Evaluation of spectral imaging for plant cell analysis

Fluorescence imaging at high spectral resolution is now a practical reality and has great promise in plant cell biology. Emission spectral curve data can be used computationally to distinguish spectrally similar fluorophores, or to remove autofluorescence, and to spectrally analyse autofluorescent molecules, which are especially abundant in plant tissues. Examples of these applications in plant cells are given, and a comparison is made between the current offerings in spectral imaging laser scanning confocal microscopes.     

The use of fluorescence lifetime imaging (FLIM) for in situ microbial detection in complex mineral substrates

The utility of fluorescence lifetime imaging microscopy (FLIM) for identifying bacteria in complex mineral matrices was investigated. Baseline signals from unlabelled Bacillus subtilis and Euglena gracilis, and Bacillus subtilis labelled with SYTO 9 were obtained using two-photon excitation at 730, 750 and 800 nm, identifying characteristic lifetimes of photosynthetic pigments, unpigmented cellular autofluorescence, and SYTO 9. Labelled and unlabelled B. subtilis were seeded onto marble and gypsum samples containing endolithic photosynthetic cyanobacteria and the ability to distinguish cells from mineral autofluorescence and nonspecific dye staining was examined in parallel with ordinary multichannel confocal imaging. It was found that FLIM enabled discrimination of SYTO 9 labelled cells from background, but that the lifetime of SYTO 9 was shorter in cells on minerals than in pure culture under our conditions. Photosynthetic microorganisms were easily observed using both FLIM and confocal. Unlabelled, nonpigmented bacteria showed weak signals that were difficult to distinguish from background when minerals were present, though cellular autofluorescence consistent with NAD(P)H could be seen in pure cultures, and phasor analysis permitted detection on rocks. Gypsum and marble samples showed similar autofluorescence profiles, with little autofluorescence in the yellow-to-red range. Lifetime or time-gated imaging may prove a useful tool for environmental microbiology. LAY DESCRIPTION : The standard method of bacterial enumeration is to label the cells with a fluorescent dye and count them under high-power fluorescence microscopy. However, this can be difficult when the cells are embedded in soil and rock due to fluorescence from the surrounding minerals and dye binding to ambiguous features of the substrate. The use of fluorescence lifetime imaging (FLIM) can disambiguate these signals and allow for improved detection of bacteria in environmental samples.     

The Large-Scale Digital Cell Analysis System: an open system for nonperturbing live cell imaging

The Large-Scale Digital Cell Analysis System (LSDCAS) was designed to provide a highly extensible open source live cell imaging system. Analysis of cell growth data has demonstrated a lack of perturbation in cells imaged using LSDCAS, through reference to cell growth data from cells growing in CO₂ incubators. LSDCAS consists of data acquisition, data management and data analysis software, and is currently a Core research facility at the Holden Comprehensive Cancer Center at the University of Iowa. Using LSDCAS analysis software, this report and others show that although phase-contrast imaging has no apparent effect on cell growth kinetics and viability, fluorescent image acquisition in the cell lines tested caused a measurable level of growth perturbation using LSDCAS. This report describes the current design of the system, reasons for the implemented design, and details its basic functionality. The LSDCAS software runs on the GNU/Linux operating system, and provides easy to use, graphical programs for data acquisition and quantitative analysis of cells imaged with phase-contrast or fluorescence microscopy (alone or in combination), and complete source code is freely available under the terms of the GNU Public Software License at the project website ( http://lsdcas.engineering.uiowa.edu ).     

Single- and two-photon spectral imaging of intrinsic fluorescence of transformed human hepatocytes

Autofluorescence (AF) originating from the cytoplasmic region of mammalian cells has been thoroughly investigated; however, AF from plasma membranes of viable intact cells is less well known, and has been mentioned only in a few older publications. Herein, we report results describing single- and two-photon spectral properties of a strong yellowish-green AF confined to the plasma-membrane region of transformed human hepatocytes (HepG2) grown in vitro as small three-dimensional aggregates or as monolayers. The excitation-emission characteristics of the membrane AF indicate that it may originate from a flavin derivative. Furthermore, the AF was closely associated with the plasma membranes of HepG2 cells, and its presence and intensity were dependent on cell metabolic state, membrane integrity and presence of reducing agents. This AF could be detected both in live intact cells and in formaldehyde-fixed cells.     

Total internal reflection fluorescence imaging using an upconverting cover slip for multicolour evanescent excitation

Total internal reflection fluorescence microscopy is well known as a means of studying surface‐bound structures in cell biology. It is usually measured either by coupling a light source to the sample using a prism or with a special objective where light passing through the periphery of the lens illuminates the contact region beyond the critical angle. In this study we present a new and simple approach to total internal reflection fluorescence microscopy where the sample is mounted on a cover slip prepared from a high‐index upconverting glass‐ceramic. Excitation of the cover slip with a low‐cost near‐infrared laser diode generates intense narrow‐band visible emission within the cover slip, some of which is totally internally reflected. This emission gives rise to an evanescent wave at the interface and hence can excite surface‐bound fluorescent species. Depending on the excitation conditions the cover slip can generate violet, green and red emission and hence can excite a wide range of fluorescent labels. Fluorescence emission from the sample can be detected in spectral regions where the direct emission from the cover slip is very weak. The advantages and limitations of the technique are discussed in comparison with conventional total internal reflection fluorescence microscopy measurements and prospects for novel total internal reflection fluorescence microscopy geometries are considered.     

Curtailed light sheet microscopy for rapid imaging of macroscopic biological specimens

We study the feasibility of volume imaging from a few angular views/scans in a light sheet fluorescence microscopy. Two‐dimensional (2D) images (angular views) were acquired at an angular separation of 10° and volume images were constructed with merely 18, 9, and 6 views. We study the structural changes in a 5‐day old Zebrafish embryo labeled with Phalloidin TRITC that binds to F‐Actin of embryo cell. To collect the data, the specimen is rotated (for varying sampling angles Δθ) with respect to a fixed vertical axis passing through the volume‐of‐interest (yolk sac). In the proposed realization of selective plane illumination microscopy (SPIM) technique, the translation is completely avoided. Analysis shows rich structural information with marginal reduction in contrast. Comparison with the state‐of‐the‐art SPIM shows appreciable volume reconstruction (from an order less 2D scans) that may be good enough for rapid monitoring of macroscopic specimens. Microsc. Res. Tech. 79:455–458, 2016. © 2016 Wiley Periodicals, Inc.     

Dual-mode reflectance and fluorescence near-video-rate confocal microscope for architectural, morphological and molecular imaging of tissue

We have developed a near‐video‐rate dual‐mode reflectance and fluorescence confocal microscope for the purpose of imaging ex vivo human specimens and in vivo animal models. The dual‐mode confocal microscope (DCM) has light sources at 488, 664 and 784 nm, a frame rate of 15 frames per second, a maximum field of view of 300 × 250 μm and a resolution limit of 0.31 μm laterally and 1.37 μm axially. The DCM can image tissue architecture and cellular morphology, as well as molecular properties of tissue, using reflective and fluorescent molecular‐specific optical contrast agents. Images acquired with the DCM demonstrate that the system has the sub‐cellular resolution needed to visualize the morphological and molecular changes associated with cancer progression and has the capability to image animal models of disease in vivo. In the hamster cheek pouch model of oral carcinogenesis, the DCM was used to image the epithelium and stroma of the cheek pouch; blood flow was visible and areas of dysplasia could be distinguished from normal epithelium using 6% acetic acid contrast. In human oral cavity tissue slices, DCM reflectance images showed an increase in the nuclear‐to‐cytoplasmic ratio and density of nuclei in neoplastic tissues as compared to normal tissue. After labelling tissue slices with fluorescent contrast agents targeting the epidermal growth factor receptor, an increase in epidermal growth factor receptor expression was detected in cancerous tissue as compared to normal tissue. The combination of reflectance and fluorescence imaging in a single system allowed imaging of two different parameters involved in neoplastic progression, providing information about both the morphological and molecular expression changes that occur with cancer progression. The dual‐mode imaging capabilities of the DCM allow investigation of both morphological changes as well as molecular changes that occur in disease processes. Analyzing both factors simultaneously may be advantageous when trying to detect and diagnose disease. The DCM's high resolution and near‐video‐rate image acquisition and the growing inventory of molecular‐specific contrast agents and disease‐specific molecular markers holds significant promise for in vivo studies of disease processes such as carcinogenesis.     

Digital scanned laser light-sheet fluorescence lifetime microscopy with wide-field time-gated imaging

We develop a multidimensional fluorescence imaging technique by implementing a wide-field time-gated fluorescence lifetime imaging into digital scanned laser light-sheet microscopy (FLIM-DSLM) to measure 3D fluorescence lifetime distribution in mesoscopic specimens with high resolution. This is achieved by acquiring a series of time-gated images at different relative time delays with respect of excitation pulses at different depths. The lifetime is determined for each voxel by iteratively fitting to single exponential decay. The performance of the developed system is evaluated with the measurements of a lifetime reference Rhodamine 6G solution and a subresolution fluorescent bead phantom. We also demonstrate the application performances of this system to ex vivo and in vivo imaging of Tg(kdrl:EGFP) transgenic zebrafish embryos, illustrating the lifetime differences between the GFP signal and the autofluorescence signal. The results show that FLIM-DSLM can be used for sample size up to a few millimetres and can be utilised as a powerful and robust method for biomedical research, for example as a readout of protein–protein interactions via Förster resonance energy transfer.     

Comparison of three-dimensional imaging properties between two-photon and single-photon fluorescence microscopy

The imaging performance in single-photon (1-p) and two-photon (2-p) fluorescence microscopy is described. Both confocal and conventional systems are compared in terms of the three-dimensional (3-D) point spread function and the 3-D optical transfer function. Images of fluorescent sharp edges and layers are modelled, giving resolution in transverse and axial directions. A comparison of the imaging properties is also given for a 4Pi confocal system. Confocal 2-p 4Pi fluorescence microscopy gives the best axial resolution in the sense that its 3-D optical transfer function has the strongest response along the axial direction.     

Three-dimensional imaging in fluorescence by confocal scanning microscopy

The improved resolution and sectioning capability of a confocal microscope make it an ideal instrument for extracting three-dimensional information especially from extended biological specimens. The imaging properties, also with finite detection pinholes are considered and a number of biological applications demonstrated.     

Exploiting advances in imaging technology to study biofilms by applying multiphoton laser scanning microscopy as an imaging and manipulation tool

Biofilms are an important element of the natural ecosystems but can be detrimental in health care and industrial settings. To improve our ability to combat biofilms, we need to understand the processes that facilitate their formation and dispersal. One approach that has proven to be invaluable is to image biofilms as they grow. Here we describe tools and protocols to visualize biofilms with multiphoton laser scanning microscopy, compare this with single photon laser scanning confocal microscopy and highlight best working procedures. Furthermore, we describe how with multiphoton laser scanning microscopy the laser can be used to manipulate the biofilm, specifically to achieve localized bleaching, killing or ablation within the biofilm biomass. These applications open novel ways to study the dynamics of biofilm formation, regeneration and dispersal.     

An open‐source deconvolution software package for 3‐D quantitative fluorescence microscopy imaging

Deconvolution techniques have been widely used for restoring the 3‐D quantitative information of an unknown specimen observed using a wide‐field fluorescence microscope. Deconv , an open‐source deconvolution software package, was developed for 3‐D quantitative fluorescence microscopy imaging and was released under the GNU Public License. Deconv provides numerical routines for simulation of a 3‐D point spread function and deconvolution routines implemented three constrained iterative deconvolution algorithms: one based on a Poisson noise model and two others based on a Gaussian noise model. These algorithms are presented and evaluated using synthetic images and experimentally obtained microscope images, and the use of the library is explained. Deconv allows users to assess the utility of these deconvolution algorithms and to determine which are suited for a particular imaging application. The design of Deconv makes it easy for deconvolution capabilities to be incorporated into existing imaging applications.     

Fluorescence microscopy: established and emerging methods, experimental strategies, and applications in immunology

Cutting-edge biophysical technologies including total internal reflection fluorescence microscopy, single molecule fluorescence, single channel opening events, fluorescence resonance energy transfer, high-speed exposures, two-photon imaging, fluorescence lifetime imaging, and other tools are becoming increasingly important in immunology as they link molecular events to cellular physiology, a key goal of modern immunology. The primary concern in all forms of microscopy is the generation of contrast; for fluorescence microscopy contrast can be thought of as the difference in intensity between the cell and background, the signal-to-noise ratio. High information-content images can be formed by enhancing the signal, suppressing the noise, or both. As improved tools, such as ICCD and EMCCD cameras, become available for fluorescence imaging in molecular and cellular immunology, it is important to optimize other aspects of the imaging system. Numerous practical strategies to enhance fluorescence microscopy experiments are reviewed. The use of instrumentation such as light traps, cameras, objectives, improved fluorescent labels, and image filtration routines applicable to low light level experiments are discussed. New methodologies providing resolution well beyond that given by the Rayleigh criterion are outlined. Ongoing and future developments in fluorescence microscopy instrumentation and technique are reviewed. This review is intended to address situations where the signal is weak, which is important for emerging techniques stressing super-resolution or live cell dynamics, but is less important for conventional applications such as indirect immunofluorescence. This review provides a broad integrative discussion of fluorescence microscopy with selected applications in immunology.     

Advanced spinning disk‐TIRF microscopy for faster imaging of the cell interior and the plasma membrane

Understanding the cellular processes that occur between the cytosol and the plasma membrane is an important task for biological research. Till now, however, it was not possible to combine fast and high‐resolution imaging of both the isolated plasma membrane and the surrounding intracellular volume. Here, we demonstrate the combination of fast high‐resolution spinning disk (SD) and total internal reflection fluorescence (TIRF) microscopy for specific imaging of the plasma membrane. A customised SD‐TIRF microscope was used with specific design of the light paths that allowed, for the first time, live SD‐TIRF experiments at high acquisition rates. A series of experiments is shown to demonstrate the feasibility and performance of our setup.     

MRT letter: A unified accelerated maximum likelihood technique for widefield,confocal, and super‐resolution 4Pi microscopy

We propose an algorithmic technique for accelerating maximum likelihood (ML) algorithm for image reconstruction in fluorescence microscopy. This is made possible by integrating Biggs–Andrews (BA) method with ML approach. The results on widefield, confocal, and super‐resolution 4Pi microscopy reveal substantial improvement in the speed of 3D image reconstruction (the number of iterations has reduced by approximately one‐half). Moreover, the quality of reconstruction obtained using accelerated ML closely resembles with nonaccelerated ML method. The proposed technique is a step closer to realize real‐time reconstruction in 3D fluorescence microscopy. Microsc. Res. Tech. 78:331–335, 2015. © 2015 Wiley Periodicals, Inc.     

Frequency domain lifetime and spectral imaging microscopy

In the femtoliter observation volume of a two-photon microscope, multiple fluorophores can be present and complex photophysics can take place. Combined detection of the fluorescence emission spectra and lifetimes can provide deeper insight into specimen properties than these two imaging modalities taken separately. Therefore, we have developed a detection scheme based on a frequency-modulated multichannel photomultiplier, which measures simultaneously the spectrum and the lifetime of the emitted fluorescence. Experimentally, the efficiency of the frequency domain lifetime measurement was compared to a time domain set-up. The performance of this spectrally and lifetime-resolved microscope was evaluated on reference specimens and living cells labeled with three different stains targeting the membrane, the mitochondria, and the nucleus.     

Confocal laser scanning microscopy: using cuticular autofluorescence for high resolution morphological imaging in small crustaceans

The utility of cuticular autofluorescence for the visualization of copepod morphology by means of confocal laser scanning microscopy (CLSM) was examined. Resulting maximum intensity projections give very accurate information on morphology and show even diminutive structures such as small setae in detail. Furthermore, CLSM enables recognition of internal structures and differences in material composition. Optical sections in all layers and along all axes of the specimens can be obtained by CLSM. The facile and rapid preparation method bears no risk of artefacts or damage occurring to the preparations and the visualized specimens can be used for later analyses allowing for the investigation of irreplaceable type specimens or parts of them. These features make CLSM a very effective tool for both taxonomical and ecological studies in small crustaceans; however, the maximum thickness of the specimens is limited to a few hundred micrometers. Three‐dimensional models based on CLSM image stacks allow observation of the preparations from all angles and can permit, improve and speed up studies on functional morphology. The visualization method described has a strong potential to become a future standard technique in aquatic biology due to its advantages over conventional light microscopy and scanning electron microscopy.     

Techniques for reducing the interfering effects of autofluorescence in fluorescence microscopy: improved detection of sulphorhodamine B-labelled albumin in arterial tissue

Measurements of the transport of circulating sulphorhodamine B-labelled albumin into the arterial wall, made by applying digital imaging fluorescence microscopy to sections of arteries fixed in situ , are limited in sensitivity by the low levels of tracer fluorescence and high levels of autofluorescence emitted from the tissue. Three attempts to improve these ratios are described. In the first, spectra of the tracer in solution and of arterial autofluorescence were used to design novel microscope filters for rhodamine-like dyes. By exciting with the rarely used yellow lines of the mercury arc lamp and detecting a narrow band of emission with Stokes shifts as small as 15 nm, the ratio of tracer fluorescence to autofluorescence was tripled. In the second, effects of different fixatives were investigated. Using a model system, it was confirmed that Karnovsky's fixative gives good tracer immobilization but elevates autofluorescence, whereas fixative-free buffer solutions give low autofluorescence but do not retain the tracer. It was further found that simple formaldehyde-based fixatives, hitherto considered to be poor fixatives of albumin, immobilized the tracer as well as the glutaraldehyde-based fixative, whilst giving autofluorescence levels comparable to those seen with buffer alone; they therefore give excellent tracer fluorescence to autofluorescence ratios. In the third, lowering specimen temperature by 50 °C was found to increase the intensity of tracer fluorescence by 30% whilst autofluorescence was unaffected. These data may have relevance to microscopical studies using other tissues and fluorescent tracers.     

Light sheet based imaging flow cytometry on a microfluidic platform

We propose a light sheet based imaging flow cytometry technique for simultaneous counting and imaging of cells on a microfluidic platform. Light sheet covers the entire microfluidic channel and thus omits the necessity of flow focusing and point scanning based technology. Another advantage lies in the orthogonal detection geometry that totally cuts‐off the incident light, thereby substantially reducing the background in the detection. Compared to the existing state‐of‐art techniques the proposed technique shows marked improvement. Using fluorescently‐coated Saccharomyces cerevisiae cells we have recorded cell counting with throughput as high as 2,090 cells/min in the low flow rate regime and were able to image the individual cells on‐the‐go. Overall, the proposed system is cost‐effective and simple in channel geometry with the advantage of efficient counting in operational regime of low laminar flow. This technique may advance the emerging field of microfluidic based cytometry for applications in nanomedicine and point of care diagnostics. Microsc. Res. Tech. 76:1101–1107, 2013. © 2013 Wiley Periodicals, Inc.     

Compact multiphoton/single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging

An inverted fluorescence microscope was upgraded into a compact three-dimensional laser scanning microscope (LSM) of 65 x 62 x 48 cm dimensions by means of a fast kHz galvoscanner unit, a piezodriven z-stage, and a picosecond (ps) 50 MHz laser diode at 405 nm. In addition, compact turn-key near infrared femtosecond lasers have been employed to perform multiphoton fluorescence and second harmonic generation (SHG) microscopy. To expand the features of the compact LSM, a time-correlated single photon counting unit as well as a Sagnac interferometer have been added to realize fluorescence lifetime imaging (FLIM) and spectral imaging. Using this unique five-dimensional microscope, TauMap, single-photon excited (SPE), and two-photon excited (TPE) cellular fluorescence as well as intratissue autofluorescence of water plant leaves have been investigated with submicron spatial resolution, <270 ps temporal resolution, and 10 nm spectral resolution.