Adaptive harmonic generation microscopy of mammalian embryos

Abstract

Adaptive optics is implemented in a harmonic generation microscope using a wavefront sensorless correction scheme. Both the second-and third-harmonic intensity signals are used as the optimization metric. Aberration correction is performed to compensate both system-and specimen-induced aberrations by using an efficient optimization routine based upon Zernike polynomial modes. Images of live mouse embryos show an improved signal level and resolution.Harmonic generation microscopy (HGM) was proposed as a method for label-free imaging of biological specimens [1]. Using the nonlinear optical properties of the tissue, third-and second-harmonic generation (THG, SHG) are produced at the focus of a short-pulsed laser beam, revealing cellular structure with three-dimensional resolution. Implementations of combined SHG and THG imaging have often used excitation wavelengths around 1200 nm [2][3][4]. This choice was influenced by the relatively low absorption of this wavelength by biological tissues and the convenient placement of the SHG and THG around 600 and 400 nm, respectively, permitting the use of standard optics and detectors. A disadvantage of this excitation wavelength is that high-numerical-aperture objectives required for such imaging are not typically specified for this wavelength. One should therefore expect reduced performance both in terms of transmission and aberrations. Furthermore, aberrations are introduced by the specimens themselves, particularly by thick specimens to which HGM is often applied [5]. As the harmonic generation is nonlinearly dependent on the excitation intensity, these factors could have a significant effect on the imaging performance.One application area where HGM has shown promise is in imaging for developmental biology [3,2,4]. In early-stage mouse embryos, THG images reveal cellular structure and sub-cellular features, whereas SHG has shown mitotic spindles and the zona pellucida. It has also been shown that large aberrations are induced when focussing through embryos [5]. When combined with the system aberrations arising from the nonoptimal objective lenses, the effects can cause a significant reduction in signal level and resolution. Adaptive optics has been used to compensate for aberrations in various microscopes [6]. In this Letter we demonstrate the use of adaptive optics for the correction of system and specimen-induced aberrations in the HGM of live mouse embryos. OCIS codes: 180.0180, 180.4315, 110.1080, 170.3880, 180.6900, 190.4160. A schematic of the adaptive microscope is shown in Fig. 1. The chromium forsterite laser (Mavericks, Del Mar Photonics) emits 65 fs pulses at a repetition rate of 100 MHz, center wavelength λ=1235 nm and output power around 200 mW. The expanded beam was steered by two-axis galvanometer mirrors that are imaged onto a deformable membrane mirror (MIRAO 52-e, Imagine Eyes). The deformable mirror (DM) was imaged onto the pupil plane of the microscope objective. The illumination power in the focus was approximately 30 mW. The harmonic emiss...

Optical Property Analyses of Plant Cells for Adaptive Optics Microscopy

“…[6,7] AO microscopy has successfully been used to cancel the disturbances caused by cellular structures in wide-field microscopy, [8] laser-scanning confocal microscopy, [9,10] multi-photon microscopy, [11][12][13] harmonic generation microscopy, [14,15] and superresolution stimulated emission depletion microscopy, [16,17] with or without the wavefront sensor. [18] So far, most AO microscopy has observed artificial samples and animal tissues; plant cells and tissues have not been examined by AO microscopy yet.…”

Optimization-based wavefront sensorless adaptive optics for multiphoton microscopy

“…Following [33], we employ the mean image intensity as a metric to correct aberrations in our second-harmonic microscope. Our experiments show successful aberration correction using this metric (see Subsection 4.G).…”

“…For a certain setting of the DM r, the region is scanned using the xyz stage. The corresponding value of the image quality metricỹ is measured as the mean image intensity [32][33][34][35], i.e., the mean pixel value recorded over the region. The pixel dwell time is 0.5 ms and the sampling is 24 pixels × 24 pixels.…”

“…Due to the variations, we were not able to apply the aberration correction algorithm using a single set of parameters, e.g., by correcting aberrations in regions B and C using the parameters computed from region A. Variations in the parameters represent a challenge for model-based aberration correction algorithms, since the parameters are computed once only using a calibration experiment [25][26][27][28][29][30][31][32][33][34][35][36][37].…”

“…Reducing the number of trial aberrations is critical in achieving short image acquisition times and in limiting side effects, such as photobleaching and phototoxicity. For small aberrations, the response of the image quality metric can be approximated using a simple model, such as a quadratic polynomial [25][26][27][28][29][30][31][32][33][34][35]. Model-based aberration correction algorithms [25][26][27][28][29][30][31][32][33][34][35][36][37][38] exploit the knowledge about the model of the metric to quicken the aberration correction, thus curtailing the aforementioned side effects.…”