The limits of conventional light microscopy (“Abbe-Limit“) depend critically on the numerical aperture (NA) of the objective lens. Imaging at large working distances or a large field-of-view typically requires low NA objectives, thereby reducing the optical resolution to the multi micrometer range. Based on numerical simulations of the intensity field distribution, we present an illumination concept for a super-resolution microscope which allows a three dimensional (3D) optical resolution around 150 nm for working distances up to the centimeter regime. In principle, the system allows great flexibility, because the illumination concept can be used to approximate the point-spread-function of conventional microscope optics, with the additional benefit of a customizable pupil function. Compared with the Abbe-limit using an objective lens with such a large working distance, a volume resolution enhancement potential in the order of 10 4 is estimated. Due to novel developments in optical technology and photophysics it has become possible to radically overcome the classical diffraction limit for high NA objective lenses (ca.
Objective Working Distance 30.8mm 20X Infinity Super-Long Working Distance Plan Apochromatic NIR Objective IR18091141: Amazon.ca: Electronics. Design and Technology. Current off-the-shelf 100X microscope objective’s working distance is only 12 mm. The objective has a Parfocal distance of 95mm which is different from the usual 45 mm on the current market. Its extra long working distance enable customers to achieve high level diffraction limit.
200 nm laterally, 600 nm along the optical axis; also called the Abbe-limit) of conventional far-field microscopy. These discoveries which promise to revolutionize Biology and Medicine have been honored by the 2014 Nobel Prize in Chemistry to Eric Betzig and William Moerner, for developing single fluorophore detection as the basis for single molecule localization microscopy using photoactivated proteins; and to Stefan Hell for the development of Stimulated Emission Depletion (STED) Microscopy, a “focused nanoscopy” method. Using these approaches, both optical resolution (smallest distance detectable between two adjacent point sources) and structural resolution (smallest structural detail determined based on the density of point sources resolved) has been enhanced very substantially.
At the present state of the art, they allow a light-optical resolution of biostructures down to about 5 nm, corresponding to 1/100th of the excitation wavelength λ exc.However, due to the high NA objective lenses used in these studies, the thickness of an object which can be analyzed in 3D with such a high resolution in many approaches is presently restricted to a maximum of several tens of µm. This means that in most cases, only individual cells arranged in monolayers on glass substrates, or thin tissue sections can be studied at highest resolution.For many biological and medical applications, this limitation of present super-resolution methods (SRM), to a relatively small field of view, typically in the order of 100 µm diameter, and to thin objects poses a severe road block to developmental biology as well as to biomedical research: This limitation has hampered the full use of SRM methods to study e.g. The distribution of viruses, proteins or DNA/RNA sequences in three dimensional cellular arrangements, or to study microscopically disease correlated epigenetic changes on the single cell level in the organismic context. In many applications a field of view many times larger than 100 µm and a specimen thickness in the millimeter to centimeter range should be highly desirable.One solution for large field-of-view deep tissue imaging has been to design specialized objective lenses which implement a set of correction methods to compensate for aberrations. At a numerical aperture of NA = 0.47 and a working distance of 3 mm, it provides a field-of-view of ca. 6 mm across, thus allowing rapid data acquisition of large sample volumes.
However, the lateral resolution is presently limited to ca. 1.3 λ (excitation wavelength in vacuum), and correspondingly the axial depth-of-focus is much larger than what can be obtained using high-NA objective lenses. In contrast to this existing system, illuminating the sample with light originating from an even larger solid angle (or a higher NA of the illumination scheme) would allow further reduction of the illumination spot. Another solution to study large fields of view of thin objects with high NA objective lenses has been to perform multiple acquisitions at different locations,. For example, one might scan the object by multiple beams, e.g.
10,000 scanning beams, each scanning a field of view of 100 µm in diameter; in this case imaging could be parallelized, corresponding to a total field of view of 1 cm 2. Such multiple beam scanning devices may be realized by using diffractive elements. (1)where λ exc is the fluorescence excitation wavelength, NA = n sin( α) is the numerical aperture (refractive index n and half-angle α of the light acceptance cone), I STED is the intensity of the doughnut focused STED beam, and I sat is the saturation intensity of the fluorophore used for STED imaging. This formula predicts that it should be possible to achieve any STED resolution also at low NA (i.e. At large working distances) by an appropriate increase of the STED beam intensity; according to this relation, assuming the same wavelength and STED resolution, the required STED beam intensity scales inversely with NA 2; this means that with an objective lens of numerical aperture NA = 0.2, an about 50 times higher STED beam intensity (1.4/0.2) 2 would be required to achieve the same lateral resolution as with NA = 1.4; to what extent this will be practically possible and compatible with specimen conservation or live cell imaging is not known.
Bleaching and phototoxicity already now appear to produce disadvantageous effects in many STED applications; to overcome them at very large working distances probably would require the use of novel dyes with appropriately lowered saturation intensities I sat. Recent STED developments on making the depletion beam quasi-degenerate with the excitation beam in principle facilitate the operation at much lower disexcitation powers by using a depletion wavelength closer to the peak in the emission spectra; nonetheless, for many dyes this advanced procedure is hampered by the increased cross-excitation due to the STED beam, resulting in a higher switching fatigue of the dye. Additionally, the localization precision in Single Molecule Localization Microscopy could be enhanced by using STED illumination.Alternatively, it remains highly desirable to consider the development of super-resolution techniques for very large working distances with substantially lower illumination intensities. Such techniques have been described for fluorescence microscopy approaches based on structured illumination with two excitation beams passing an objective lens,; at a given numerical aperture, they provide an optical resolution enhanced by a factor two; in the example given above for NA = 0.2, this would result in a theoretical optical resolution of about 0.75 µm laterally and 12.5 µm axially; for NA = 0.1, the achievable lateral optical resolution would be d SIM (NA = 0.1) = 0.61 λ/NA/2 = 1.5 µm.
Proof-of-principle experiments using Retina cells with a structured illumination microscope featuring a working distance of about 4.5 cm indicated an optical resolution around 1.6 µm (as obtained from the spatial cut-off frequency), in accordance with the theoretical estimate.The above mentioned theoretical and practical restrictions of optical resolution at large working distances are due to the low numerical aperture of the objective lenses used; however, these limits may be circumvented (i.e. The resolution can be enhanced many times more) by a scanning approach using a structured illumination concept with multiple beams focused constructively, thereby approximating the far-field of a spherical wave. The best approximation of the far field of a spherical wave is achieved in a “4π” geometry, which means that the light sources producing the individual beams are distributed over an area covering a full solid stereo angle of 4π as closely as possible. The basic idea to achieve SRM at large working distances by constructive focusing of multiple beams in such a 4π-geometry has been put forward already in the 1970s; but so far numerical calculations of its feasibility have been lacking. In this report, we provide such numerical simulations; the results indicate that using an appropriate array of multiple collimated laser beams, an illumination focus with a Full-Width-at-half-Maximum (FWHM) around 140 nm in all directions can be produced (λ = 488 nm; n = 1.518) in a homogeneous, transparent medium.
Since each of the coherent light beams is collimated, the distance of the sources is in principle arbitrary, i.e. This distance can be varied within large limits (e.g. Up to several cm); this, however, is equivalent to the possibility to realize a joint ‘focal spot’ (similar to the illumination point-spread-function PSF ill in conventional lens based illumination) for scanning based imaging. As discussed below, the joint ‘focal spot’ thus obtained can be made substantially smaller than possible with low NA objective lenses appropriate to realize the same large working distance; hence an enhanced resolution compared to the Rayleigh formula (using the same low NA) may be obtained.Producing such a small focal diameter at a very large working distance is necessary in order to generate a strong signal response from within the object. But this is only the first requirement for enhanced resolution imaging: A second requirement is the detection of the generated signal, e.g. Fluorescence or scattered light. Since at else equal conditions the fluorescence signal I det detected is directly proportional to the area covered by the front lens of the detector system used, I det scales inversely with the square of the working distance L.
To give an example, if the photon flux entering the front lens of an NA = 1.4 objective at a working distance L 1.4 = 0.2 mm is assumed to be I flux1.4, then for an equally sized front lens in the distance of L = 10 mm (assumed NA = 0.2) and the same refraction index, the photon flux I flux0.2 would scale inversely with L 2 and hence be smaller by the factor 10/0.2 2 = 2500; and correspondingly we obtain for the localization accuracy σ loc achievable in localization microscopy σ loc 1/N det 0.5, where N det = number of detected photons I flux. As a consequence, the localization accuracy σ loc would be 50 times worse, e.g. 1 µm nm instead of 20 nm, and the optical (two point) resolution would hence be around 2.35 µm (FWHM = 2.35 σ loc) instead of around 50 nm. We shall discuss how to avoid such a deterioration of the fluorescence signal without having to sacrifice the advantages obtained by the small laser focus.To produce a very small focal diameter for point-by-point-scanning of the object at a large working distance and to efficiently detect the generated signal (e.g. Fluorescence or scattering) would already allow some highly interesting biophysical studies, e.g. To measure by Fluorescence Correlation Spectroscopy (FCS), the mobility and concentration of fluorophores in a very small cellular volume inside a large cellular aggregate, or a small model organism or entire organs (made suitably transparent).
For example, using FCS at a large working distance with a numerical aperture of NA = 0.2 would monitor the fluorescence variation in an observation volume of V obs = 30 µm 3; in the 4π distributed aperture microscope (“4π-DAM”) described below, it should be possible to achieve at equivalent large working distances as for NA = 0.2 an estimated observation volume (for assumptions see above) around V obs,4π = 4/3 × π × 0.07 × 0.07 × 0.07 µm 3 ≈ 0.0014 µm 3 i.e. Many thousand times smaller. Another interesting application would be the possibility to introduce very small lesions inside a large cellular object, e.g. A chromatin damage inside a nucleus of a large cellular cluster; or to perform a corresponding optical stimulation e.g. Of a neuron inside a thick specimen; or to facilitate the introduction of high resolution optical inspection into production lines.To make possible imaging in the DAM, the object has to be scanned point-by-point with the ‘focal spot’ created. To realize this, either the beam has to be moved, or the specimen has to be moved.
For simplicity, in this report we shall discuss only a stage scanning solution: Both the condition to move the stage and to optimize the fluorescence detection requires to use a beam array with some spacing between the beams; we shall present numerical calculations indicating that this requirement has only a slight effect on the achievable resolution.As stated above, for the sake of simplicity of presentation, in the following conceptual study we typically assumed a vacuum excitation wavelength of λ = 488 nm and a refraction index n =.
The AmScope IN480TC-FL-BWF digital inverted epifluorescence trinocular microscope has interchangeable pairs of 10x22mm and WH25x10mm plan high-eyepoint super-widefield eyepieces, an under-mounted quintuple nosepiece, eight DIN long working-distance infinity plan objectives, fluorescence 100W mercury lamp and Brightfield 30W halogen illumination, a 0.3 NA Kohler condenser with iris diaphragm, and a double-layer mechanical stage with a stage stop to protect slides and objectives from damage. The 1.4MP Peltier-cooled fluorescence camera has a CMOS color sensor, image capture and editing software, and USB 2.0 output to capture or display still or video images on a computer or projector. The microscope has a color-corrected infinity optical system (CCIS) that provides color correction and improved focus of the intermediate image across the entire focal plane. The trinocular head has a Siedentopf binocular mount with 50 to 76mm interpupillary adjustment and a fixed 45-degree vertical inclination to reduce eye and neck strain. A Siedentopf binocular head enables the viewer to change the interpupillary distance without changing the tube length, eliminating the need to refocus the image. Anti-mold coatings protect the microscope in high-humidity environments. The microscope includes interchangeable pairs of high-eyepoint super-widefield WH10x22mm plan and WH25x10mm eyepieces.
Plan eyepieces provide improved focus over the entire field of view (FOV). High-eyepoint eyepieces ease viewing for viewers who wear glasses.
Dioptric adjustment accommodates individual eye-strength differences. The vertical trinocular port accepts a 23mm or C-Mount camera. The under-mounted quintuple nosepiece accepts up to five 20mm objectives at a time.
The microscope includes five Brightfield long working-distance infinity plan objectives (4x, 10x, 20x, 40x, and 60x) and three phase-contrast long working-distance infinity plan objectives (10x, 20x, and 40x). Long working-distance plan objectives provide improved focus over the entire range of the viewing field and a longer working distance that is required when viewing specimens in dishes and large containers. Phase-contrast provides high contrast and visibility without the use of stains, allowing specimens to be observed in their natural state without being killed or fixed. An inverted microscope has its light source above the stage pointing downward and the objectives and turret below the stage pointing upward. It is most often used for observing organisms in a large container, such as a well plate or petri dish.
It can also be used when specimens require manipulation, or in metallurgical applications. A digital fluorescence microscope is used in biological and metallurgical research and in industrial inspection applications to observe details illuminated by ultraviolet light, and where image capture, detailed records, or documentation is required.The included AmScope MT5800-CCD 1.4MP Peltier-cooled fluorescence camera has a 2/3' Sony black and white CCD sensor for displaying still microscopy images and streaming live videos to a computer monitor or digital TV. The camera is suitable for low-light applications, including fluorescence and darkfield microscopy. The camera can be mounted in any standard C-Mount. The Peltier-cooled sensor combined with black and white background provides low-noise fluorescence image acquisition. The sensor has a progressive scan mode, an RGB Bayer pattern filter, and a 1300mV G-sensitivity.
The camera includes image capture and editing software that provides still image and live video capture and editing capability, including measurement functions. User-defined parameters include brightness, gain, RGB, and automatic or manual white balance and exposure selection. Exposure duration has a range of 0.1 to 60 minutes. The software supports JPG, TIFF, GIF, PSD, WMF, and BMP file formats and is compatible with Windows XP, Vista, 7, and 8. The camera supports Twain, DirectShow, Amcap, and Minisee file formats. The camera has a USB 2.0 data port (cable included). The camera includes an aluminum case to protect the camera during storage or transport.The microscope has Brightfield, phase-contrast, and fluorescence illumination.
Brightfield and phase-contrast illumination is mounted above the stage and has transmitted 30W halogen illumination. Brightfield (BF) illumination allows the specimen to absorb light, resulting in a dark image on a light background. The phase-contrast element aligns the light source with the phase-contrast objectives to optimize high contrast and visibility without the use of stains.
Halogen illumination provides bright light in a concentrated path, and a rheostat controls the amount of light emanating from the lamp. The objectives are mounted on a turret to ease magnification changes. Often used in biological and metallurgical research, fluorescence microscopy uses a xenon or mercury lamp to create ultraviolet light and excitation filters to filter unwanted wavelengths, allowing users to see details undetectable to other light sources. The Kohler condenser has a 0.3 NA to provide a long working-distance of 72mm and 28mm, respectively. Kohler illumination focuses and centers the light path using two iris diaphragms, providing optimum contrast and resolution. The horizon can be rotated 0 to 300 degrees. The double-layer mechanical stage with 1mm stage divisions and 0.1mm vernier resolution locks the slide into place and provides precise slide manipulation along the X- and Y-axes to allow coordinates to be recorded, enabling the viewer to return to a specific location on the slide.
The oversize stage is 6.3 x 10 inches (160 x 250mm) (W x D, where W is width, the horizontal distance from left to right; D is depth, the horizontal distance from front to back). It has a traveling range of 4-3/4 x 3-1/8 inches (X- direction x Y-direction). A petri dish holder secures specimens in place. Nested coaxial coarse and fine focus has tension-adjustable coarse focusing and an adjustable lock ring stopper that limits stage range to protect slides and objectives from damage. The focus system has a 38mm working distance, 2mm fine focus, and 0.002mm precision. The total working distance is 6.5mm upward and 2.5mm downward from focus.
All-metal mechanical parts, solid-metal frame construction, and a stain-resistant enamel finish provide durability.
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