LUNA-based platform for CSR single-cell imaging and illustration of optical principle.

a, Schematic of hypothesized single-cell bacterial CSR inferred from population-level OD analysis. The red dashed line indicates the time of cold shock (CS) application. After CS, OD ceases to increase, entering an acclimation phase, after which it resumes increase. At the single-cell level, possible cellular size changes following CS include decrease, stasis, or continued increase. b, Schematic of our bespoke LUNA-based platform. Diagram includes the phase-contrast imaging unit, microfluidic-compatible temperature control device and LUNA optical path. Circulating water is indicated by the color cyan. In LUNA, laser beam is reflected into the main optical train by a dichroic mirror. Red dashed line: reflected light. FC, fiber collimator; CL, cylindrical lens; DL, detector lens; FLE, focal length extender. The implementation of LUNA follows this design. c, Computed intensity distribution in the dimensionless space under 100× objective. Computations were performed using the optical model: An off-axis Gaussian beam propagates through an objective lens of radius a and focal length f. Coordinates (x’, y’, z’) equals (kβx, kβy, kβ2z), where k is wavenumber, β = a / f. Images at each axial position were individually normalized. Triangle markers indicate the crescent spots. d, Computed intensity distribution in x-z space. The dashed line indicates the centroid locations of the brightest crescent spots along the z-axis. Image contrast was adjusted for better exhibition (Gamma = 0.5). e, Magnified defocus distance on the detection plane in the ray tracing simulation. Linear regression with slope (95% confidence intervals) and fit goodness is shown. Scale bar, 60 µm. f, Working principle of LUNA. Set Plane1 (Plane2) as the interested focal plane by specifying the centroid location of the line spot on the detector as the origin. The defocus distance Δz between the target and defocused Plane2 (Plane1) is inferred by the displacement Δd. Autofocusing is then achieved by repositioning the spot to the origin. RI, reflection interface; Obj., objective.

Comprehensive performance metrics of LUNA.

a, Focus drift monitoring at 1 Hz in the laboratory environment. Right histogram shows the focal plane positions after each autofocusing procedure. b, Focusing accuracy and precision assessment. Dots and error bars represent the mean and s.d. values of the focus errors. Color palette in b, c, different set thresholds. c, Focusing time measurement for autofocusing procedures at different intervals. Lines those marked with dots emphasize the representative constraints. d, System states over time when imaging bacteria. CS occurs at t = 0. T, temperature of cell environment; RD, relative drift of sample stage before autofocusing; FT, corresponding focusing time. Data in scatter form represent the average of all imaged sites (n = 45). Shaded region indicates the rapid temperature drop period. Error bands in c, d, s.e.m. e, Representative images and onset of autofocusing procedure. Scale bar, 4 µm. Intervals between consecutive procedures indicate the time consumption of phase-contrast imaging and stage motion.

Investigation of single-cell dynamics in CSR using LUNA.

a, Temporal montage of a single microchannel in the microfluidic chip. CS occurs at t = 0. Image was superimposed by cell contours with identified colors according to their generation marks. Timeline unit, hour. Intervals in the left and right panels are 6 and 10 mins, respectively. Scale bar, 4 µm. b, Average growth dynamics of cells. Arrhenius range of growth is shaded in inset. c, Growth deceleration as function of time. The time window of the three Phases is 0-3 min, 3-10 min and 10-120 min, respectively. Error bands in b, c, s.e.m. d, Temperature dependence of growth rate during the dynamic cooling process. Linear fitting to the reciprocal temperature and logarithm of λ is shown as line. Dot lines indicate the 95% prediction bounds. Arrhenius range of growth is shaded. e, An illustration of the allocation of different proteins synthesis at different stage during CSR. Percentage data were retrieved from reference5. f, Average elongation rate across generations as function of normalized interdivision time. g, Average growth rate across generations as function of normalized interdivision time. Error bands in f, g, s.e.m. The color code in f and g marks the same generation as in a.

Cell size regulation and growth synchronism under CS.

a, Notations in cell cycle. ξ, relative CS onset time since cell birth; τd, interdivision time. Dashed cells, expected exponential growth in the steady-state. b, Percentage change of division length of each generation after CS compares to GN. Data are presented as box plots with the edges at the 0.25 and 0.75 quantiles, the central line at the median, and the whiskers at the minima and maxima within 1.5× interquartile range from the box. Circles, outliers outside the whiskers. Each box is located at the average division time of that generation. c, Percentage change of division length of each group between G0 and their corresponding predecessors. Data are presented as box plots as in b. d, Relationship between length extension and birth length across generations (rescaled by the expectation of Lb). Gradient colors indicate data density of each generation. Linear regressions of all the measured cycles and Kendall’s τ are shown. n represents cycle number. e, Lag matrix for distinct groups of ξ. Values were computed by locating the cross-correlation maximum. f, Representative lineages with synchronized elapsed cycles (ξ is 0.10 and 0.90, respectively). e.c., elapsed cycles. g, Elapsed cycle numbers of all lineages as functions of time. Errors shown in Figure S14b.

Analysis of CS experiments of batch culture.

a, Measurements with temperature dropped to 14 ℃. Measured and computed OD600 values (top). Computed data was scaled at t0. Bottom: cell concentrations and average volumes extracted from the flow cytometer, normalized to t0 and average value before CS, respectively. Relative cell density ρdry was computed and scaled for present. Shaded region indicates the acclimation. b, Theoretical σ across a range of equivalent cell volumes of simulated cells. Power exponent was determined by linear regression on the logarithmically transformed data (R2 = 0.996, confidence interval is ±0.01). c, Computed σ from experimental data as function of relative volume. Power exponent was obtained as same process in b (R2= 0.971, confidence interval is ±0.12). Dashed lines indicate the 95% prediction bounds. a.u., arbitrary units. d, Computed total dry mass plotted as a function of OD, together with linear fittings. Fitting for the acclimation has a R2 of -0.263 (Pearson correlation coefficient -0.00). Mass increment during the acclimation is indicated as the height of the shaded area and marked in the inset. Error bars in a, c, d, s.d. e, Total protein measurement of MG1655 strain. Temperature drops to 12 ℃ were performed.