Optical sectioning by two-pinhole confocal fluorescence microscopy

Martı́nez-Corral, Manuel; Caballero, María Teresa Caballero; Ibáñez-López, Cristina; Sarafis, V.

doi:10.1016/s0968-4328(03)00057-x

Cited by 17 publications

(8 citation statements)

References 7 publications

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“…An electronically controlled translation table controls the axial movement of a plane mirror to measure the axial optical sectioning curve of the confocal system, plotted in Figure 4b. The normalized axial optical sectioning curve indicates the axial optical transmission efficiency of fluorescence for every axial crosssection because defocus fluorescence signal is filtered by the pinhole [28]. The full width at half-maximum(z c ) is used to estimate the thickness at which diamond fluorescence is detected: z c = 62 µm.…”

Section: Sensitivity Analysismentioning

confidence: 99%

All Fiber Vector Magnetometer Based on Nitrogen-Vacancy Center

et al. 2023

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Magnetometers based on nitrogen-vacancy (NV) centers in diamonds have promising applications in fields of living systems biology, condensed matter physics, and industry. This paper proposes a portable and flexible all-fiber NV center vector magnetometer by using fibers to substitute all conventional spatial optical elements, realizing laser excitation and fluorescence collection of micro-diamond with multi-mode fibers simultaneously and efficiently. An optical model is established to investigate multi-mode fiber interrogation of micro-diamond to estimate the optical performance of NV center system. A new analysis method is proposed to extract the magnitude and direction of the magnetic field, combining the morphology of the micro-diamond, thus realizing μm-scale vector magnetic field detection at the tip of the fiber probe. Experimental testing shows our fabricated magnetometer has a sensitivity of 0.73 nT/Hz1/2, demonstrating its feasibility and performance in comparison with conventional confocal NV center magnetometers. This research presents a robust and compact magnetic endoscopy and remote-magnetic measurement approach, which will substantially promote the practical application of magnetometers based on NV centers.

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Section: Sensitivity Analysismentioning

confidence: 99%

All Fiber Vector Magnetometer Based on Nitrogen-Vacancy Center

et al. 2023

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“…We note that the advantage of using information from differences in confocal pinhole size has long been recognized (Heintzmann et al, 2003;Kakade et al, 2015;Martinez-Corral et al, 2003;Wang et al, 2013) and the virtual adaptable aperture system (VAAS) from Nikon (Okugawa, 2008) represents a commercial implementation based on the same principle. However, most of the proposed techniques are based on the weighted subtraction of two images.…”

Section: Split-pin Imaging Of Subcellular Structuresmentioning

confidence: 99%

A phasor‐based approach to improve optical sectioning in any confocal microscope with a tunable pinhole

D'Amico

Franco

Cerutti

et al. 2022

Microscopy Res & Technique

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Confocal fluorescence microscopy is a well‐established imaging technique capable of generating thin optical sections of biological specimens. Optical sectioning in confocal microscopy is mainly determined by the size of the pinhole, a small aperture placed in front of a point detector. In principle, imaging with a closed pinhole provides the highest degree of optical sectioning. In practice, the dramatic reduction of signal‐to‐noise ratio (SNR) at smaller pinhole sizes makes challenging the use of pinhole sizes significantly smaller than 1 Airy Unit (AU). Here, we introduce a simple method to “virtually” perform confocal imaging at smaller pinhole sizes without the dramatic reduction of SNR. The method is based on the sequential acquisition of multiple confocal images acquired at different pinhole aperture sizes and image processing based on a phasor analysis. The implementation is conceptually similar to separation of photons by lifetime tuning (SPLIT), a technique that exploits the phasor analysis to achieve super‐resolution, and for this reason we call this method SPLIT‐pinhole (SPLIT‐PIN). We show with simulated data that the SPLIT‐PIN image can provide improved optical sectioning (i.e., virtually smaller pinhole size) but better SNR with respect to an image obtained with closed pinhole. For instance, two images acquired at 2 and 1 AU can be combined to obtain a SPLIT‐PIN image with a virtual pinhole size of 0.2 AU but with better SNR. As an example of application to biological imaging, we show that SPLIT‐PIN improves confocal imaging of the apical membrane in an in vitro model of the intestinal epithelium. Research Highlights We describe a method to boost the optical sectioning power of any confocal microscope. The method is based on the sequential acquisition of multiple confocal images acquired at different pinhole aperture sizes. The resulting image series is analyzed using the phasor‐based separation of photons by lifetime tuning (SPLIT) algorithm. The SPLIT‐pinhole (SPLIT‐PIN) method produces images with improved optical sectioning but preserved SNR. This is the first time that the phasor analysis and SPLIT algorithms are used to exploit the spatial information encoded in a tunable pinhole size and to improve optical sectioning of the confocal microscope.

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“…Other publications showed the edge effect with defocus of the sample, and due to their finite tube systems, a defocus of the light is at the pinhole plane, respectively (Sheppard & Heaton, 1984; Wilson & Carlini, 1988b). To improve performances including optical sectioning, techniques, such as two‐pinhole confocal fluorescence, parallel confocal microscopy using high‐order axially symmetric polarized beams and differential confocal microscopy (DCM), are used (Lee & Wang, 1997; Martínez‐Corral, Caballero, Ibáñez‐López, & Sarafis, 2003; Zhou & Zhu, 2015). Nevertheless, all those methods need additional components, extra image postprocessing or more image acquisition time, or defocus the sample and thus decrease the resolving power of the system.…”

Section: Introductionmentioning

confidence: 99%

Realization of noncontact confocal optical microsphere imaging microscope

Zhou

Hong

2021

Microscopy Res & Technique

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A novel noncontact confocal optical microsphere imaging microscope (COMIM) is proposed to achieve high contrast optical nano‐imaging at a high scanning speed. The developed setup obtains images with significantly higher contrast when compared with conventional bright‐field microsphere nano‐imaging, as well as with a commercial confocal laser scanning microscope (CLSM). With the image‐by‐image scanning scheme, COMIM takes only 11% time to complete a three‐dimensional (3D) scanning image than using a commercial CLSM, while still offers an outstanding resolving power for the nano‐features on the sample.

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Optical sectioning by two-pinhole confocal fluorescence microscopy

Cited by 17 publications

References 7 publications

All Fiber Vector Magnetometer Based on Nitrogen-Vacancy Center

All Fiber Vector Magnetometer Based on Nitrogen-Vacancy Center

A phasor‐based approach to improve optical sectioning in any confocal microscope with a tunable pinhole

Realization of noncontact confocal optical microsphere imaging microscope

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