Determination of particle number and brightness using a laser scanning confocal microscope operating in the analog mode

We describe a method to obtain the brightness and number of molecules at each pixel of an image stack obtained with a laser scanning microscope. The method is based on intensity fluctuations due to the diffusion of molecules in a pixel. For a detector operating in the analog mode, the variance must be proportional to the intensity. Once this constant has been calibrated, we use the ratio between the variance and the intensity to derive the particle brightness. Then, from the ratio of the intensity to the brightness we obtain the average number of particles in the pixel. We show that the method works with molecules in solution and that the results are comparable to those obtained with fluctuation correlation spectroscopy. We compare the results obtained with the detector operating in the analog and photon counting mode. Although the dynamic range of the detector operating in the photon counting mode is superior, the performance of the analog detector is acceptable under common experimental conditions. Since most commercial laser scanning microscopes operate in the analog mode, the calculation of brightness and number of particles can be applied to data obtained with these instruments, provided that the variance is proportional to the intensity. We demonstrate that the recovered brightness of mEGFP, independent of concentration, is similar whether measured in solution or in two different cell types. Furthermore, we distinguish between mobile and immobile components, and introduce a method to correct for slow variations in intensity.     

Evaluation of a femtosecond fiber laser for two-photon fluorescence correlation spectroscopy

This work evaluates a femtosecond fiber laser for use in two-photon fluorescence fluctuation spectroscopy. Fiber lasers present an attractive alternative to Ti:Sapphire systems because of their compact size and portability. Autocorrelation of the second harmonic generation signal from the laser demonstrates that its stability is sufficient for two-photon fluorescence correlation spectroscopy. Fluorescence correlation spectroscopy autocorrelation traces were well fit by a Gaussian-Lorentzian squared model with a beam waist near the diffraction limit for the 810 nm wavelength. A photon counting histogram collected with this system also fit nicely to a single-species model, further demonstrating the quality of the focal shape. The authors conclude that the output from the femtosecond fiber laser is sufficiently stable and has a high enough quality beam shape for fluctuation fluorescence methods, and thus represents an effective, compact, readily portable two-photon excitation source.     

Quantitative measurement of the resolution and sensitivity of confocal microscopes using line-scanning fluorescence correlation spectroscopy

Spatial resolution and the sensitivity to detect a fluorophore are the two most important optical parameters that characterize a confocal microscope. However, these are rather difficult to estimate quantitatively. We show that fluorescence correlation spectroscopy (FCS) provides an easy and reliable measure of these quantities. We modify existing schemes for performing FCS on a commercial confocal microscope to carry out these measurements, and provide an analysis routine that can yield the relevant quantities. Our method does not require any modification of the confocal microscope, yet it yields a robust measure of the resolution and sensitivity of the instrument.     

A method to compensate for light attenuation with depth in three-dimensional DNA image cytometry using a confocal scanning laser microscope

A method to compensate for attenuation of detected light with increased depth of the collected optical section, and its application in three-dimensional (3-D) DNA image cytometry is described. The method is based on studying the stack of 2-D histograms that can be formed from each consecutive pair of sections in a stack of optical serial sections. An attenuation factor is calculated interactively and a new compensated section series is computed. Formalin-fixed paraffin-embedded rat tissue was stained with propidium iodide. Each cell nucleus is extracted by thresholding and its total intensity is calculated. The coefficient of variation (CV) of the total intensity of all cells in each stack is computed. For comparison the CV of the same cells is computed in the uncompensated stacks. This study shows a significantly lower CV for the compensated data, thus contributing to the accuracy of DNA quantification in 3-D DNA image cytometry.     

A simple method for overcoming some problems when observing thick reflective biological samples with a confocal scanning laser microscope

A simple device is described, which allows the range of depth of scanning to be reduced when observing thick reflecting biological samples with a confocal scanning laser microscope (CSLM). Thick histological sections of human skin and rat brain stem were mounted between two coverslips (‘sandwich’ style) and the optical tomography was performed from both sides by turning the ‘sandwich’ upside-down. The samples were impregnated using standard Golgi–Cox, ‘rapid Golgi’ or other silver methods. The ability to turn the ‘sandwich’ upside-down is particularly useful when the reflective structure inspected is deep inside the section, i.e. near the lower surface of the specimen, or when it is opaque to the laser beam or excessively reflective.     

A system for gas sensing employing correlation spectroscopy and wavelength modulation techniques with a multimode diode laser

A tunable multi-mode diode laser system based on correlation spectroscopy and wavelength modulation spectroscopy (TMDL–COSPEC–WMS) is designed and demonstrated for the concentration measurements of oxygen using A-band absorption lines of oxygen around 760 nm. The O₂ concentrations are conversed from the relation between the normalized WMS-2f signal peak heights of the measurement and reference signals which selected based on high signal to noise ratio and correlation coefficient. The correlation and the fitted slope between the measured and actual O₂ concentration are 0.9987 and of 1.025 ± 0.012 respectively over the tested range, which indicate the high linearity and accuracy of the system. A sensitivity of 350 ppm m is approved using 30 successive measurements with each measurement time taking ∼20 s during 30 min. A continuous measurement for oxygen in ambient air during approximately 200 min confirms the stability and the capability of the system.     

The use of Lucifer yellow,bodipy, FITC,TRITC, RITC and Texas red for dual immunofluorescence visualized with a confocal scanning laser microscope

A new method of comparing the relative merits of different fluorophores that undergo relatively rapid irreversible photo-inactivation is described. This method showed that the levels of fluorescent emission seen with both fluorescein isothiocyanate (FITC) and bodipy fl conjugated to streptavidin were similar when examined under conditions where they exhibited equal rates of irreversible photo-inactivation. Bodipy fl and FITC give lower levels of cross-talk into images of cells immunofluorescently stained with either rhodamine isothiocyanate (RITC) or tetramethyl rhodamine isothiocyanate (TRITC) than into images of cells stained with Texas red, under conditions where the three red fluorophores exhibited an equal level of sensitivity. Furthermore, bodipy fl gave much lower levels of cross-talk into images of RITC-stained cells than either FITC or Lucifer yellow. TRITC, but not RITC or Texas red, gave significant levels of cross-talk into the green band-pass filters used to visualize FITC and bodipy fl. From these results it seems that a combination of bodipy fl and RITC provides the best contrast when visualizing dual immunofluorescence with a confocal scanning laser microscope if the 488-nm line of an argon ion laser is used as the excitation source.     

Polyethylene glycol (PEG) embedding and subsequent de-embedding as a method for the correlation of light microscopy,scanning electron microscopy,and transmission electron microscopy

The polyethylene glycol (PEG) embedding and subsequent deembedding method was applied to the observation of general tissues in scanning electron microscopy (SEM). Resulting SEM images were of high quality. It was demonstrated that intermicroscopic correlation of images between light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) is easily and reliably done by means of the PEG method. In particular, the exact correlation of immuno-LM with SEM is shown to be of potential value.     

A precise light attenuation correction for confocal scanning microscopy with O(n4/3) computing time and O(n) memory requirements for n voxels

Some dyes and tissues observed by confocal fluorescence microscopy show remarkable opacity, caused by absorption and scattering within the scanned volume. The efficiency of the excitation and fluorescence process may vary by a factor of 10 from top to bottom regions even in not very extended scan volumes. Based on known attenuation properties of a specimen, the amount of damping is computed by integrating the attenuation along all light paths within the numerical aperture of the objective, Thus, to correct a single volume element (voxel), one has to take into account the damping within the whole extended conical volume between the lens and the focus, resulting in intolerable execution times. Common approaches reduce resolution or simplify the integration paths, thereby resulting in either a loss of fine resolution or showing a low resolution versus computation time ratio. This paper presents a more efficient reformulation of this spatial integration process without simplifying the physical background. This algorithm requires computing times slightly longer than those of programs using simplified physical and statistical approaches. However, the algorithm may be tuned to achieve a precision and stability comparable to exhaustive integration.     

Atomic force microscope (AFM) combined with the ultramicrotome: a novel device for the serial section tomography and AFM/TEM complementary structural analysis of biological and polymer samples

A new device (NTEGRA Tomo) that is based on the integration of the scanning probe microscope (SPM) (NT‐MDT NTEGRA SPM) and the Ultramicrotome (Leica UC6NT) is presented. This integration enables the direct monitoring of a block face surface immediately following each sectioning cycle of ultramicrotome sectioning procedure. Consequently, this device can be applied for a serial section tomography of the wide range of biological and polymer materials. The automation of the sectioning/scanning cycle allows one to acquire up to 10 consecutive sectioned layer images per hour. It also permits to build a 3‐D nanotomography image reconstructed from several tens of layer images within one measurement session. The thickness of the layers can be varied from 20 to 2000 nm, and can be controlled directly by its interference colour in water. Additionally, the NTEGRA Tomo with its nanometer resolution is a valid instrument narrowing and highlighting an area of special interest within volume of the sample. For embedded biological objects the ultimate resolution of SPM mostly depends on the quality of macromolecular preservation of the biomaterial during sample preparation procedure. For most polymer materials it is comparable to transmission electron microscopy (TEM). The NTEGRA Tomo can routinely collect complementary AFM and TEM images. The block face of biological or polymer sample is investigated by AFM, whereas the last ultrathin section is analyzed with TEM after a staining procedure. Using the combination of both of these ultrastructural methods for the analysis of the same particular organelle or polymer constituent leads to a breakthrough in AFM/TEM image interpretation. Finally, new complementary aspects of the object's ultrastructure can be revealed.