Objectives: Super Apochromatic Microscope Objectives Microscopy Objectives, Dry Microscopy Objectives, Oil Immersion Physiology Objectives, Water Dipping or Immersion Long Working Distance Objectives Reflective Microscopy Objectives UV Focusing Objectives 532 nm and 1064 nm Objectives: Scan Lenses and Tube Lenses: Scan Lenses F-Theta Scan Lenses.
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. 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 =.
NIR Objective Objective Optical SystemInfiniteObjective Optical Magnification20XObjective TypePlan Apochromatic ObjectiveObjective Parfocal Distance95mmObjective Focal Length10mmObjective Working Distance30.8mmNumerical Aperture (N.A.)N.A. 0.29Objective Resolution1μmObjective Wavelength400-1200nmObjective Cover Glass Thickness/0Objective Immersion MediaDry ObjectiveObjective Screw ThreadM26x1/36 in.Objective Outer DiameterDia. 36.5mmNIR Transmittance770-790nm 90% or moreDiameter of Image Focal PlaneDia. 24mmSurface TreatmentPolished ChromeMaterialMetalColorSilverNet Weight0.37kg (0.82lbs). Objective Close Λ The objective (lens) is the first set of optical systems that image the object being observed, and is also the most important imaging component in the microscope. Depending on the application, objective is usually classified into the following categories: Biological Objective Metallurgical Objective Phase Contrast Objective Polarizing Objective Dark/Bright Field Objective Stereo Objective Monocular Video Microscope Objective Infinity-Corrected Long Working Distance Objective NIR Objective NUV Objective UV Objective Telecentric Objective Lens Some objectives are mounted directly on the microscope body, some separate from the body and are installed when needed.
Different types of microscope objectives are generally not interchangeable. However, when ofthe same type and parameter design the same or similar, the objectives of different models and manufacturers are interchangeable, provided that attention shouldbe paid to the change in magnification, working distance, field of view and image quality. Usually, on the objective outer casing, there are signs of the following parameters: Objective Magnification: for example 10X, 40X Numerical Aperture (N.A.): for example, /1.30 Objective Immersion Media: Oil represents oil, W represents water, and Glyc represents glycerin Mechanical Tube Length and Objective Cover Glass Thickness: the two parameters are usually written together and separated by /.
The finite mechanical is usually 160, 195, etc., and infinite is represented by '∞'; objective cover glass thickness (thickness / mm) is expressed after/, for example /0.17; for specimen that does not use objective cover glass, it is represented by 0, for example, '/0'; for those that do not use objective cover glass or the objective cover glass thickness is smaller than 0.17, it is represented by '/-'. Phase Contrast Objective: represented by PH, for example, PH2, the digit after PH represents the associated ring diaphragm. Polarizing Objective: represented by POL. Plan Objective: represented by PLAN or PL Achromatic: generally, achromatic objective does not require identification Apochromatic: represented by APO Long Working Distance: represented by L There are also objectives that are unique in magnification and medium, and their difference is indicated by color circle. For objectives that do not have mark, it is necessary to refer to the microscope body for judgment, or refer to the product manual. Usually, the objective has very fine mounting threads.
When there is a need to install the objective /objective frame, be careful to install it. Align the nosepiece installation position, keep it completely “flat”. When it is blocked, remove it and reinstall it. Do not force it in.
Note: although between different manufacturers, some objectives can be used universally, they may still bring magnification error and image quality degradation. Infinite Close Λ Microscopes and components have two types of optical path design structures.
One type is finite optical structural design, in which light passing through the objective lens is directed at the intermediate image plane (located in the front focal plane of the eyepiece) and converges at that point. The finite structure is an integrated design, with a compact structure, and it is a kind of economical microscope. Another type is infinite optical structural design, in which the light between the tube lens after passing the objective lens becomes 'parallel light'.
Within this distance, various kinds of optical components necessary such as beam splitters or optical filters call be added, and at the same time, this kind of design has better imaging results. As the design is modular, it is also called modular microscope. The modular structure facilitates the addition of different imaging and lighting accessories in the middle of the system as required. The main components of infinite and finite, especially objective lens, are usually not interchangeable for use, and even if they can be imaged, the image quality will also have some defects. The separative two-objective lens structure of the dual-light path of stereo microscope (SZ/FS microscope) is also known as Greenough. Parallel optical microscope uses a parallel structure (PZ microscope), which is different from the separative two-object lens structure, and because its objective lens is one and the same, it is therefore also known as the CMO common main objective.
Objective Optical Magnification Close Λ The finite objective is the lateral magnification of the primary image formed by the objective at a prescribed distance. Infinite objective is the lateral magnification of the real image produced by the combination of the objective and the tube lens. Infinite objective magnification = tube lens focal length (mm) / objective focal length (mm) Lateral magnification of the image, that is, the ratio of the size of the image to the size of the object. The larger the magnification of the objective, the higher the resolution, the smaller the corresponding field of view, and the shorter the working distance. Objective Type Close Λ In the case of polychromatic light imaging, the aberration caused by the light of different wavelengths becomes chromatic aberration. Achromatic aberration is to correct the axial chromatic aberration to the two line spectra (C line, F line); apochromatic aberration is to correct the three line spectra (C line, D line, F line).
The objective is designed according to the achromaticity and the flatness of the field of view. It can be divided into the following categories.
Achromatic objective: achromatic objective has corrected the chromatic aberration, spherical aberration, and comatic aberration. The chromatic portion of the achromatic objective has corrected only red and green, so when using achromatic objective, yellow-green filters are often used to reduce aberrations. The aberration of the achromatic objective in the center of the field of view is basically corrected, and as its structure is simple, the cost is low, it is commonly used in a microscope.
Semi-plan achromatic objective: in addition to meeting the requirements of achromatic objective, the curvature of field and astigmatism of the objective should also be properly corrected. Plan achromatic objective: in addition to meeting the requirements of achromatic objectives, the curvature of field and astigmatism of the objective should also be well corrected. The plan objective provides a very good correction of the image plane curvature in the field of view of the objective, making the entire field of view smooth and easy to observe, especially in measurement it has achieved a more accurate effect. Plan semi-apochromatic objective: in addition to meeting the requirements of plan achromatic objective, it is necessary to well correct the secondary spectrum of the objective (the axial chromatic aberration of the C line and the F line).
Plan apochromatic objective: in addition to meeting the requirements of plan achromatic objective, it is necessary to very well correct the tertiary spectrum of the objective (the axial chromatic aberration of the C line, the D line and the F line) and spherochromatic aberration. The apochromatic aberration has corrected the chromatic aberration in the range of red, green and purple (basically the entire visible light), and there is basically no limitation on the imaging effect of the light source. Generally, the apochromatic aberration is used in a high magnification objective.
Objective Parfocal Distance Close Λ Objective parfocal distance refers to the imaging distance between the objective shoulder and the uncovered object surface (referred to as the “object distance). It conforms to the microscope design, usually 45mm.
The objective of different magnifications of the compound microscope has different lengths; when the distance between the objective shoulder and the object distance is the same, the focal length may not be adjusted when converting to objectives of different magnifications. Objective Working Distance Close Λ The objective working distance is the vertical distance from the foremost surface end of the objective of the microscope to the object surface to be observed. Generally, the greater the magnification, the higher the resolution of the objective, and the smaller the working distance, the smaller the field of view. Conversely, the smaller the magnification, the lower the resolution of the objective, and the greater the working distance, and greater the field of view. High-magnification objectives (such as 80X and 100X objectives) have a very short working distance. Be very careful when focusing for observation.
Generally, it is after the objective is in position, the axial limit protection is locked, then the objective is moved away from the direction of the observed object. The relatively greater working distance leaves a relatively large space between the objective and the object to be observed. It is suitable for under microscope operation, and it is also easier to use more illumination methods. The defect is that it may reduce the numerical aperture of the objective, thereby reducing the resolution. Numerical Aperture (N.A.) Close Λ Numerical aperture, N.A. For short, is the product of the sinusoidal function value of the opening or solid angle of the beam reflected or refracted from the object into the mouth of the objective and the refractive index of the medium between the front lens of the objective and the object.
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Simply speaking, it is the magnitude of the luminous flux that can be brought in to the mouth of the objective adapter, the closer the objective to the specimen for observation, the greater the solid angle of the beam entering the mouth of the objective adapter, the greater the N.A. Value, and the higher the resolution of the objective. When the mouth of the objective adapter is unchanged and the working distance between the objective and the specimen is constant, the refractive index of the medium will be of certain meaning.
For example, the refractive index of air is 1, water is 1.33, and cedar oil is 1.515, therefore, when using an aqueous medium or cedar oil, a greater N.A. Value can be obtained, thereby improving the resolution of the objective. Formula is: N.A. = refractive index of the medium X sin solid angle of the beam of the object entering the front lens frame of the objective/ 2 Numerical aperture of the objective.
Usually, there is a calculation method for the magnification of the microscope. That is, the magnification of the microscope cannot exceed 1000X of the objective. For example, the numerical aperture of a 100X objective is 1.25, when using a 10X eyepiece, the total magnification is 1000X, far below 1.25 X 1000 = 1250X, then the image seen in the eyepiece is relatively clear; if a 20X eyepiece is used, the total magnification will reach 2000X, much higher than 1250X, then eventhoughthe image actually seen by the 20X eyepiece is relatively large, the effect will be relatively poor. Objective Resolution Close Λ Objective resolution is the distance that can be distinguished between the two mass points on the object plane, or the number of pairs that can be distinguished within 1mm of the image place. Usually, its unit is expressed as the number of pairs/mm.
In general, the greater the magnification, the higher the resolution. Under the same objective magnification, the greater the numerical aperture (N.A.) of the objective, the higher the resolution of the objective. Numerical aperture (N.A.) is the most important technical index reflecting the resolution of the objective. The objective is located at the forefront of the object being observed. When the objective magnifies and forms an image, the rear eyepieces and other equipment are to magnify again. When the eyepiece magnifies enough, one may only get a large enough but blurred image.
Therefore, if the front-end objective cannot distinguish, neither can the rear device or equipment distinguish againmore information. The objective is the most important part of a microscope. Objective Immersion Media Close Λ The use of different media between the objective and the object to be observed is to change and improve the resolution. For example, the refractive index of air is 1, water is 1.33, and cedar oil is 1.515. Therefore, when using an aqueous medium or cedar oil, a greater N.A.
Value can be obtained, thereby increasing the resolution of the objective. Air medium is called dry objective, where oil is used as medium iscalled oil immersion objective, and water medium is called water immersion objective. However, because of the working distance of the objective, when the working distance of the objective is too long, the use of liquid medium will be relatively more difficult, and it is generally used only on high magnification objective having a shorter working distance, such as objectives of 60X, 80X and 100X. When using oil immersion objective, first add a drop of cedar oil (objective oil) on the cover glass, then adjust the focus (fine adjustment) knob, and carefully observe it from under the side of the objective of the microscope, until the oil immersion objective is immersed in the cedar oil and close to the cover glass of the specimen, then use the eyepiece to observe, and use the fine focus knob to lift the tube until the clear imageof the specimen is clearly seen.
The cedar oil should be added in an appropriate amount. After the oil immersion objective is used, it is necessary to use a piece of lens wiping tissue to dip xylene to wipe off the cedar oil, and then wipe dry the lens thoroughly with a lens wiping tissue. Packaging Close Λ After unpacking, carefully inspect the various random accessories and parts in the package to avoid omissions. In order to save space and ensure safety of components, some components will be placed outside the inner packaging box, so be careful of their inspection. For special packaging, it is generally after opening the box, all packaging boxes, protective foam, plastic bags should be kept for a period of time. If there is a problem during the return period, you can return or exchange the original. After the return period (usually 10-30 days, according to the manufacturer’s Instruction of Terms of Service), these packaging boxes may be disposed of if there is no problem.
We will match any price online Information.My Account.About UsBoli Optics sells professional, high quality microscopes, microscope accessories, and magnifying lamps. We supply research laboratories, medical centers, universities, industrial manufactures, factories, students, and hobbyists.
Our parts and accessories are compatible with Leica, Olympus, Nikon, and Zeiss. Contact Info. 2131 S Hellman Ave Unit POntario, CALIFORNIA 91761USA. (909) 930-3933. [email protected] With Us.
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