A High Precision Laser-Based Autofocus Method Using Biased Image Plane for Microscopy

Gu, Chenglin; Cheng, Hao; Wu, Kaijie; Liang-jun, Zhang; Guan, Xinchun

doi:10.1155/2018/8542680

Cited by 13 publications

(11 citation statements)

References 10 publications

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“…Such calculated metrics typically do not distinguish whether the defocus is positive or negative. This may be addressed by offsetting the focusing of the back-reflected laser beam at the autofocus camera such that any drift of the sample relative to the microscope objective does not cause the reflected laser beam to pass through focus (minimum spot size) on the autofocus camera 8 . Unfortunately, this decreases the operating range of the optical autofocus.…”

Section: Introductionmentioning

confidence: 99%

Robust, single-shot, optical autofocus system utilizing cylindrical lenses to provide high precision and long range of operation

Lightley

Kumar

García

et al. 2022

Preprint

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We present a robust, "real-time" optical autofocus system for microscopy that provides high accuracy (<230 nm) and long range (~130 μm) with a 1.4 numerical aperture oil immersion objective lens. This autofocus can operate in a closed loop, single-shot functionality over a range of ±37.5 μm and can also operate as a 2-step process up to ±68 μm. A real-time autofocus capability is useful for experiments with long image data acquisition times, including single molecule localization microscopy, that may be impacted by defocusing resulting from drift of components, e.g., due to changes in temperature or mechanical drift. It is also vital for automated slide scanning or multiwell plate imaging where the sample may not be in the same horizontal plane for every field of view during the image data acquisition. To realise high precision and long range, we implement orthogonal optical readouts using cylindrical lenses. We demonstrate the performance of this new optical autofocus system with automated multiwell plate imaging and single molecule localisation microscopy and illustrate the benefit of using a superluminescent diode as the autofocus light source.

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Section: Introductionmentioning

confidence: 99%

Robust, single-shot, optical autofocus system utilizing cylindrical lenses to provide high precision and long range of operation

Lightley

Kumar

García

et al. 2022

Preprint

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“…Methods based on image processing need to calculate multiple pictures to estimate the focal position 15 . These methods are rather time-consuming because the requirements of a full-range searching process 16 . Meanwhile, methods based on reflected spot signal are widely used because of their high processing efficiency and low cost.…”

Section: Introductionmentioning

confidence: 99%

Defocusing detection of high-speed imaging for samples with arbitrary pattern structures

Gao

Wei

2022

Thirteenth International Conference on Information Optics and Photonics (CIOP 2022)

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High-speed imaging is very important in the semiconductor optical mask field and biology and life sciences. There have been countless auto-focusing methods based on reflected light for improving the imaging efficiency. However, these methods are not suitable for transparency or semi-transparency samples. There are some common characteristics for the samples, such as disorder and irregular for the structures on the sample surfaces transparency or semi-transparency. Focusing errors would occur due to the uneven reflectivity of the sample surface. This work presents a transmitted light spot method to solve the auto-focusing problem for the transparency or semi-transparency samples. The trace of transmitted light is simulated, and the expression of the focusing error signal is derived and given. The relationships between the defocus amount and focus error signal are calculated, and the results are experimentally demonstrated, accordingly. This method is expected to be applied to high-speed optical imaging.

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“…This results in an inherent compromise between achieving high precision (which requires short depth of focus comparable to the imaging DOF) or longer range (which results in lower precision). Furthermore, to distinguish whether the defocus is positive or negative, they may be set to measure distance from an offset position 10 rather than when the autofocus laser beam is at focus, and this further reduces the range of the autofocus.…”

Section: Introductionmentioning

confidence: 99%

Robust deep learning optical autofocus system applied to automated multiwell plate single molecule localization microscopy

Lightley

Görlitz

Kumar

et al. 2021

Journal of Microscopy

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We presenta robust, long‐range optical autofocus system for microscopy utilizing machine learning. This can be useful for experiments with long image data acquisition times that may be impacted by defocusing resulting from drift of components, for example due to changes in temperature or mechanical drift. It is also useful for automated slide scanning or multiwell plate imaging where the sample(s) to be imaged may not be in the same horizontal plane throughout the image data acquisition. To address the impact of (thermal or mechanical) fluctuations over time in the optical autofocus system itself, we utilize a convolutional neural network (CNN) that is trained over multiple days to account for such fluctuations. To address the trade‐off between axial precision and range of the autofocus, we implement orthogonal optical readouts with separate CNN training data, thereby achieving an accuracy well within the 600 nm depth of field of our 1.3 numerical aperture objective lens over a defocus range of up to approximately +/–100 μm. We characterize the performance of this autofocus system and demonstrate its application to automated multiwell plate single molecule localization microscopy.

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A High Precision Laser-Based Autofocus Method Using Biased Image Plane for Microscopy

Cited by 13 publications

References 10 publications

Robust, single-shot, optical autofocus system utilizing cylindrical lenses to provide high precision and long range of operation

Robust, single-shot, optical autofocus system utilizing cylindrical lenses to provide high precision and long range of operation

Defocusing detection of high-speed imaging for samples with arbitrary pattern structures

Robust deep learning optical autofocus system applied to automated multiwell plate single molecule localization microscopy

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