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. 2020 Jul 23;182(2):515–530.e17. doi: 10.1016/j.cell.2020.05.051

Figure 1.

Figure 1

Design and performance of the super-resolution fluorescence microscope CryoSIM

(A and B) Widefield (WF) (A) and SIM lateral point spread (B) of a 175 nm diameter, 505/515 nm wavelength microsphere (PS-Speck Thermo Fischer Scientific) collected on the cryoSIM at 71 K by using 488 nm light for excitation and a 520/35 nm filter (Semrock) for emission.

(C) Plot of lateral point spread (single line profile) of (A) and (B) and their respective Gaussian fits showing clear resolution enhancement. Analysis of representative cryo-SIM data from a single mammalian U2OS cell containing structures tagged with mCherry.

(D) Z axis projection of the raw SIM data processed to produce a widefield image stack.

(E) Z axis projection of the same data deconvolved with a standard Richardson-Lucy iterative deconvolution algorithm.

(F) SIM data fully reconstructed showing the resulting resolution enhancement.

(G) Fourier information content analysis of (H)–(K) (flattened and sampled radially) showing the relative increase in information content after reconstruction. The vertical blue bars show the resolution achieved at 525 nm emission in widefield (420 nm, 1/2.38) and SIM (200 nm, 1/5).

(H and I) Reciprocal space resolution plots of the widefield data in the cell shown in (D) (Alexa488 and mCherry signal, respectively).

(J and K) Same data as (H) and (I), respectively, from the SIM reconstruction shown in (F). Comparing (J) with (H) and (K) with (I) shows the increased image resolution in SIM by the extra intensity further from the origin and therefore at higher frequencies. Concentric dashed circles denote resolution boundaries of 600 nm (small circle) and 200 nm (large circle). All image analysis was done using Fiji (Schindelin et al., 2012) and SIMcheck (Ball et al., 2015).

(L) SPEKcheck (Phillips et al., 2018) display of the cryoSIM for a representative fluorophore (Alexa488) excited in the system with an efficiency of 69% and a resulting collection of 70% of the total emitted light. Laser light is shown as a bright blue spike, excitation and emission are shown in shades of light green, and the total transmitted light is shown in bright green. The system dichroic mirrors and filters are also represented (Abbreviations are as follows: ZT, system dichroics; T560, detector splitter dichroic; EM, emission filter).