Application of a reflective microscope objective for multiphoton microscopy

Kabir, Mohammad M.; Choubal, Aakash; Toussaint, Kimani C.

doi:10.1111/jmi.12702

Cited by 6 publications

(2 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To further improve image quality, it is possible to combine the Schmidt objective with adaptive optics. Owing to their low dispersion and large wavelength range, reflective objectives have previously been used for multi-photon microscopy 23 , 24 but not as fully immersed systems. We think that our work opens an unexplored design space for building immersion microscope objectives.…”

Section: Discussionmentioning

confidence: 99%

Reflective multi-immersion microscope objectives inspired by the Schmidt telescope

et al. 2023

View full text Add to dashboard Cite

Imaging large, cleared samples requires microscope objectives that combine a large field of view (FOV) with a long working distance (WD) and a high numerical aperture (NA). Ideally, such objectives should be compatible with a wide range of immersion media, which is challenging to achieve with conventional lens-based objective designs. Here we introduce the multi-immersion ‘Schmidt objective’ consisting of a spherical mirror and an aspherical correction plate as a solution to this problem. We demonstrate that a multi-photon variant of the Schmidt objective is compatible with all homogeneous immersion media and achieves an NA of 1.08 at a refractive index of 1.56, 1.1-mm FOV and 11-mm WD. We highlight its versatility by imaging cleared samples in various media ranging from air and water to benzyl alcohol/benzyl benzoate, dibenzyl ether and ethyl cinnamate and by imaging of neuronal activity in larval zebrafish in vivo. In principle, the concept can be extended to any imaging modality, including wide-field, confocal and light-sheet microscopy.

show abstract

Section: Discussionmentioning

confidence: 99%

Reflective multi-immersion microscope objectives inspired by the Schmidt telescope

et al. 2023

View full text Add to dashboard Cite

show abstract

“…Obscuration refers to mechanical light attenuation from mirror design, where a significant portion of light entering the objective fails to reflect to a secondary mirror, and an additional fraction is obstructed by thin suspensions used to mount the primary mirror. Encouragingly, simulation has shown that back-reflections may be minimized by the adoption of annular-shaped beams in favor of Gaussian profiles [13]. Moreover, reflective objectives' transmission properties (>99% from 450 nm to 20 μm) vastly outperform traditional refractive objectives with NIR coatings, including optimized multiphoton objectives (~70% from 1100 to 1400 nm) [14].…”

mentioning

confidence: 99%

Improved nondegenerate multiphoton microscopy and axial registration with a reflective objective

2019

View full text Add to dashboard Cite

Conventional, degenerate multiphoton microscopy (D-MPM) requires the use of a high-numericalaperture (NA) objective. Nondegenerate MPM (ND-MPM) imposes the additional demand for precise spatiotemporal overlap of two distinct excitation sources. We demonstrate that the axial focal shift introduced by refractive objective chromatic aberration hinders the spatial requirement of ND-MPM, whereas the use of a reflective objective overcomes this challenge and allows for improved ND excitation efficiency in spite of a lower NA. Moreover, we demonstrate that reflective objective focusing eliminates the axial misregistration of volumetric stacks in traditional D-MPM experiments when multiple excitation wavelengths are used. Conventional, or degenerate, multiphoton microscopy (D-MPM) relies on the absorption of two or more spatiotemporally overlapped photons of identical energies [1]. Nondegenerate multiphoton microscopy (ND-MPM) combines synchronized pulses from two lasers of different wavelengths (λ 1 and λ 2 ) to excite a fluorophore transition [2,3]. Provided the two pulses arrive to the same location at the same time, the energies of the photons add at an effective excitation wavelength given by λ 3 = 2 λ 1 −1 + λ 2 −1 −1 [4]. ND-MPM has severalunique advantages, including simultaneous multicolor imaging capabilities [4,5], improved signal-to-background ratio [6], and the ability to excite fluorophores with substrateincompatible absorptions at the virtual λ 3 wavelength. Notably, the total excitation of the combined beams is given by the cross-correlation term I 1 + I 2 2 = I 1 2 + I 2 2 + 2I 1 I 2 , where I 1 2 is the excitation profile (i.e., point spread function, or PSF) at λ 1 , I 2 2 dictates the PSF at λ 2 , and 2I 1 I 2 dictates the PSF at λ 3 [4]. The ability to target a fluorophore at λ 3 with ND-MPM, yet probabilistically stimulate fluorescent events at all three excitation pathways, has been shown to achieve a significant increase in excitation efficiency relative to D-MPM [7].Traditional multiphoton objectives are composed of a series of refractive lenses that compensate for one another's aberrations. High-quality objectives are commonly corrected to improve image quality by minimization of spherical aberration, coma, astigmatism, and distortion. Unfortunately, chromatic aberration is unavoidable with refractive objectives, meaning that distinct wavelengths are focused at different optical z-planes. Achromat objectives tailored to minimize this undesired effect can only provide chromatic correction *

show abstract

Demonstration of flat-top beam illumination in widefield multiphoton microscopy

et al. 2019

Self Cite

View full text Add to dashboard Cite

Multiphoton microscopy provides a suitable technique for imaging biological tissues with submicrometer resolution. Usually a Gaussian beam (GB) is used for illumination, leading to a reduced power efficiency in the multiphoton response and vignetting for a square-shaped imaging area. A flat-top beam (FTB) provides a uniform spatial intensity distribution that equalizes the probability of a multiphoton effect across the imaging area. We employ a customized widefield multiphoton microscope to compare the performance of a squareshaped FTB illumination with that based on using a GB, for both two-photon fluorescence (TPF) and second-harmonic generation (SHG) imaging. The variation in signal-to-noise ratio across TPF images of fluorescent dyes spans ∼5.6 dB for the GB and ∼1.2 dB for the FTB illumination, respectively. For the GB modality, TPF images of mouse colon and Convallaria root, and SHG images of chicken tendon and human breast biopsy tissue showcase ∼20% area that are not imaged due to either insufficient or lack of illumination. For quantitative analysis that depends on the illuminated area, this effect can potentially lead to inaccuracies. This work emphasizes the applicability of FTB illumination to multiphoton applications.

show abstract

Application of a reflective microscope objective for multiphoton microscopy

Cited by 6 publications

References 27 publications

Reflective multi-immersion microscope objectives inspired by the Schmidt telescope

Reflective multi-immersion microscope objectives inspired by the Schmidt telescope

Improved nondegenerate multiphoton microscopy and axial registration with a reflective objective

Demonstration of flat-top beam illumination in widefield multiphoton microscopy

Contact Info

Product

Resources

About