there seems to be a labelled region/area (or contamination??) stretching from 
one island to another one. What is happening here? 
 
 
Minor points: 
- Figure 2e and 2f are described in the text as 3e and 3f. 
- In figure 7d, please add the names of the compounds. It is now a bit hard to follow. 
 
 
 
Reviewer #3: 
Remarks to the Author: 
To clarify simultaneously the different response behaviors in cells treated by chemical or physical 
treatment, the authors of this manuscript developed a technique, Multiplexed Optical Sensors in 
Arrayed Islands of Cells (MOSAIC). This method enables parallel sensing for different signaling or 
metabolism species or events utilizing the patterned cells encoded with different fluorescent sensor 
with microarray printing the sensor encoding lentiviral vectors. The special design for motile and 
nonmoyile cells has also been displayed. This technique is a new advance in synchronous sensing 
of cell events in a number of living cells, which provides more accurate same culture conditions 
and timing sequence. It is desirable the rapid publication in this journal. Before the acceptance for 
publication, the following concerns should be addressed. 
1. The marker detection for the proposed autophagy in HEK293 cells upon nutrient deprivation 
should be carried out to confirm the hypothesis. Otherwise, the related discussion is questionable. 
2. What is exact reason for the different plasmid number in different islands, especially the islands 

for same sensing target? And how to guarantee the desired number in practical experiments. 
3. The immobilization of motile cells might be discussed further, how to evaluate the effect of 
immobilization and is there any other measures to improve the immobilization of the cells?