Commercial Laser Scanning Microscope Research Articles

Segmented fluorescence correlation spectroscopy (FCS) on a commercial laser scanning microscope

Performing accurate Fluorescence Correlation Spectroscopy (FCS) measurements in cells can be challenging due to cellular motion or other intracellular processes. In this respect, it has recently been shown that analysis of FCS data in short temporal segments (segmented FCS) can be very useful to increase the accuracy of FCS measurements inside cells. Here, we demonstrate that segmented FCS can be performed on a commercial laser scanning microscope (LSM), even in the absence of the dedicated FCS module. We show how data can be acquired on a Leica SP8 confocal microscope and then exported and processed with a custom software in MATLAB. The software performs segmentation of the data to extract an average ACF and measure the diffusion coefficient in specific subcellular regions. First of all, we measure the diffusion of fluorophores of different size in solution, to show that good-quality ACFs can be obtained in a commercial LSM. Next, we validate the method by measuring the diffusion coefficient of GFP in the nucleus of HeLa cells, exploiting variations of the intensity to distinguish between nucleoplasm and nucleolus. As expected, the measured diffusion coefficient of GFP is slower in the nucleolus relative to nucleoplasm. Finally, we apply the method to HeLa cells expressing a PARP1 chromobody to measure the diffusion coefficient of PARP1 in different subcellular regions. We find that PARP1 diffusion is slower in the nucleolus compared to the nucleoplasm.

Improvement of image resolution by combining enhanced confocal microscopy and quantum dot triexciton imaging.

Super‐resolution fluorescence imaging provides critically improved information about the composition, organization, and dynamics of subcellular structures. Quantum dot triexciton imaging (QDTI) has been introduced as an easy‐to‐use sub‐diffraction imaging method that achieves an almost 2‐fold improvement in resolution when used with conventional confocal microscopes. Here, we report an overall 3‐fold increase in lateral and axial resolution compared to conventional confocal microscopes by combining QDTI with state‐of‐the‐art commercial laser scanning microscope systems.

Super-resolution fluorescence microscopy by line-scanning with an unmodified two-photon microscope.

Fluorescence-based microscopy as one of the standard tools in biomedical research benefits more and more from super-resolution methods, which offer enhanced spatial resolution allowing insights into new biological processes. A typical drawback of using these methods is the need for new, complex optical set-ups. This becomes even more significant when using two-photon fluorescence excitation, which offers deep tissue imaging and excellent z-sectioning. We show that the generation of striped-illumination patterns in two-photon laser scanning microscopy can readily be exploited for achieving optical super-resolution and contrast enhancement using open-source image reconstruction software. The special appeal of this approach is that even in the case of a commercial two-photon laser scanning microscope no optomechanical modifications are required to achieve this modality. Modifying the scanning software with a custom-written macro to address the scanning mirrors in combination with rapid intensity switching by an electro-optic modulator is sufficient to accomplish the acquisition of two-photon striped-illumination patterns on an sCMOS camera. We demonstrate and analyse the resulting resolution improvement by applying different recently published image resolution evaluation procedures to the reconstructed filtered widefield and super-resolved images.This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)'.

Femtosecond Stimulated Raman microscopy: home-built realization and a case study of biological imaging

The progresses of femtosecond laser physics have had a significant impact on research and development of nonlinear microscopy techniques, such as Stimulated Raman scattering microscopy. The main advantages of this technique, based on vibrational spectroscopy, is its abilities to perform label-free imaging with high sensitivity, high spatial and spectral resolution, 3D sectioning capability and fast time of image acquisition (typically a few seconds). The main aim of this paper is to demonstrates the home-built realization of an imaging system based on Stimulated Raman Scattering (SRS) powered by femtosecond laser sources, using a National Instrument data acquisition (NI-DAQ) card. The SRS microscope is obtained by the integration of an SRS experimental set-up with a commercial laser scanning microscope. The successful implementation of the imaging system is demonstrated reporting label free images of polystyrene beads with diameter of 3 microns and evaluating their signal to noise ratio. Finally, the reliability of our system in a biological environment is tested. Taking advantage of SRS microscopy, subcellular localizations of lipid droplets inside adipocyte cells are demonstrated.

Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering.

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. SRS imaging modality can be obtained using commercial laser-scanning microscopes by equipping with a non-descanned forward detector with proper bandpass filters and lock-in amplifier (LIA) detection scheme. A schematic layout of a typical SRS microscope includes the following: two pulsed laser beams, (i.e., the pump and probe directed in a scanning microscope), which must be overlapped in both space and time at the image plane, then focused by a microscope objective into the sample through two scanning mirrors (SMs), which raster the focal spot across an x-y plane. After interaction with the sample, transmitted output pulses are collected by an upper objective and measured by a forward detection system inserted in an inverted microscope. Pump pulses are removed by a stack of optical filters, whereas the probe pulses that are the result of the SRS process occurring in the focal volume of the specimen are measured by a photodiode (PD). The readout of the PD is demodulated by the LIA to extract the modulation depth. A two-dimensional (2D) image is obtained by synchronizing the forward detection unit with the microscope scanning unit. In this paper, the implementation of an SRS microscope is described and successfully demonstrated, as well as the reporting of label-free images of polystyrene beads with diameters of 3 µm. It is worth noting that SRS microscopes are not commercially available, so in order to take advantage of these characteristics, the homemade construction is the only option. Since SRS microscopy is becoming popular in many fields, it is believed that this careful description of the SRS microscope implementation can be very useful for the scientific community.

Resolution enhancement in laser scanning microscopy with deconvolution switching laser modes (D-SLAM).

Laser scanning microscopy is limited in lateral resolution by the diffraction of light. Superresolution methods have been developed since the 90s to overcome this limitation. However superresolution is generally achieved at the expense of a greater complexity (high power lasers, very long acquisition times, specific fluorophores) and limitations on the observable samples. In this paper we propose a method to improve the resolution of confocal microscopy by combining different laser modes and deconvolution. Two images of the same field are acquired with the confocal microscope using different laser modes and used as inputs to a deconvolution algorithm. The two laser modes have different Point Spread Functions and thus provide complementary information leading to an image with enhanced resolution compared to using a single confocal image as input to the same deconvolution algorithm. By changing the laser modes to Bessel-Gauss beams we were able to further improve the efficiency of the deconvolution algorithm and obtain images with a residual Point Spread Function having a width of 0.14 λ (72 nm at a wavelength of 532 nm). This method only requires a laser scanning microscope and is not dependent on certain specific properties of fluorescent proteins. The proposed method requires only a few add-ons to classical confocal or two-photon microscopes and can easily be retrofitted into an existing commercial laser scanning microscope.

Automated calibration and control for polarization-resolved second harmonic generation on commercial microscopes.

Polarization-resolved second harmonic generation (P-SHG) microscopy has evolved as a promising technique to reveal subresolution information about the structure and orientation of ordered biological macromolecules. To extend the adoption of the technique, it should be easily integrated onto commercial laser scanning microscopes. Furthermore, procedures for easy calibration and assessment of measurement accuracy are essential, and measurements should be fully automated to allow for analysis of large quantities of samples. In this paper we present a setup for P-SHG which is readily incorporated on commercial multiphoton microscopes. The entire system is completely automated which allows for rapid calibration through the freely available software and for automated imaging for different polarization measurements, including linear and circular polarization of the excitation beam. The results show that calibration settings are highly system dependent. We also show that the accuracy of the polarization control is easily quantified and that it varies between systems. The accuracy can be tuned by iterative alignment of optics or a more fine-grained calibration procedure. Images of real samples show that the red accuracy of the results is easily visualized with the automated setup. Through this system we believe that P-SHG could develop a wider adoption in biomedical applications.

Crosstalk-free multicolor RICS using spectral weighting.

Raster image cross-correlation spectroscopy (ccRICS) can be used to quantify the interaction affinities between diffusing molecules by analyzing the fluctuations between two-color confocal images. Spectral crosstalk compromises the quantitative analysis of ccRICS experiments, limiting multicolor implementations to dyes with well-separated emission spectra. Here, we remove this restriction by introducing raster spectral image correlation spectroscopy (RSICS), which employs statistical filtering based on spectral information to quantitatively separate signals of fluorophores during spatial correlation analysis. We investigate the performance of RSICS by testing how different levels of spectral overlap or different relative signal intensities affect the correlation function and analyze the influence of statistical filter quality. We apply RSICS in vitro to resolve dyes with very similar emission spectra, and carry out RSICS in live cells to simultaneously analyze the diffusion of molecules carrying three different fluorescent protein labels (eGFP, Venus and mCherry). Finally, we successfully apply statistical weighting to data that was recorded with only a single detection channel per fluorophore, highlighting the general applicability of this method to data acquired with any type of multicolor detection. In conclusion, RSICS enables artifact-free quantitative analysis of concentrations, mobility and interactions of multiple species labeled with different fluorophores. It can be performed on commercial laser scanning microscopes, and the algorithm can be easily extended to other image correlation methods. Thus, RSICS opens the door to quantitative multicolor fluctuation analyses of complex (bio-) molecular systems.

Quantifying transcription factor-DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy.

Probing transcription factor (TF)-DNA interactions remains challenging in complex in vivo systems such as mammalian embryos, especially when TF copy numbers and fluorescence background are high. To address this difficulty, fluorescence correlation spectroscopy (FCS) can be combined with the use of photoactivatable fluorescent proteins to achieve selective photoactivation of a subset of tagged TF molecules. This approach, termed paFCS, enables FCS measurements within single cell nuclei inside live embryos, and obtains autocorrelation data of a quality previously only attainable in simpler in vitro cell culture systems. Here, we present a protocol demonstrating the applicability of paFCS in developing mouse embryos by outlining its implementation on a commercial laser-scanning microscope. We also provide procedures for optimizing the photoactivation and acquisition parameters and determining key parameters describing TF-DNA binding. The entire procedure can be performed within ∼2 d (excluding embryo culture time), although the acquisition of each paFCS data set takes only ∼10 min. This protocol can be used to noninvasively reveal cell-to-cell variation in TF dynamics, as well as critical, fate-predicting changes over the course of early embryonic development.

Quantitative Mapping of Intranuclear Diffusion in Living Cells by Phasor Analysis of Local RICS

Image Correlation Spectroscopy (ICS) (Wiseman, Cold Spring Harb. Protoc. 2015) based techniques are quantitative tools for assessing the concentration and dynamics of proteins diffusing in living cells. A wide variety of these methods (ICS, ICCS, RICS, kICS etc.) is applicable to commercial laser scanning microscopes in order to investigate subcellular biophysical properties, for instance the study of protein mobility within the nucleus of eukaryotic cells. Although powerful, these techniques may fail to retrieve a correct value when analyzing inhomogeneous samples, given that no further assumption is made on the specific nature of the inhomogeneity. Being the cell an intrinsically heterogeneous system, a prescreening method for the characterization of the heterogeneity of the system will increase the reliability of ICS techniques for cellular applications.In this respect, we recently introduced a method, based on the phasor analysis of local ICS (PLICS), for extending ICS to heterogeneous systems without the need of a priori assumptions (Scipioni et al., Biophys. J. 2016). The phasor approach, originally developed for the identification of components in fluorescence-lifetime imaging (Digman et al., Biophys. J. 2008), is used within the PLICS algorithm for the analysis of local spatial correlation functions as a fast and unbiased alternative to the fitting procedure. Here we present application of the PLICS concept to Raster Image Correlation Spectroscopy (RICS) (Digman et al., Biophys. J. 2005) for obtaining a map of intra-nuclear diffusion of expressed EGFP in living HeLa cells. The PLICS approach is used to provide a segmentation of the image into sub-regions with homogeneous diffusion coefficient, that is successively quantified by applying Arbitrary Regions Image Correlation Spectroscopy (ARICS) (Hendrix et al., Biophys. J. 2016),a state of the art technique developed for extending the ICS analysis to non-rectangular regions.We believe that PLICS could become a widely used prescreening approach, applicable to several kinds of ICS techniques, for addressing the heterogeneity of a dataset prior to the application of the technique. Furthermore, if the statistics is significant, PLICS is capable of providing an exhaustive mapping over the entire dataset of the property we are willing to measure.

Scanning Mueller polarimetric microscopy.

A full Mueller polarimeter was implemented on a commercial laser-scanning microscope. The new polarimetric microscope is based on high-speed polarization modulation by spectral coding using a wavelength-swept laser as a source. Calibration as well as estimation of the measurement errors of the device are reported. The acquisition of Mueller images at the speed of a scanning microscope is demonstrated for the first time. Mueller images of mineral and biological samples illustrate this new polarimetric microscopy.

One-Photon Lithography for High-Quality Lipid Bilayer Micropatterns.

A relevant question in cell biology with broad implications in biomedicine is how the organization and dynamics of interacting membranes modulate signaling cascades that involve cell-cell contact. The functionalization of surfaces with supported lipid bilayers containing tethered proteins is a particularly useful method to present ligands with membrane-like mobility to cells. Here, we present a method to generate micrometer-sized patches of lipid bilayers decorated with proteins. The method uses an economic microcontact printing technique based on one-photon lithography that can be easily implemented in a commercial laser scanning microscope. We verified that both proteins and lipids freely diffuse within the patterned bilayer, as assessed by z-scan fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. These results suggest that the supported lipid bilayer patterns constitute an optimal system to explore processes involving direct interactions between cells. We also illustrate possible applications of this method by exploring the interaction of cells expressing the Fas receptor and patterns of lipid bilayers containing an agonist antibody against Fas.

A simple but precise method for quantitative measurement of the quality of the laser focus in a scanning optical microscope.

SummaryWe report a method for characterizing the focussing laser beam exiting the objective in a laser scanning microscope. This method provides the size of the optical focus, the divergence of the beam, the ellipticity and the astigmatism. We use a microscopic‐scale knife edge in the form of a simple transmission electron microscopy grid attached to a glass microscope slide, and a light‐collecting optical fibre and photodiode underneath the specimen. By scanning the laser spot from a reflective to a transmitting part of the grid, a beam profile in the form of an error function can be obtained and by repeating this with the knife edge at different axial positions relative to the beam waist, the divergence and astigmatism of the postobjective laser beam can be obtained. The measured divergence can be used to quantify how much of the full numerical aperture of the lens is used in practice. We present data of the beam radius, beam divergence, ellipticity and astigmatism obtained with low (0.15, 0.7) and high (1.3) numerical aperture lenses and lasers commonly used in confocal and multiphoton laser scanning microscopy. Our knife‐edge method has several advantages over alternative knife‐edge methods used in microscopy including that the knife edge is easy to prepare, that the beam can be characterized also directly under a cover slip, as necessary to reduce spherical aberrations for objectives designed to be used with a cover slip, and it is suitable for use with commercial laser scanning microscopes where access to the laser beam can be limited.

Creation of a three-dimensional spherical fluorescence spot for super-resolution microscopy using a two-color annular hybrid wave plate.

We fabricate a two-color annular hybrid wave plate (TAHWP) for fluorescence depletion super-resolution microscopy by combining quartz plates. Inserting the TAHWP into a commercial laser-scanning microscope together with a quarter-wave plate creates a three-dimensional spherical fluorescence spot smaller than the diffraction limit. Since a simple experimental setup easily provides a spot with a volume of ∼100 nm3, the TAHWP can improve the performance of various microspectroscopic devices.

The fast polarization modulation based dual-focus fluorescence correlation spectroscopy

We introduce two new alternative experimental realizations of dual focus fluorescence correlation spectroscopy (2fFCS), a method which allows for obtaining absolute diffusion coefficient of fast moving fluorescing molecules at nanomolar concentrations, based on fast polarization modulation of the excitation beam by a resonant electro-optical modulator. The first approach rotates every second linearly polarized laser pulse by 90 degrees to obtain independent intensity readout for both foci, similar to original design. The second approach combines polarization modulation of cw laser and fluorescence lifetime correlation spectroscopy (FLCS) like analysis to obtain clean correlation curves for both overlapping foci. We tested our new approaches with different lasers and samples, revealed a need for intensity cross-talk corrections by comparing the methods with each other and discussed experimental artifacts stemming from improper polarization alignment and detector afterpulsing. The advantages of our solutions are that the polarization rotation approach requires just one pulsed laser for each wavelength, that the polarization modulation approach even mitigates the need of pulsed lasers by using standard cw lasers and that it allows the DIC prism to be placed at an arbitrary angle. As a consequence the presented experimental solutions for 2fFCS can be more easily implemented into commercial laser scanning microscopes.

Multi-photon microscopic imaging of inflammatory fibroid polyps/Multi-Photonen-Mikroskopie entzündlich fibroider Polypen

AbstractMost applications of multi-photon microscopy (MPM) in biology and medicine focus on high-resolution imaging of physiology, morphology and cell–cell interactions in intact tissues or live animals. In this paper, our aim is to assess whether MPM has the ability to provide comparable information for stained histology slides and to identify inflammatory fibroid polyp (IFP) tissue sections without H&E staining.A tissue section without H&E staining was examined using a multi-photon microscope consisting of a mode-locked Ti: sapphire femtosecond laser (Mira 900-F; Coherent, Inc.) and a commercial laser scanning microscope (LSM 510 META; Carl Zeiss, Inc.) with a Plan-Neofluar 40×/0.75 objective. Large area images and a series of optical sectioning images were obtained using an optional fine-focusing stage (HRZ 200; Carl Zeiss, Inc.).MPM images showed the main characteristics of IFP that corresponded well with H&E microscopic results, such as a typical onion skin arrangement of reticular fibers around thin-walled blood vessels, and short spindle cells intermixed with abundant inflammatory cells. A series of optical sectioning images displayed morphological structure of tissue sections at different depths.MPM has the ability to provide comparable results to H&E staining. Its capability of optical sectioning can present the morphological structure of different optical sectioning planes, making MPM an alternative for those cases where the sample is very thick or cannot be stained.

Super-resolution microscope using a two-color phase plate for generating quasi-Laguerre–Gaussian beam

To achieve high lateral resolution overcoming the diffraction limit, a two-color phase plate (TPP) for generating a quasi-Laguerre–Gaussian beam was applied to super-resolution microscopy (SRM) based on fluorescence depletion. Putting the TPP into a robust optical path in a commercial laser-scanning microscope, we obtained a point spread function with a full width at half maximum three times smaller than diffraction limit. The measured contrast transfer function shows that line and space patterns finer than the diffraction limit were clearly resolved and the image contrast was improved. Since the TPP can easily improve lateral resolution in SRM without any precise adjustment, our setup provides a practical super-resolution microscope.

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.

Accurate Determination of Membrane Dynamics with Line-Scan FCS

Here we present an efficient implementation of line-scan fluorescence correlation spectroscopy (i.e., one-dimensional spatio-temporal image correlation spectroscopy) using a commercial laser scanning microscope, which allows the accurate measurement of diffusion coefficients and concentrations in biological lipid membranes within seconds. Line-scan fluorescence correlation spectroscopy is a calibration-free technique. Therefore, it is insensitive to optical artifacts, saturation, or incorrect positioning of the laser focus. In addition, it is virtually unaffected by photobleaching. Correction schemes for residual inhomogeneities and depletion of fluorophores due to photobleaching extend the applicability of line-scan fluorescence correlation spectroscopy to more demanding systems. This technique enabled us to measure accurate diffusion coefficients and partition coefficients of fluorescent lipids in phase-separating supported bilayers of three commonly used raft-mimicking compositions. Furthermore, we probed the temperature dependence of the diffusion coefficient in several model membranes, and in human embryonic kidney cell membranes not affected by temperature-induced optical aberrations.

Detecting Protein Complexes in Living Cells from Laser Scanning Confocal Image Sequences by the Cross Correlation Raster Image Spectroscopy Method

We describe a general method for detecting molecular complexes based on the analysis of single molecule fluorescence fluctuations from laser scanning confocal images. The method detects and quantifies complexes of two different fluorescent proteins noninvasively in living cells. Because in a raster scanned image successive pixels are measured at different times, the spatial correlation of the image contains information about dynamic processes occurring over a large time range, from the microseconds to seconds. The correlation of intensity fluctuations measured simultaneously in two channels detects protein complexes that carry two molecules of different colors. This information is obtained from the entire image. A map of the spatial distribution of protein complexes in the cell and their diffusion and/or binding properties can be constructed. Using this cross correlation raster image spectroscopy method, specific locations in the cell can be visualized where dynamics of binding and unbinding of fluorescent protein complexes occur. This fluctuation imaging method can be applied to commercial laser scanning microscopes thereby making it accessible to a large community of scientists.