Figure 1.

Optical setup for mpFCS/FLIM. (A) Schematic drawing of the mpFCS/FLIM optical setup. The 482 nm laser beam with elliptical cross-section is transformed into a circular beam using an anamorphic prism pair and expanded using a Kepler telescope setup (L1 and L2) with a pinhole in its focus. The expanded circular laser beam is focused by the focusing lens (L3) mounted on an xyz translation stage, which is positioned in front of the diffractive optical element (DOE) that can be translated along and rotated around the z-axis. The illumination matrix consisting of 16 × 16 (256) spots, which are generated in the image plane of the back port of the microscope, is imaged by the microscope relay optics (L4) and the objective lens to the object plane. Fluorescence is detected by a single-photon avalanche diode (SPAD) camera that can be translated along the z-axis and tilted at two angles (pitch and yaw) or a digital single-lens reflex (DSLR) camera. (B₁) Image of the illumination matrix visualized by the DSLR camera using a thin fluorescence layer as a specimen. (B₂) Enlarged image of a single illumination spot shown in (B₁). Inset: Fluorescence intensity (FI) distribution through the center of the spot (white dashed line) and the best-fit Gaussian curve (red solid line). Spot roundness, assessed by measuring the spot radius in different directions: horizontal (0°; white dashed line), 45, 90, and 135°, showed that the ratio of spot radius over the spot radius at 0° was 1.00, 1.02, 0.96, and 1.04, respectively. (B₃) Histogram of spot radii for all 256 spots in the confocal image of the illumination matrix is shown in (B₁). The average spot radius, r_spot = (17 ± 2) μm, was determined from a half of the full width at half-maximum (FWHM) of the best-fit Gaussian curve. (B₄) Histogram of peak fluorescence intensity for all spots in the confocal image of the illumination matrix is shown in (B₁). The average peak fluorescence intensity, FI_MAX = (90 ± 5) au. (C₁) Scanning-free confocal image of the same specimen as in (B₁) acquired using the SPC³ SPAD camera. Here, each SPAD in addition to being a photodetector also acts as a 30 nm pinhole. Of note, every other SPAD in the centrally positioned 32 × 32 SPADs of the 64 × 32 SPC³ SPAD camera was used. Unilluminated SPADs (dark blue), on the sides and in-between the illuminated ones (yellow–red ones), are clearly distinguishable by fluorescence intensity. (C₂) Histogram of fluorescence intensity, i.e., photon count rates (CR) measured in all illuminated SPADs shown in (C₁). The average fluorescence intensity was determined, CR = (440 ± 35) kHz. (C₃) Scatter plot showing spot peak intensity measured using the SPC³ SPAD camera (C₁) as compared to the spot intensity measured using the DSLR camera (B₁). While a unimodal distribution is observed, six SPADs with disparate values were identified. (D₁) 256 single-SPAD autocorrelation curves (ACCs) recorded in an aqueous buffer solution of eGFP, c_eGFP = 4 nM, with the corresponding average ACC (black). (D₂) ACCs acquired in the same solution as in D₁ by mpFCS (black; same as in D₁) and spFCS ACC (red). The dashed gray line shows G(τ) = 1. (D₃) ACCs shown in D₂ normalized to the same amplitude, G(10 μs) = 1 at τ = 10 μs, acquired using the spFCS (red) and the mpFCS (black) systems. The dashed gray line shows G_n(τ) = 0. In all images, scale bar is 10 μm.