Commercial Confocal Laser Scanning Microscope Research Articles

Realization of noncontact confocal optical microsphere imaging microscope.

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.

Labeling cellular structures in vivo using confined primed conversion of photoconvertible fluorescent proteins.

The application of green-to-red photoconvertible fluorescent proteins (PCFPs) for in vivo studies in complex 3D tissue structures has remained limited because traditional near-UV photoconversion is not confined in the axial dimension, and photomodulation using axially confined, pulsed near-IR (NIR) lasers has proven inefficient. Confined primed conversion is a dual-wavelength continuous-wave (CW) illumination method that is capable of axially confined green-to-red photoconversion. Here we present a protocol to implement this technique with a commercial confocal laser-scanning microscope (CLSM); evaluate its performance on an in vitro setup; and apply primed conversion for in vivo labeling of single cells in developing zebrafish and mouse preimplantation embryos expressing the green-to-red photoconvertible protein Dendra2. The implementation requires a basic understanding of laser-scanning microscopy, and it can be performed within a single day once the required filter cube is manufactured.

Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity.

It is important to determine the most effective method of delivering light onto a specimen for minimal light induced damage. Assays are presented to measure photo-bleaching of fluorophores and photo-toxicity to living cells under different illumination conditions. Turning the light off during part of the experimental time reduced photo-bleaching in a manner proportional to the time of light exposure. The rate of photo-bleaching of EGFP was reduced by 9-fold with light pulsing on the micro-second scale. Similarly, in living cells, rapid line scanning resulted in reduced cell stress as measured by mitochondrial potential, rapid cell protrusion and reduced cell retraction. This was achieved on a commercial confocal laser scanning microscope, without any compromise in image quality, by using rapid laser scan settings and line averaging. Therefore this technique can be implemented broadly without any software or hardware upgrades. Researchers can use the rapid line scanning option to immediately improve image quality on fixed samples, reduce photo-bleaching for large high resolution 3D datasets and improve cell health in live cell experiments. The assays developed here can be applied to other microscopy platforms to measure and optimize light delivery for minimal sample damage and photo-toxicity.

Eu/Tb codoped spindle-shaped fluorinated hydroxyapatite nanoparticles for dual-color cell imaging.

Lanthanide doped fluorinated hydroxyapatite (FAp) nanoparticles are promising cell imaging nanomaterials but they are excited at wavelengths which do not match the light sources usually found in a commercial confocal laser scanning microscope (CLSM). In this work, we have successfully prepared spindle-shaped Eu/Tb codoped FAp nanoparticles by a hydrothermal method. Compared with single Eu doped FAp, Eu/Tb codoped FAp can be excited by a 488 nm laser, and exhibit both green and red light emission. By changing the amounts of Eu and Tb peaks, the emission in the green region (500-580 nm) can be decreased to the benefit of the emission in the red region (580-720 nm), thus reaching a balanced dual color emission. Using MC3T3-E1 cells co-cultured with Eu/Tb codoped FAp nanoparticles, it is observed that the nanoparticles are cytocompatible even at a concentration as high as 800 μg ml(-1). The Eu/Tb codoped FAp nanoparticles are located in the cytoplasm and can be monitored by dual color-green and red imaging with a single excitation light at 488 nm. At a concentration of 200 μg ml(-1), the cytoplasm is saturated in 8 hours, and Eu/Tb codoped FAp nanoparticles retain their fluorescence for at least 3 days. The cytocompatible Eu/Tb codoped FAp nanoparticles with unique dual color emission will be of great use for cell and tissue imaging.

Image correlation spectroscopy for measurements of particle densities and colocalization.

Cells interact with their environment through receptor proteins expressed at their plasma membrane, and protein-protein interactions govern the transduction of signals across the membrane into the cell. Therefore, the ability to measure receptor densities and protein colocalization within the membrane of intact cells is of paramount importance. This unit describes a technique to extract these parameters from fluorescence microscopy images obtained using a commercial confocal laser scanning microscope (CLSM) and other similar types of microscopes. It is based on the analysis of spatial fluorescence intensity fluctuations in the images, which can then be related to particle density and aggregation state via calculation of a spatial autocorrelation function, or used to measure particle colocalization via calculation of a spatial cross-correlation function from dual-color images of proteins tagged with two different fluorophores and imaged in two detection channels. These parameters offer key insights on the interaction of the cell with its environment.

Detailed three‐dimensional visualization of resilin in the exoskeleton of arthropods using confocal laser scanning microscopy

Resilin is a rubber-like protein found in the exoskeleton of arthropods. It often contributes large proportions to the material of certain structures in movement systems. Accordingly, the knowledge of the presence and distribution of resilin is essential for the understanding of the functional morphology of these systems. Because of its specific autofluorescence, resilin can be effectively visualized using fluorescence microscopy. However, the respective excitation maximum is in the UV range, which is not covered by the lasers available in most of the modern commercial confocal laser scanning microscopes. The goal of this study was to test the potential of confocal laser scanning microscopy (CLSM) in combination with a 405 nm laser to visualize and analyse the presence and distribution of resilin in arthropod exoskeletons. The results clearly show that all resilin-dominated structures, which were visualized successfully using wide-field fluorescence microscopy (WFM) and a 'classical' UV excitation, could also be visualized efficiently with the proposed CLSM method. Furthermore, with the application of additional laser lines CLSM turned out to be very appropriate for studying differences in the material composition within arthropod exoskeletons in great detail. As CLSM has several advantages over WFM with respect to detailed morphological imaging, the application of the proposed CLSM method may reveal new information about the micromorphology and material composition of resilin-dominated exoskeleton structures leading to new insights into the functional morphology and biomechanics of arthropods.

Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish.

Fluorescence correlation spectroscopy, raster image correlation spectroscopy, and number and brightness on a commercial confocal laser scanning microscope with analog detectors (Nikon C1)

Fluorescence correlation spectroscopy (FCS) was developed in 1972 by Magde, Elson and Webb. Photon counting detectors and avalanche photodiodes have become standards in FCS to the point that there is a widespread belief that these detectors are essential to perform FCS experiments, despite the fact that FCS was developed using analog detectors. Spatial and temporal intensity fluctuation correlations using analog detection on a commercial Olympus Fluoview 300 microscope have been reported by Brown et al. (2008). However, each analog instrument has its own idiosyncrasies that need to be understood before using the instrument for FCS. In this work, we explore the capabilities of the Nikon C1, a low-cost confocal microscope, to obtain single point FCS, Raster-scan image correlation spectroscopy (RICS), and Number and Brightness data both in solution and incorporated into the membrane of giant unilamellar vesicles. We show that it is possible to obtain dynamic information about fluorescent molecules from single point FCS, RICS, and Number and Brightness using the Nikon C1. We highlighted the fact that care should be taken in selecting the acquisition parameters to avoid possible artifacts due to the detector noise. However, due to relatively large errors in determining the distribution of digital levels for a given microscope setting, the system is probably only adequate for determining relative brightness within the same image.

Diffusion Measurements inside Biofilms by Image-Based Fluorescence Recovery after Photobleaching (FRAP) Analysis with a Commercial Confocal Laser Scanning Microscope

Research about the reactional and structural dynamics of biofilms at the molecular level has made great strides, owing to efficient fluorescence imaging methods in terms of spatial resolution and fast acquisition time but also to noninvasive conditions of observation consistent with in situ biofilm studies. In addition to conventional fluorescence intensity imaging, the fluorescence recovery after photobleaching (FRAP) module can now be routinely implemented on commercial confocal laser scanning microscopes (CLSMs). This method allows measuring of local diffusion coefficients in biofilms and could become an alternative to fluorescence correlation spectroscopy (FCS). We present here an image-based FRAP protocol to improve the accuracy of FRAP measurements inside "live" biofilms and the corresponding analysis. An original kymogram representation allows control of the absence of perturbing bacterial movement during image acquisition. FRAP data analysis takes into account molecular diffusion during the bleach phase and uses the image information to extract molecular diffusion coefficients. The fluorescence spatial intensity profile analysis used here for the first time with biofilms is supported both by our own mathematical model and by a previously published one. This approach was validated to FRAP experiments on fluorescent-dextran diffusion inside Lactococcus lactis and Stenotrophomonas maltophilia biofilms, and the results were compared to previously published FCS measurements.

Analysis of the diffusion of Ras2 in Saccharomyces cerevisiae using fluorescence recovery after photobleaching

Binding, lateral diffusion and exchange are fundamental dynamic processes involved in protein association with cellular membranes. In this study, we developed numerical simulations of lateral diffusion and exchange of fluorophores in membranes with arbitrary bleach geometry and exchange of the membrane-localized fluorophore with the cytosol during fluorescence recovery after photobleaching (FRAP) experiments. The model simulations were used to design FRAP experiments with varying bleach region sizes on plasma membrane-localized wild-type GFP-Ras2 with a dual lipid anchor and mutant GFP-Ras2C318S with a single lipid anchor in live yeast cells to investigate diffusional mobility and the presence of any exchange processes operating in the time scale of our experiments. Model parameters estimated using data from FRAP experiments with a 1 µm × 1 µm bleach region-of-interest (ROI) and a 0.5 µm × 0.5 µm bleach ROI showed that GFP-Ras2, single or dual lipid modified, diffuses as single species with no evidence of exchange with a cytoplasmic pool. This is the first report of Ras2 mobility in the yeast plasma membrane. The methods developed in this study are generally applicable for studying diffusion and exchange of membrane-associated fluorophores using FRAP on commercial confocal laser scanning microscopes.

Single Point FCS on a Commercial Confocal Laser Scanning Microscope with Analog Detectors

Fluorescence Correlation Spectroscopy is a technique invented in the early 1970s to measure diffusion coefficient, chemical reaction rates and photo physical processes. It is a common belief that in order to obtain single point FCS data, one needs either a sophisticated FCS instrument with photon counting detectors or avalanche photon detectors or an instrument custom made for this type of experiments. Here we show that we can obtain single point FCS data on a commercial confocal laser scanning microscope without any modifications (Nikon C1).

Measuring Diffusion of Lipid-like Probes in Artificial and Natural Membranes by Raster Image Correlation Spectroscopy (RICS): Use of a Commercial Laser-Scanning Microscope with Analog Detection

The heterogeneity in composition and interaction within the cellular membrane translates into a wide range of diffusion coefficients of its constituents. Therefore, several complementary microfluorimetric techniques such as fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP) and single-particle tracking (SPT) have to be applied to explore the dynamics of membrane components. The recently introduced raster image correlation spectroscopy (RICS) offers a much wider dynamic range than each of these methods separately and allows for spatial mapping of the dynamic properties. RICS is implemented on a confocal laser-scanning microscope (CLSM), and the wide dynamic range is achieved by exploiting the inherent time information carried by the scanning laser beam in the generation of the confocal images. The original introduction of RICS used two-photon excitation and photon counting detection. However, most CLSM systems are based on one-photon excitation with analog detection. Here we report on the performance of such a commercial CLSM (Zeiss LSM 510 META) in the study of the diffusion of the fluorescent lipid analog 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indodicarbocyanine perchlorate (DiI-C(18)(5)) both in giant unilamellar vesicles and in the plasma membrane of living oligodendrocytes, i.e., the myelin-producing cells of the central nervous system. It is shown that RICS on a commercial CLSM with analog detection allows for reliable results in the study of membrane diffusion by removal of unwanted correlations introduced by the analog detection system. The results obtained compare well with those collected by FRAP and FCS.

Diffusion and Exchange of Non-Integral Membrane Associated Fluorophores During Fluorescence Recovery After Photobleaching with the Confocal Laser Scanning Microscope: ROI Size Analysis of EGFP:Ras2 Plasma Membrane Diffusion in Saccharomyces cerevisiae

Binding, lateral diffusion and exchange are fundamental dynamic processes involved in protein localization in cellular membranes. In this study, we developed numerical simulations of lateral diffusion of a fluorophore in a membrane with arbitrary bleach geometry and exchange of the fluorophore with cytosol during Fluorescence Recovery after Photobleaching (FRAP) experiments. Based on our model simulations, we designed and performed FRAP experiments with varying bleach region sizes on plasma-membrane localized EGFP:Ras2 in live yeast cells to investigate the mobility and the presence of any exchange processes operating in the time scale of our experiments. Model parameters estimated from a 1 micron x 1 micron bleach region-of-interest (ROI) size successfully predicted the 0.5 micron x 0.5 micron bleach ROI experiment without additional fitting. Successful prediction of the second experiment without data fitting shows the agreement of the experiment with the theory and excluded alternative models including both diffusion and binding, which were also tested against the experimental data. We also performed Fluorescence Correlation Spectroscopy (FCS) experiments as an independent method to measure the mobility of EGFP:Ras2. We show that simulation of FRAP experiments based on the mobilities and fluorophore fractions derived from FCS model fits enables the validation of the FCS model. The methods developed in this study are generally applicable for studying diffusion and exchange of membrane associated fluorophores using FRAP on widely available commercial confocal laser scanning microscopes.

Noninvasive monitoring of mesenchymal stem cells by 1.2 GHz acoustic microscopy

Cell‐based therapies can benefit from noninvasive and marker‐free monitoring techniques for living cells. For this purpose a phase‐sensitive scanning acoustic microscope operating at a frequency of 1.2 GHz was combined with a commercial confocal laser scanning microscope. The system is equipped with a live‐support system for the long‐term observation of living cells. Confocal acoustic imaging with phase and magnitude contrast and confocal laser scanning microscopy can be performed simultaneously. Both techniques are used in reflection from opposing sides of the object. Time‐lapsed acoustic microscope images of ovine mesenchymal stem cells are presented. For this purpose, a pseudo‐3D representation is generated by encoding the unwrapped phase in the height and the magnitude in the brightness. In the case of highly reflective substrates and sufficiently low reflection from the interface between the cells and the surrounding fluid the echo from the top of the cells can be neglected and the phase contrast image can be transformed to a time‐of‐flight image. In the same approximation the magnitude image provides information about the gradual extinction of the echo signal due to absorption in the cells. The two images can be combined to generate a new form of contrast representing the product of the absorption coefficient and the velocity of sound inside the observed cells.

Multifunctional fluorescence correlation microscope for intracellular and microfluidic measurements

A modified fluorescence correlation microscope (FCM) was built on a commercial confocal laser scanning microscope (CLSM) by adding two sensitive detectors to perform fluorescence correlation spectroscopy (FCS). A single pinhole for both imaging and spectroscopy and a simple slider switch between the two modes thus facilitate the accurate positioning of the FCS observation volume after the confocal image acquisition. Due to the use of a single pinhole for CLSM and FCS the identity of imaged and spectroscopically observed positions is guaranteed. The presented FCM system has the capability to position the FCS observation volume at any point within the inner 30% of the field of view without loss in performance and in the inner 60% of the field of view with changes of FCS parameters of less than 10%. A single pinhole scheme for spatial fluorescence cross correlation spectroscopy performed on the FCM system is proposed to determine microfluidic flow angles. To show the applicability and versatility of the system, we measured the translational diffusion coefficients on the upper and lower membranes of Chinese hamster ovary cells. Two-photon excitation FCS was also realized by coupling a pulsed Ti: sapphire laser into the microscope and used for flow direction characterization in microchannels.

A mems electromagnetic optical scanner for a commercial confocal laser scanning microscope

A MEMS electromagnetic optical scanner for horizontal scanning of a commercial confocal laser scanning microscope has been developed. The purpose is to replace the currently used commercially available scanner with our new MEMS scanner in an existing microscope product, and therefore, the scanner specifications have to be compatible with those of the current one. Electromagnetic actuation is selected because of the millimeter-sized mirror, and a single crystal silicon hinge is used for realizing high-speed scanning with sufficient scan angle. In order to maintain mirror flatness for high quality optics requirement, the whole wafer thickness (300 μm) is used as the mirror, resulting in a large moment of inertia, and this has been taken into consideration in the actuator design. Although few MEMS actuators have been commercialized to date, it has successfully satisfied all the specifications including not only the fundamentals such as resonant frequency and scan angle but also those for the commercial product such as scanning stability and reliability. It has been commercialized as a part of our product, Olympus OLS1100 (remodeled as OLS1200 in August 2002).

High Sensitivity Spectrograph for Use in Fluorescence Microscopy

In this paper a versatile, high sensitivity spectrograph is presented for use in fluorescence microscopy. The high sensitivity is achieved by using a prism for the dispersion in combination with a state-of-the-art back illuminated charge-coupled device (CCD) camera. The spectrograph, including the CCD camera, has a detection efficiency of 0.77 ± 0.05 at 633 nm. Full emission spectra with a 1–5 nm spectral resolution can be recorded at a maximum rate of 800 spectra per second. Two applications are shown, in which the spectrograph is fiber-coupled to a commercial confocal laser scanning microscope. In the first example, Förster resonance energy transfer imaging experiments were carried out on double-labeled actin filaments in the in vitro motility assay. A 160 × 160 point image was recorded in 1.5 min at 3 ms dwell time per image point. In the second application, a time-resolved study of single quantum dots is presented at 5.2 ms time resolution.