UV photonic integrated circuits for far-field structured illumination autofluorescence microscopy

Abstract

Ultra-violet (UV) light has still a limited scope in optical microscopy despite its potential advantages over visible light in terms of optical resolution and of interaction with a wide variety of biological molecules. The main challenge is to control in a robust, compact and cost-effective way UV light beams at the level of a single optical spatial mode and concomitantly to minimize the light propagation loss. To tackle this challenge, we present here photonic integrated circuits made of aluminum oxide thin layers that are compatible with both UV light and high-volume manufacturing. These photonic circuits designed at a wavelength of 360 nm enable super-resolved structured illumination microscopy with conventional wide-field microscopes and without modifying the usual protocol for handling the object to be imaged. As a biological application, we show that our UV photonic chips enable to image the autofluorescence of yeast cells and reveal features unresolved with standard wide-field microscopy.

Fig. 1. Working principle of the UV-PIC SIM.

a Picture of a UV photonic integrated circuit for structured illumination microscopy. MMI: multimodal interferometer (blue), GC: grating out-coupler (violet) and PS: thermal-phase shifter (orange). D₁, D₂, and D₃ indicate three different optical paths corresponding to structured illumination directions turned at an angle of 120 degrees. Light purple path: D₂. b Working principle of the UV-PIC-based SIM technique. W_D: working distance of the microscope objective, W_c distance between the top surface of the photonic chip and the sample. The blue and violet arrows illustrate the fluorescence and the UV excitation light, respectively. c Schematic of the chip-based far-field SIM set-up including a conventional microscope. d Picture of the photonic chip mounted on an electric printed circuit board with gold wire connections.

Fig. 2. Fluorescent sector star target.

a Schematic of the metal sector star target on borosillicate microscope slide with green fluorescent dyes coated on top. Thickness of the coated dye layer: h = 300 nm. b Scanning electron micrograph of the metal sector target. The orange solid circle points out the position where the grating pitch along the circle equals to 100 nm. The imaging is independently repeated five times. c Optical spectra of the UV exciting beam and the fluorescent dyes. λ_ex: excitation wavelength, λ_em: emission wavelength. d to f Fluorescence images of the sector target illuminated by structured light for the D₁, D₂, and D₃ orientations, respectively. Λ_ex : modulation period of the fringe pattern.

Fig. 3. UV-PIC SIM with NA_ex= 0.5.

a Standard (raw data) wide-field (WF) image and b reconstructed super-resolved SIM image of the fluorescent sector star target in the case of an excitation numerical aperture NA_ex = 0.5. c and d Magnified images of the area inside the dashed orange box in a and d, respectively. The blue and red circles have a radius r = 2.2 μm, which corresponds to a grating pitch of 192 nm. e Intensity profiles extracted along the circles in c and d. Λ: spatial period. f Fast Fourier transform of the intensity profiles extracted from the WF (blue), SIM (red), and SEM (black) images along circles of radii r = 3.5 μm, 3.0 μm, 2.2 μm, and 2.05 μm. The theoretical profiles of the optical transfer function (OTF) for the WF and SIM configurations are plotted at the top where the dashed purple line sets the intensity threshold over which the signal is detectable. The vertical dashed black lines locate the maximum spatial frequencies: $K_{\mathrm{Max}}^{\mathrm{WF}}$ for the WF configuration, $K_{\mathrm{Max1}}^{\mathrm{SIM}}\mathrm{and}{K}_{\mathrm{Max2}}^{\mathrm{SIM}}$ for the SIM configuration, where the two values result from the anisotropy of the OTF.

Fig. 4. UV-PIC SIM with NA_ex =  0.9.

a–c Optical images of the UV interference fringe patterns in the case of NA_ex =  0.9 for the D₁, D₃, and D₂ illumination orientations, respectively. d Normalized intensity profile along the dashed line in c. Λ_ex: modulation period of the fringe pattern. e and f Standard wide-field (WF) image and reconstructed SIM image of the fluorescent sector target excited with the patterns in a–c. The radii of the circles r are equal to 1.9 μm, which corresponds to a period of the spatial modulation Λ of 166 nm. g Intensity profiles along the circles in e and f.

Fig. 5. UV-PIC SIM on yeast cells.

a–c Autofluorescence image of NADH in yeast cells under UV structured illumination with D₁, D₂, and D₃ orientations, respectively. The insert is a three times magnified zoom of the area defined by the orange dotted contour to highlight the modulation patterns of the fluorescence intensity resulting from the structured illumination. d Standard wide-field and e reconstructed SIM images of NADH in yeast cells. The experiment is independently repeated three times for a–e on different samples. f and g zoomed images of the area delimited by the orange rectangle in d and e, respectively. h Cross-section profiles along the segment [AB] in f and g.