Phase imaging and synthetic aperture super-resolution via total internal reflection microscopy

Abstract : Total internal reflection microscopy is mainly used in its fluorescence mode and is the reference technique to image fluorescent proteins in the vicinity of cell membranes. Here, we show that this technique can easily become a phase microscope by simply detecting the coherent signal resulting from the interference between the field scattered by the probed sample and the total internal reflection. Moreover, combining several illumination angles permits generating synthetic aperture reconstructions with improved resolutions compared to standard label-free micro-scopy techniques. Total internal reflection fluorescence (TIRF) microscopy is the reference technique to study the membrane dynamics and organization in biological cells [1,2]. Its main advantage is the ability to illuminate with a high axial sectioning the sample, typically over a thin slice of about 100 nm, by taking benefit from a total internal reflection (TIR) configuration. The sample is deposited on a glass substrate, and by illuminating through the substrate above the critical angle, an evanescent wave is created at the interface to locally probe the sample. Compared to standard wide-field fluorescence microscopy, images with highly improved contrast are provided, since the background signal stemming from the volume of the sample can be suppressed. The preferred TIRF configuration nowadays uses a high numerical aperture (NA) immersion objective both to illuminate above the critical angle and collect the fluorescence signal. Using fluorophores as contrast agents, however, presents some drawbacks, as their addition is invasive and induces the increased risk of photobleaching and phototoxicity to the cell [3]. Long-term studies are thus difficult to achieve. The spe-cificity of labeling is, moreover, at the same time the main advantage and the main limitation of fluorescence imaging, since the lack of structural images can be detrimental to the interpretation of the results. Therefore, there is a clear need for a high-resolution technique able to probe cell membranes without the constraint of incorporating a fluorescent label. Two main approaches have been used so far to perform label free imaging in TIR with a high NA objective. The first approach uses a spatially incoherent source (such as a light bulb or LED) to illuminate the sample simultaneously with all angles above the critical value imposed by the glass substrate and the sample immersion medium (usually water) [4,5]. As the sample refractive index is higher than that of water, TIR is frustrated at locations where the sample is close to the interface, which modulates the reflected intensity and is the source of contrast in the resulting image. The second approach uses a laser beam focused in the back focal plane (BFP) of the objective at the edge of the NA [6,7]. The focused beam is scanned along a circle in the BFP, so that the sample is illuminated in TIR by a rotating collimated beam, and the integration time of the camera is set to match the rotation period of the beam. The totally reflected beam is blocked in a plane conjugated with the BFP to detect only the scattered intensity and obtain dark-field imaging. Both approaches have a high temporal resolution, and the second one claims an improved resolution at the price of some sample distortions in the image. In this Letter, we propose a label-free TIR microscopy technique that keeps the same setup simplicity as the previous approaches, but permits besides to perform phase imaging and improve the resolution through synthetic aperture generation. The core idea of the technique is that the intensity detected in TIR microscopy can directly give access to the phase and the amplitude of the field scattered by the probed sample. Assuming the scalar approximation, the detected intensity I can be written as I ˆ jE r ‡ E s j 2 ˆ jE r j 2 ‡ jE s j 2 ‡ E r E s ‡ E r E s , (1) where E r is the field reflected by the interface in TIR, and E s is the field backscattered by the sample. As in TIR the sample is illuminated over a very thin slice and is usually of very weak contrast in biology, we can assume E r is very strong compared to E s , and neglect jE s j 2 in Eq. (1). Since E r is close to the edge Letter Vol. 43, No. 9 /
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Guillaume Maire, Hugues Giovannini, Anne Talneau, Patrick Chaumet, Kamal Belkebir, et al.. Phase imaging and synthetic aperture super-resolution via total internal reflection microscopy. Optics Letters, Optical Society of America, 2018, 43 (9), pp.2173. ⟨10.1364/ol.43.002173⟩. ⟨hal-02023424⟩



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