High-speed digital microscopy

High-speed imaging is an ideal technique to accurately resolve the temporal and spatial characteristics of rapid events at either the molecular or cellular level. In this article, the digital imaging techniques used to simultaneously acquire transillumination phase-contrast images, at 240 images s(-1) (high-speed), to characterize ciliary beat frequency, and fluorescence images, at 30 images s(-1) (fast), to measure intracellular calcium concentration ([Ca2+]i), are described. With this technique, a precise correlation between the changes in ciliary beat frequency with changes in [Ca2+]i can be made. Simultaneous imaging is achieved by using different wavelengths of light to form the phase-contrast and fluorescent images and selectively directing these light wavelengths to different cameras with dichroic mirrors and bandpass filters. High-speed images compatible with standard video recording equipment are obtained by prematurely resetting the raster scan of a CCD camera with additional vertical synchronization pulses. The fast [Ca2+]i images are determined using the ratiometric dye fura-2 and a recording technique that monitors rapid changes in fluorescence at a single wavelength and uses intermittent reference images for calibration.     

Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror

Confocal microscopy is routinely used for high-resolution fluorescence imaging of biological specimens. Most standard confocal systems scan a laser across a specimen and collect emitted light passing through a single pinhole to produce an optical section of the sample. Sequential scanning on a point-by-point basis limits the speed of image acquisition and even the fastest commercial instruments struggle to resolve the temporal dynamics of rapid cellular events such as calcium signals. Various approaches have been introduced that increase the speed of confocal imaging. Nipkov disk microscopes, for example, use arrays of pinholes or slits on a spinning disk to achieve parallel scanning which significantly increases the speed of acquisition. Here we report the development of a microscope module that utilises a digital micromirror device as a spatial light modulator to provide programmable confocal optical sectioning with a single camera, at high spatial and axial resolution at speeds limited by the frame rate of the camera. The digital micromirror acts as a solid state Nipkov disk but with the added ability to change the pinholes size and separation and to control the light intensity on a mirror-by-mirror basis. The use of an arrangement of concave and convex mirrors in the emission pathway instead of lenses overcomes the astigmatism inherent with DMD devices, increases light collection efficiency and ensures image collection is achromatic so that images are perfectly aligned at different wavelengths. Combined with non-laser light sources, this allows low cost, high-speed, multi-wavelength image acquisition without the need for complex wavelength-dependent image alignment. The micromirror can also be used for programmable illumination allowing spatially defined photoactivation of fluorescent proteins. We demonstrate the use of this system for high-speed calcium imaging using both a single wavelength calcium indicator and a genetically encoded, ratiometric, calcium sensor.     

Nouveautés en cardiologie nucléaire : une gamma caméra à semi-conducteurs dédiée à l’imagerie cardiaque

During the last few years, cardiac imaging made important breakthroughs thanks to the development of various techniques allowing the risk stratification of patients with coronary artery disease. The well-established single photon emission computed tomography (SPECT) as a myocardial imaging technique made an important progress with the recent improvement of high-speed volumic acquisition, using dedicated semi-conductor gamma camera. These cameras bring significant improvement to the image quality and the image acquisition time, which is now seven times lower. New type of artefacts is expected because of the geometry of detection, thus studies are still needed to assess the exact performance of this revolutionary technology.     

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to investigate mechanisms governing cell adhesion under dynamic flow conditions. For instance, cancer cells labeled with multiple fluorophores can be perfused over a potentially reactive substrate to model mechanisms of cancer metastasis. However, multi-channel single camera systems and color cameras exhibit shortcomings in image acquisition for real-time live cell analysis. To overcome these limitations, we used a dual camera emission splitting system to simultaneously capture real-time image sequences of fluorescently labeled cells in the flow chamber. Dual camera emission splitting systems filter defined wavelength ranges into two monochrome CCD cameras, thereby simultaneously capturing two spatially identical but fluorophore-specific images. Subsequently, psuedocolored one-channel images are combined into a single real-time merged sequence that can reveal multiple target molecules on cells moving rapidly across a region of interest.     

High-speed multicolor microscopy of repeating dynamic processes

Images of multiply labeled fluorescent samples provide unique insights into the localization of molecules, cells, and tissues. The ability to image multiple channels simultaneously at high speed without cross talk is limited to a few colors and requires dedicated multichannel or multispectral detection procedures. Simpler microscopes, in which each color is imaged sequentially, produce a much lower frame rate. Here, we describe a technique to image, at high frame rate, multiply labeled samples that have a repeating motion. We capture images in a single channel at a time over one full occurrence of the motion then repeat acquisition for other channels over subsequent occurrences. We finally build a high-speed multichannel image sequence by combining the images after applying a normalized mutual information-based time registration procedure. We show that this technique is amenable to image the beating heart of a double-labeled embryonic quail in three channels (brightfield, yellow, and mCherry fluorescent proteins) using a fluorescence wide-field microscope equipped with a single monochrome camera and without fast channel switching optics. We experimentally evaluate the accuracy of our method on image series from a two-channel confocal microscope.     

Experimental Comparison of the High-Speed Imaging Performance of an EM-CCD and sCMOS Camera in a Dynamic Live-Cell Imaging Test Case

The study of living cells may require advanced imaging techniques to track weak and rapidly changing signals. Fundamental to this need is the recent advancement in camera technology. Two camera types, specifically sCMOS and EM-CCD, promise both high signal-to-noise and high speed (>100 fps), leaving researchers with a critical decision when determining the best technology for their application. In this article, we compare two cameras using a live-cell imaging test case in which small changes in cellular fluorescence must be rapidly detected with high spatial resolution. The EM-CCD maintained an advantage of being able to acquire discernible images with a lower number of photons due to its EM-enhancement. However, if high-resolution images at speeds approaching or exceeding 1000 fps are desired, the flexibility of the full-frame imaging capabilities of sCMOS is superior.     

A mercury arc lamp-based multi-color confocal real time imaging system for cellular structure and function

Multi-point scanning confocal microscopy using a Nipkow disk enables the acquisition of fluorescent images with high spatial and temporal resolutions. Like other single-point scanning confocal systems that use Galvano meter mirrors, a commercially available Nipkow spinning disk confocal unit, Yokogawa CSU10, requires lasers as the excitation light source. The choice of fluorescent dyes is strongly restricted, however, because only a limited number of laser lines can be introduced into a single confocal system. To overcome this problem, we developed an illumination system in which light from a mercury arc lamp is scrambled to make homogeneous light by passing it through a multi-mode optical fiber. This illumination system provides incoherent light with continuous wavelengths, enabling the observation of a wide range of fluorophores. Using this optical system, we demonstrate both the high-speed imaging (up to 100 Hz) of intracellular Ca(2+) propagation, and the multi-color imaging of Ca(2+) and PKC-gamma dynamics in living cells.     

Digital video combined with conventional radiography creates an excellent high-speed X-ray video system

Analysing rapid internal movement in animals requires high-speed X-ray imaging to visualise motion of bony and soft body structures. The frame rate of X-ray cinematic camera is limited to 200 frames s(-1) with a shutter speed of 1/3000s, due to the low X-ray kV, required for small animals. These rates are often too slow for analysing the rapid accelerations of which small animals are capable. Here, we present three alternative digital-video systems that can be combined with a conventional X-ray intensifier to yield excellent slow-motion images of internal body parts of small animals. Each system shows high performance for different requirements of radiographic motion analysis.     

Single-shot optical projection tomography for high-speed volumetric imaging of dynamic biological samples

A single-shot adaptation of Optical Projection Tomography (OPT) for high-speed volumetric snapshot imaging of dynamic mesoscopic biological samples is presented. Conventional OPT has been applied to in vivo imaging of animal models such as D. rerio, but the sequential acquisition of projection images typically requires samples to be immobilized during the acquisition. A proof-of-principle system capable of single-shot tomography of a ~1 mm³ volume is presented, demonstrating camera-limited rates of up to 62.5 volumes/s, which has been applied to 3D imaging of a freely swimming zebrafish embryo. This is achieved by recording eight projection views simultaneously on four low-cost CMOS cameras. With no stage required to rotate the sample, this single-shot OPT system can be implemented with a component cost of under £5000. The system design can be adapted to different sized fields of view and may be applied to a broad range of dynamic samples, including high throughput flow cytometry applied to model organisms and fluid dynamics studies.     

Real-time multi-wavelength fluorescence imaging of living cells

We describe a new real-time fluorescence video microscope design for capturing intensified images of cells containing dual wavelength "ratio" dyes or multiple dyes. The microscope will perform real-time capture of two separate fluorescence emission images simultaneously, improving the time resolution of spatial distribution of fluorescence to video frame rates (30 frames/s or faster). Each emission wavelength is imaged simultaneously by one of two cameras, then digitized, background corrected and appropriately combined at standard video frame rates to be stored at high resolution on tape or digital video disk for further off-line analysis. Use of low noise, high sensitivity image intensifiers, coupled to CCD cameras produce stable, high contrast images using ultra low light levels with little persistence or bloom. The design has no moving parts in its optical train, which overcomes a number of technical difficulties encountered in the present filter wheel designs for multiple imaging. Coupled to compatible image processing software utilizing PC-AT computers, the new design can be built for a significantly lower cost than many presently available commercial machines. The system is ideal for ratio imaging applications; the software can calculate the ratio of fluorescence intensities pixel by pixel and provide the information to generate false-color images of the intensity data as well as other calculations based on the two images. Thus, it provides a powerful, inexpensive tool for studying the real-time kinetics of changes in living cells. Examples are presented for the kinetics of rapidly changing intracellular calcium detected by the calcium indicator probe indo-1 and the redistribution kinetics of multiple vital dyes placed in cells undergoing cell fusion.     

Rapid assessment of forest canopy and light regime using smartphone hemispherical photography

Hemispherical photography (HP), implemented with cameras equipped with “fisheye” lenses, is a widely used method for describing forest canopies and light regimes. A promising technological advance is the availability of low‐cost fisheye lenses for smartphone cameras. However, smartphone camera sensors cannot record a full hemisphere. We investigate whether smartphone HP is a cheaper and faster but still adequate operational alternative to traditional cameras for describing forest canopies and light regimes. We collected hemispherical pictures with both smartphone and traditional cameras in 223 forest sample points, across different overstory species and canopy densities. The smartphone image acquisition followed a faster and simpler protocol than that for the traditional camera. We automatically thresholded all images. We processed the traditional camera images for Canopy Openness (CO) and Site Factor estimation. For smartphone images, we took two pictures with different orientations per point and used two processing protocols: (i) we estimated and averaged total canopy gap from the two single pictures, and (ii) merging the two pictures together, we formed images closer to full hemispheres and estimated from them CO and Site Factors. We compared the same parameters obtained from different cameras and estimated generalized linear mixed models (GLMMs) between them. Total canopy gap estimated from the first processing protocol for smartphone pictures was on average significantly higher than CO estimated from traditional camera images, although with a consistent bias. Canopy Openness and Site Factors estimated from merged smartphone pictures of the second processing protocol were on average significantly higher than those from traditional cameras images, although with relatively little absolute differences and scatter. Smartphone HP is an acceptable alternative to HP using traditional cameras, providing similar results with a faster and cheaper methodology. Smartphone outputs can be directly used as they are for ecological studies, or converted with specific models for a better comparison to traditional cameras.     

Developing a multi-Kinect-system for monitoring in dairy cows: object recognition and surface analysis using wavelets

Camera-based systems in dairy cattle were intensively studied over the last years. Different from this study, single camera systems with a limited range of applications were presented, mostly using 2D cameras. This study presents current steps in the development of a camera system comprising multiple 3D cameras (six Microsoft Kinect cameras) for monitoring purposes in dairy cows. An early prototype was constructed, and alpha versions of software for recording, synchronizing, sorting and segmenting images and transforming the 3D data in a joint coordinate system have already been implemented. This study introduced the application of two-dimensional wavelet transforms as method for object recognition and surface analyses. The method was explained in detail, and four differently shaped wavelets were tested with respect to their reconstruction error concerning Kinect recorded depth maps from different camera positions. The images’ high frequency parts reconstructed from wavelet decompositions using the haar and the biorthogonal 1.5 wavelet were statistically analyzed with regard to the effects of image fore- or background and of cows’ or persons’ surface. Furthermore, binary classifiers based on the local high frequencies have been implemented to decide whether a pixel belongs to the image foreground and if it was located on a cow or a person. Classifiers distinguishing between image regions showed high (⩾0.8) values of Area Under reciever operation characteristic Curve (AUC). The classifications due to species showed maximal AUC values of 0.69.     

Approaches to improving breast cancer diagnosis using a high resolution,breast specific gamma camera

Mammography remains the gold standard in breast cancer detection, although there remains a challenge for improvement in sensitivity of breast cancer detection and diagnosis. Although mammography is the most frequently utilized examination to screen for breast cancer, which has resulted in a reduction of breast cancer mortality, still some cancers are unable to be visualized on mammographic images. Mammography films are interpreted using an anatomic approach. A new approach to breast cancer diagnosis utilizes a breast specific gamma camera to measure radiotracer uptake of abnormal tissue in the breast in patients with an abnormal mammogram or palpble mass using technetium sestamibi. Images are taken using the same positioning techniques as mammography for comparison of both types of images. Multi-Institutional trials using a traditional gamma camera demonstrated potential for this approach. However, the inability of traditional gamma camera intrinsic resolution and non-optinuized breast imaging limited scintimammography. Therefore, a breast specific, high resolution gamma camera was developed to overcome these limitations.Results of clinical studies evaluating BSGI are promising and are increasingly being used. Additionally, means for minimally invasive imaging-guided acquisition of tissue are being developed so that biopsies will be avaibles for areas of interest based on radiotracer uptake.Breast specific gamma camera nuclear imaging of the breast is a developing and increasingly utilized approach to improving breast cancer detection and diagnosis.     

Assessing the capabilities of a 4kx4k CCD camera for electron cryo-microscopy at 300kV

CCD cameras have numerous advantages over photographic film for detecting electrons; however the point spread function of these cameras has not been sufficient for single particle data collection to subnanometer resolution with 300kV microscopes. We have adopted spectral signal to noise ratio (SNR) as a parameter for assessing detector quality for single particle imaging. The robustness of this parameter is confirmed under a variety of experimental conditions. Using this parameter, we demonstrate that the SNR of images of either amorphous carbon film or ice embedded virus particles collected on a new commercially available 4kx4k CCD camera are slightly better than photographic film at low spatial frequency (<1/5 Nyquist frequency), and as good as photographic film out to half of the Nyquist frequency. In addition it is slightly easier to visualize ice embedded particles on this CCD camera than on photographic film. Based on this analysis it is realistic to collect images containing subnanometer resolution data (6-9A) using this CCD camera at an effective magnification of approximately 112000x on a 300kV electron microscope.     

Investigation of an SFOV hybrid gamma camera for thyroid imaging

The Hybrid Compact Gamma Camera (HCGC) is a small field of view (SFOV) portable hybrid gamma-optical camera intended for small organ imaging at the patient bedside. In this study, a thyroid phantom was used to determine the suitability of the HCGC for clinical thyroid imaging through comparison with large field of view (LFOV) system performance.A direct comparison with LFOV contrast performance showed that the lower sensitivity of the HCGC had a detrimental effect on image quality. Despite this, the contrast of HCGC images exceeded those of the LFOV cameras for some image features particularly when a high-resolution pinhole collimator was used.A clinical simulation showed that thyroid morphology was visible in a 5 min integrated image acquisition with an expected dependency on the activity within the thyroid. The first clinical use of the HCGC for imaging thyroid uptake of ¹²³I is also presented.Measurements indicate that the HCGC has promising utility in thyroid imaging, particularly as its small size allows it to be brought into closer proximity with a patient. Future development of the energy response of the HCGC is expected to further improve image detectability.     

Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera

We demonstrate the calculation of particle brightness and concentration from fluorescence-fluctuation photon-counting statistics using an electron-multiplied charge-coupled device (EMCCD) camera. This technique provides a concentration-independent measure of particle brightness in dynamic systems. The high sensitivity and highly parallel detection of EMCCD cameras allow for imaging of dynamic particle brightness, providing the capability to follow aggregation reactions in real time. A critical factor of the EMCCD camera is the presence of nonlinearity at high intensities. These nonlinearities arise due to limited capacity of the CCD well and to the analog-to-digital converter maximum range. However, we show that the specific camera we used (with a 16-bit analog-to-digital converter) has sufficient dynamic range for most microscopy applications. In addition, we explore the importance of camera timing behavior as it is affected by the vertical frame transfer speed of the camera. Although the camera has microsecond exposure time for illumination of a few pixels, the exposure time increased to milliseconds for full-field illumination. Finally, we demonstrate the ability of the technique to follow concentration changes and measure single-molecule brightness in real time in living cells.     

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.     

Optimised generalized polarisation analysis of C-laurdan reveals clear order differences between T cell membrane compartments

Heterogenous packing of plasma membrane lipids is important for cellular processes like signalling, adhesion and sorting of membrane components. Solvatochromic membrane fluorophores that respond to changes from liquid-ordered (l_o) phase to liquid-disordered (l_d) by red shifts in their emission spectra are often used to assess lipid packing. Their response can be quantified using generalized polarisation (GP) using fluorescence microscopy images from two emission ranges, preferably from a region of interest (ROI) limited to a specific membrane compartment. However, image quality is limited by Poisson noise and convolution by the point spread function of the imaging system. Examining GP-analysis of C-laurdan labelled T cells using the image restoration procedure deconvolution, we demonstrate that deconvolution substantially improves the image resolution by making the plasma membrane clearly discernible and facilitating plasma membrane ROI selection. We conclude that automatic ROI selection has advantages over manual ROI selection when it comes to reproducibility and speed, but reliable GP-measurements can also be obtained by manually demarcated ROIs. We find that deconvolution enhances the difference in GP-values between the plasma and intracellular membranes and demonstrate that moving an intensity defined plasma membrane ROI outwards from the cell further improves this differentiation. By systematically changing the key deconvolution regularization parameter signal to noise, we establish a protocol for deconvolution optimisation applicable to any solvatochromic dye and imaging system. The image processing and ROI selection protocol presented improves both the resolution and precision of GP-measurement and will enable detection of smaller changes in membrane order than is currently achievable.     

99mTc thyroid imaging system using multiple imaging plates

A system for taking static thyroid ^99mTc images was devised by using multiple imaging plates (IPs) and a low-energy high resolution collimator. System spatial resolution of the IP systems and the gamma camera was determined by referring to standards set by the National Electrical Manufacturers Association. Sensitivity was represented by using lower detection limits (LDLs). The sensitivity and resolution of IP systems using 16 IP probes connecting two collimators and 9 IPs were determined by using a 20 ml thyroid phantom, and compared with the sensitivity of gamma cameras. The sensitivity of the IP systems increased in proportion to the number of IPs. The sensitivity and resolution of a probe using 6 IPs and a high resolution collimator were equivalent to or superior to the gamma camera for taking static thyroid ^99mTc images. IP systems can be applied clinically as mobile static nuclear imaging devices. The performance of IP systems should be thoroughly investigated for combinations of various collimators and the number of IPs in order to verify their efficacy for imaging all organs.     

Dual light‐emitting diode‐based multichannel microscopy for whole‐slide multiplane,multispectral and phase imaging

We report the development of a multichannel microscopy for whole‐slide multiplane, multispectral and phase imaging. We use trinocular heads to split the beam path into 6 independent channels and employ a camera array for parallel data acquisition, achieving a maximum data throughput of approximately 1 gigapixel per second. To perform single‐frame rapid autofocusing, we place 2 near‐infrared light‐emitting diodes (LEDs) at the back focal plane of the condenser lens to illuminate the sample from 2 different incident angles. A hot mirror is used to direct the near‐infrared light to an autofocusing camera. For multiplane whole‐slide imaging (WSI), we acquire 6 different focal planes of a thick specimen simultaneously. For multispectral WSI, we relay the 6 independent image planes to the same focal position and simultaneously acquire information at 6 spectral bands. For whole‐slide phase imaging, we acquire images at 3 focal positions simultaneously and use the transport‐of‐intensity equation to recover the phase information. We also provide an open‐source design to further increase the number of channels from 6 to 15. The reported platform provides a simple solution for multiplexed fluorescence imaging and multimodal WSI. Acquiring an instant focal stack without z‐scanning may also enable fast 3‐dimensional dynamic tracking of various biological samples.