Multicolor multifocal 3D microscopy using in-situ optimization of a spatial light modulator.

Multifocal microscopy enables high-speed three-dimensional (3D) volume imaging by using a multifocal grating in the emission path. This grating is typically designed to afford a uniform illumination of multifocal subimages for a single emission wavelength. Using the same grating for multicolor imaging results in non-uniform subimage intensities in emission wavelengths for which the grating is not designed. This has restricted multifocal microscopy applications for samples having multicolored fluorophores. In this paper, we present a multicolor multifocal microscope implementation which uses a Spatial Light Modulator (SLM) as a single multifocal grating to realize near-uniform multifocal subimage intensities across multiple wavelength emission bands. Using real-time control of an in-situ-optimized SLM implemented as a multifocal grating, we demonstrate multicolor multifocal 3D imaging over three emission bands by imaging multicolored particles as well as Escherichia coli (E. coli) interacting with human liver cancer cells, at [Formula: see text] multicolor 3D volumes per second acquisition speed. Our multicolor multifocal method is adaptable across SLM hardware, emission wavelength band locations and number of emission bands, making it particularly suited for researchers investigating fast processes occurring across a volume where multiple species are involved.

Leveraging lifetime information to perform real-time 3D single-particle tracking in noisy environments.

A microscopy platform that leverages the arrival time of individual photons to enable 3D single-particle tracking of fast-moving (translational diffusion coefficient of ≃3.3 µm2/s) particles in high-background environments is reported here. It combines a hardware-based time-gating module, which enables the rate of photon processing to be as high as 100 MHz, with a two-photon-excited 3D single-particle tracking confocal microscope to enable high sample penetration depth. Proof-of-principle experiments where single quantum dots are tracked in solutions containing dye-stained cellulose, are shown with tracking performance markedly improved using the hardware-based time-gating module. Such a microscope design is anticipated to be of use to a variety of communities who wish to track single particles in cellular environments, which commonly have high fluorescence and scattering background.

Uniform intensity in multifocal microscopy using a spatial light modulator.

Multifocal microscopy (MFM) offers high-speed three-dimensional imaging through the simultaneous image capture from multiple focal planes. Conventional MFM systems use a fabricated grating in the emission path for a single emission wavelength band and one set of focal plane separations. While a Spatial Light Modulator (SLM) can add more flexibility as a replacement to the fabricated grating, the relatively small number of pixels in the SLM chip, cross-talk between the pixels, and aberrations in the imaging system can produce non-uniform intensity in the different axially separated image planes. We present an in situ iterative SLM calibration algorithm that overcomes these optical- and hardware-related limitations to deliver near-uniform intensity across all focal planes. Using immobilized gold nanoparticles under darkfield illumination, we demonstrate superior intensity evenness compared to current methods. We also demonstrate applicability across emission wavelengths, axial plane separations, imaging modalities, SLM settings, and different SLM manufacturers. Therefore, our microscope design and algorithms provide an alternative to the use of fabricated gratings in MFM, as they are relatively simple and could find broad applications in the wider research community.

CAOS-CMOS camera.

Proposed and experimentally demonstrated is the CAOS-CMOS camera design that combines the coded access optical sensor (CAOS) imager platform with the CMOS multi-pixel optical sensor. The unique CAOS-CMOS camera engages the classic CMOS sensor light staring mode with the time-frequency-space agile pixel CAOS imager mode within one programmable optical unit to realize a high dynamic range imager for extreme light contrast conditions. The experimentally demonstrated CAOS-CMOS camera is built using a digital micromirror device, a silicon point-photo-detector with a variable gain amplifier, and a silicon CMOS sensor with a maximum rated 51.3 dB dynamic range. White light imaging of three different brightness simultaneously viewed targets, that is not possible by the CMOS sensor, is achieved by the CAOS-CMOS camera demonstrating an 82.06 dB dynamic range. Applications for the camera include industrial machine vision, welding, laser analysis, automotive, night vision, surveillance and multispectral military systems.

Multi-image acquisition-based distance sensor using agile laser spot beam.

We present a novel laser-based distance measurement technique that uses multiple-image-based spatial processing to enable distance measurements. Compared with the first-generation distance sensor using spatial processing, the modified sensor is no longer hindered by the classic Rayleigh axial resolution limit for the propagating laser beam at its minimum beam waist location. The proposed high-resolution distance sensor design uses an electronically controlled variable focus lens (ECVFL) in combination with an optical imaging device, such as a charged-coupled device (CCD), to produce and capture different laser spot size images on a target with these beam spot sizes different from the minimal spot size possible at this target distance. By exploiting the unique relationship of the target located spot sizes with the varying ECVFL focal length for each target distance, the proposed distance sensor can compute the target distance with a distance measurement resolution better than the axial resolution via the Rayleigh resolution criterion. Using a 30 mW 633 nm He-Ne laser coupled with an electromagnetically actuated liquid ECVFL, along with a 20 cm focal length bias lens, and using five spot images captured per target position by a CCD-based Nikon camera, a proof-of-concept proposed distance sensor is successfully implemented in the laboratory over target ranges from 10 to 100 cm with a demonstrated sub-cm axial resolution, which is better than the axial Rayleigh resolution limit at these target distances. Applications for the proposed potentially cost-effective distance sensor are diverse and include industrial inspection and measurement and 3D object shape mapping and imaging.

Smart laser scanning sampling head design for image acquisition applications.

A smart laser scanning sampling head design is presented using an electronically controlled variable focal length lens to achieve the smallest sampling laser spot possible at target plane distances reaching 8 m. A proof-of-concept experiment is conducted using a 10 mW red 633 nm laser coupled with beam conditioning optics that includes an electromagnetically actuated deformable membrane liquid lens to demonstrate sampling laser spot radii under 1 mm over a target range of 20-800 cm. Applications for the proposed sampling head are diverse and include laser machining and component inspection.