Fourier analysis allows for treating optical processes in terms of spatial frequencies [11]. Therefore this type of analysis can be used for modeling the aperture and objective lens in a confocal scanning system. A pictorial representation of Fourier analysis in a lens system is shown in Figure 3.
Figure 3: Illustration of Fourier analysis in a lens system. Since optical systems generally involve two-dimensional components and light, the Fourier transform pair can be generalized in two dimensions as Equation 1 and its FT as Equation 2 , where the quantities kx and ky are the angular spatial frequencies along the two corresponding axes.
The object scatters waves, which are collected by the lens, and the parallel bundles of rays are brought to convergence at the back focal plane [12]. The pupil plane mask is known as the aperture function: 1 and the object plane distribution is the Fourier transform of the pupil field distribution: Proc. In addition, the model evaluated two pupil configurations: 1 full-pupil configuration [Figure 4] and 2 divided-pupil configuration [Figure 5]. Figure 4: Full-Pupil Configuration Figure 5: Divided-Pupil Configuration The full-pupil configuration allows for illumination and detection with the entire pupil of the objective lens.
This configuration leads to good resolution; however, there is greater acceptance of multiply scattered light []. The divided-pupil configuration illuminates half of the objective lens and collects from the other half. The Gaussian approximation is good for small values of h as Figure 7 shows good correlation of on-axis image irradiance for the Gaussian source …. For large values of h, the uniform source correlates well to the point-scan image irradiance, however, there is a great loss of power in the image plane.
Figure 7: Graph shows the on-axis image irradiance for computed line-scan, Gaussian, and uniform sources. The pupil is slightly overfilled to produce the highest on-axis irradiance in the image plane. The plot of the on-axis image irradiance signal for the divided-pupil point-scan is shown in Figure We presented optimum parameters for four confocal microscopy configurations: 1 full-pupil point-scanning, 2 full- pupil line-scanning, 3 divided-pupil point-scanning, and 4 divided-pupil line-scanning.
We have shown the Proc. In future work, additional parameters we will consider are the optimal location of the point-source beam in the divided-pupil configuration, the optimal line width for the line-source, and the width of the aperture in the divided- pupil configuration. Special thanks to Dr. Elder, ed. Chapter 9, Hilger, London, Brakenhoff, and J. Micromanipulation by "multiple" optical traps created by a single fast scanning trap integrated with the bilateral confocal scanning laser microscope.
Born, E. Principles of optics. Cambridge University Press, Cambridge. Addison-Wesley, New York. Related Papers. There are a variety of compounds for mounting fixed tissues that have different refractive indices, chemicals to increase the lifespan, slow photobleaching of the sample, etc.
For details on sample preparation methods, see Smith, or Galdeen, While the majority of confocal microscopes are based upon the Minsky principle, there have been several advances to improve their functionality. As discussed above, the spinning disk and swept field confocal microscopes, and resonant scanning mirrors increase the speed of acquiring confocal data.
However, because speed is a historically limiting factor for confocal microscopy, increasing the rate of data collection continues to be a focus for technical improvements.
One recent configuration is the ribbon scanning confocal microscope Watson, et al, This functions by employing resonant scanners and a high-precision x, y stage to continuously acquire strips across the sample that are stitched together. The primary advantage of this system is the speed with which high-resolution multi-stack images can be acquired. For large, fixed samples, this technology reduces to the time to data collection for 3D stacks. As with every microscopy technique in the last decade, there is a push to increase the resolution to enable imaging of ever smaller features.
In the realm of confocal microscopy, the Airyscan technology provides 1. The Airyscan has a channel detector array with a hexagonal array of micro lenses that act as a system of very small pinholes. In this system, the primary improvement is in the signal to noise ratio SNR via pixel reassignment and summation of the collected images from all of the detectors.
The re-scan confocal microscope RCM is a recently commercialized confocal technology that improves lateral resolution by 1. The RCM includes a re-scanning unit consisting of a pair of re-scanning mirrors between the pinhole and detector that allows for de-coupling of the magnification of the object and scanning spot De Luca, et al, In this system, the re-scanning mirrors can be set to double the angular amplitude before directing the light to a CCD or sCMOS detector, which increases the scanning size and the apparent distance between spots.
Here, the lateral resolution is independent of the pinhole, but the axial resolution is the same as in standard LSCM. Another area of technical advance is the method of illumination.
In addition to improvements in laser technology for visible wavelengths and the commercial implementation of multiphoton excitation, there are also white-light, or supercontinuum lasers. Selecting the individual wavelengths or wavelength ranges is accomplished with the AOTF technology mentioned above, or with an acousto-optical beam splitter AOBS. The AOBS selects small bandlets of the spectrum by applying specific frequencies and amplitudes of acoustic waves to an appropriate crystal, which causes select colors to exit the crystal at different angles.
For wavelength selection, an alternative to the AOBS is a variable bandpass filter that has spectral properties varying along the length of the filter. The highest quantum efficiency is currently provided by GaAsP detectors gallium arsenide phosphide , a high-sensitivity PMT. The increased sensitivity permits the study of dim signals, but also provides a speed boost for brighter signals because the amount of time spent collecting the signal in each spot can be shortened.
Another recent development is the hybrid detector, which is a cross between a standard PMT and an avalanche photodiode a highly sensitive semiconductor device. They are characterized by high dynamic range, low noise, and high speeds. The increased sensitivity of the GaAsP detector is particularly important here as fewer photons are being collected after splitting. The sum of technical advances in microscopy is driving a rapid rise in the amount of data being collected.
Storing and analyzing these data is a significant challenge. However, there has been a concomitant rise in the software tools available for processing and analyzing large datasets. Commercial microscopes provide suites of tools for image registration, segmentation of features within images, algorithms for particle tracking, and many others.
Future advances are likely to include further improvements to the computational side of confocal fluorescence microscopy and the introduction of more automated systems. Artificial intelligence AI and machine-learning algorithms are currently making their way into commercial software packages and many types of machine learning algorithms are available in the open source programs listed above. Confocal microscopy provides the ability to collect clear images from a thin section of a thick sample with low background and minimal out-of-focus interference.
Optical sectioning is a common application in the biomedical sciences and has been useful for materials science as well. In practice, a sample is put on the microscope stage and an image is collected at the top focal plane and then the stage or objective is moved up or down to the next focal plane and so on.
Additionally, 3D volumes can be collected over time for 4D datasets and with multiple channels for 5D datasets. It is increasingly common to use confocal microscopy for live imaging as well as with fixed samples.
One example of a live-imaging experiment with a LSCM is shown in Figure 6 , tracking muscle calcium activity during larval locomotion in Drosophila melanogaster. Time-series of forward larval locomotion in Drosophila melanogaster. Larvae expressing calcium biosensor GCaMP6s Chen, in the muscles were monitored during forward locomotion in a confocal microscope at 4x magnification with the pinhole open to collect more light.
The type of confocal microscope best suited to a given application depends largely on the prioritization of imaging speed, resolution, and field-of-view — while keeping in mind photodamage to the sample. Table 1 lists the techniques outlined above with a summary of their principle, advantages and disadvantages, and experiments for that technique. Confocal microscopy can be an exceptionally quantitative technique. However, because these instruments are widely available and relatively easy to use, they are often not optimally utilized for quantitative data collection.
It is crucial in a confocal microscopy experiment to choose the correct technique, objective, fluorophores, mounting medium, and optical components to achieve the best images.
Confocal microscopy is widely used for fluorescence imaging in the life sciences. The last decade has seen advances in illumination sources, detectors, fluorescent probes, optics, and sample preparation techniques, which provide improvements in different combinations of speed, depth, and resolution. This paper lays out the basic principles, advantages, technical considerations, and applications for confocal microscopes to guide non-experts in determining the most appropriate confocal method for the desired experiments.
The mention of any company, product, or service in this work is in no way intended as an endorsement by the National institutes of Health or the author. National Center for Biotechnology Information , U. Curr Protoc Cytom. Author manuscript; available in PMC Mar 1. Amicia D. Elliott 1. Author information Copyright and License information Disclaimer. Corresponding Author: Amicia D.
Elliott, moc. Copyright notice. The publisher's final edited version of this article is available at Curr Protoc Cytom.
See other articles in PMC that cite the published article. Abstract In light microscopy, illuminating light is passed through the sample as uniformly as possible over the field of view. Keywords: confocal microscopy, fluorescence, laser scanning, resonant scanning, spinning disk. Overview The primary functions of a confocal microscope are to produce a point source of light and reject out-of-focus light, which provides the ability to image deep into tissues with high resolution, and optical sectioning for 3D reconstructions of imaged samples.
Open in a separate window. Figure 1. Figure 2. Types of Confocal Microscopes Confocal microscopes can be distinguished by their method of scanning. Laser Scanning Confocal Microscopes As the name implies, in a laser scanning confocal microscope LSCM , a laser beam is swept over the sample by means of scanning galvanometer mirrors.
Figure 3. Hybrid Scanning Confocal Microscopes An intermediate approach between single and multi-point scanning confocal microscopes is the slit-scanning confocal, which replaces the round pinhole with a rectangular slit to reject out-of-focus light. Practical Considerations There are a number of points to consider when designing experiments for confocal microscopy, several of which are addressed below.
Objectives Modern objective lenses have components to correct for flatness of field and chromatic aberrations, which are important for confocal microscopy, as the laser beam must pass through parts of the objective lens relatively far from the optical axis during scanning.
Figure 4. Depth Penetration Deep tissue imaging is in increasing demand and strategies to retain image quality further from the coverslip continue to emerge. Sample Size Directly related to the depth in a confocal microscope is the overall size of the sample. Figure 5. Imaging Speed A critical consideration when planning confocal microscopy experiments is the desired acquisition speed. Sample Preparation The mounting method for fixed tissues and the media for live samples can affect the 3D shape of the sample and the resolution that can be obtained by confocal microscopy.
Recent Technical Advances in Confocal Microscopy While the majority of confocal microscopes are based upon the Minsky principle, there have been several advances to improve their functionality.
Applications for Confocal Microscopy Confocal microscopy provides the ability to collect clear images from a thin section of a thick sample with low background and minimal out-of-focus interference. Figure 6. Table 1. Comparison of confocal techniques discussed in this unit.
Recent developments of genetically encoded optical sensors for cell biology. Molecular Biology of the Cell. Microscopy Research and Technique has published three special issues covering the topic of two-photon microscopy.
Please note that your institution will need to have a subscription to access these journals on-line. Nonlinear magic: multiphoton microscopy in the biosciences. Warren R. Zipfel, Rebecca M. Williams and Watt W. Nature Biotechnology Please note that your institution will need to have a subscription to access this journal on-line.
Deep tissue two-photon microscopy. Helmchen F. Nature Methods David W. PLoS Biol 3 6 : e
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