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dark field fluorescence microscopy

When to Use a Dark Field Microscope?

dark field fluorescence microscopy are used in a number of different ways to view a variety of specimens that are hard to see in a light field unit. Live bacteria, for example, are best viewed with this type of microscope, as these organisms are very transparent when unstained.

There are multitudes of other ways to use dark field illumination, often when the specimen is clear or translucent. Some examples:

Dark field illumination of caffeine crystalsLiving or lightly stained transparent specimens

Single-celled organisms

Live blood samples

Aquatic environment samples (from seawater to pond water)

Living bacteria

Hay or soil samples

Pollen samples

Certain molecules such as caffeine crystals (right)

dark field fluorescence microscopy makes many invisible specimens appear visible. Most of the time the specimens invisible to bright field illumination are living, so you can see how important it is to bring them into view!

dark field fluorescence microscopy

What is dark field fluorescence microscopy?

Similar to a bright field but it is modified by a dark field stop just below the source. The dark field stop is just  the condenser, and blocks the light in the center of the lightsource so that the only light that goes through is around the edges. That light is then bent by the condenser and diffacts off the specimen. None of the light goes directly from the light source into the objective, so if there is no specimen, the image will be very dark. The specimen in this method will be illuminated against a black background.
A dark field microscope is useful because it increases the contrast of the image and does not use stains. The lack of staining means that it can be used on live specimens and that one can observe the motility of the organism as well as its correct morphology. Usually, the stains and enzymes used in labs can distort the shape of the organism, but that isn’t an issue with dark field microscopy. This method can also be used to see organisms that are hard to stain, such as Treponema pallidum, spirochetes, and mycoplasma.The one downside is that it’s not possible to see the inclusions, or internal details of the cell

dark field fluorescence microscopy

dark field fluorescence microscopy The Major Manufacturers

 

The major microscope manufacturers all have devices capable of dark field illumination. Depending on the make/model, the microscope may come with attachments or have the options for dark field accessories. The major companies are:

MAIKONG
MONKON
Nikon
Olympus
Ziess
Leica
Meiji

In addition, lesser known smaller companies that produce dark field microscopes are Quanfa Scientific Instrument Company, Proway Optics & Electronics and Dewinter Optical Inc.

The leading innovators in microscopes are Nikon and Olympus, who both offer stereo and compound microscopes with dark field capability and/or accessories.

dark field fluorescence microscopy

what is dark field fluorescence microscopy?

Use scissors or (preferably) a brass cork borer to cut a set of stops matched to all of the objectives, and glue them to a sturdy sheet of clear acetate or glass. The acetate or glass substrate should be easily mountable onto the underside of the substage condenser, either through a filter holder or by other means, such as adhesive tape. Alignment of the stop can be done by observing it through a Bertrand lens or removing the eyepiece and viewing through a phase telescope while adjusting the condenser centering screws.

Darkfield Microscopy at High Magnifications

For more precise work and blacker backgrounds, you may choose a condenser designed especially for darkfield, i.e. to transmit only oblique rays. There are several varieties: “dry” darkfield condensers with air between the top of the condenser and the underside of the slide–and immersion darkfield condensers which require the use of a drop of immersion oil (some are designed to use water instead) establishing contact between the top of the condenser and the underside of the specimen slide. The immersion darkfield condenser has internal mirrored surfaces and passes rays of great obliquity and free of chromatic aberration, producing the best results and blackest background.

Perhaps the most widely used darkfield condenser is the paraboloid, consisting of a solid piece of glass ground very accurately into the shape of a paraboloid, as illustrated in Figure 5(b). Light incident upon the reflecting surface (between the glass and condenser housing in Figure 5(b)) of a paraboloid condenser will be focused at the focal point of the reflector. Most paraboloid condensers are cut to ensure that the focal point is slightly beyond the top of the condenser so that parallel light rays will be focused at a position that maximizes illumination of the specimen. The light stop at the bottom of the glass condenser serves to block central rays from reaching the specimen. Light rays that are reflected by the condenser are angled higher than the critical angle of reflection and converge at the principal focus of the condenser. The combination of a glass slide, mounting medium, and immersion oil (between the condenser and the microscope slide) complete the optical homogeneity of the paraboloid shape.

As discussed above, the dry darkfield condenser is useful for objectives with numerical apertures below 0.75 (Figure 5(a)), while the paraboloid and cardioid immersion condensers (Figures 1 and 5(b)) can be used with objectives of very high numerical aperture (up to 1.4). Objectives with a numerical aperture above 1.2 will require some reduction of their working aperture since their maximum numerical aperture may exceed the numerical aperture of the condenser, thus allowing direct light to enter the objective. For this reason, many high numerical aperture objectives designed for use with darkfield as well as brightfield illumination are made with a built-in adjustable iris diaphragm that acts as an aperture stop. This reduction in numerical aperture also limits the resolving power of the objective as well as the intensity of light in the image. Specialized objectives designed exclusively for darkfield work are produced with a maximum numerical aperture close to the lower limit of the numerical aperture of the darkfield condenser. They do not have internal iris diaphragms, however the lens mount diameters are adjusted so at least one internal lens has the optimum diameter to perform as an aperture stop.

Table 2 lists several properties of the most common reflecting high numerical aperture darkfield condensers. This table should be used as a guide when selecting condenser/objective combinations for use with high numerical aperture darkfield applications.
High Numerical Aperture Darkfield Condenser Specifications
Condenser Type    Hollow Cone
Numerical Aperture    Objective Maximum
Numerical Aperture    Number of Reflecting
Surfaces    Optical Corrections
Paraboloid    1.00-1.40    0.85    1 Parabolic    Achromatic
Cardioid    1.20-1.30    1.05    1 Spherical
1 Cardioidal    Achromatic/
Aplanatic
Bicentric    1.20-1.30    1.05    1 Cardioidal
1 Spherical    Aplanatic
Bispheric    1.20-1.30    1.05    2 Spherical    Aplanatic
Cassegrain    1.40-1.50    1.30    1 Aspheric
1 Spherical    Aplanatic
Spot Ring
(Bicentric)    1.40-1.50    1.30    2 Spherical    Aplanatic
Nelson
Cassegrain    1.30-1.45    1.20    1 Aspheric
1 Spherical    Aplanatic

The condensers illustrated in Figure 5 are designed specifically to produce oblique hollow light cones of high numerical aperture for darkfield illumination. In both instances, the upper surface of the condenser is planar and perpendicular to the optical axis of the microscope. The condenser on the left (Figure 5(a)) is designed to be used “dry” with no oil between the condenser and the underside of the microscope slide. In contrast, the paraboloid condenser in Figure 5(b) is intended to be “oiled” to the bottom of the microscope slide, directly underneath the specimen. Omission of immersion oil when using this condenser (or any of the other condensers listed in Table 2) will preclude any light from reaching the specimen. The oblique hollow cone of light rays emitted by these condensers cannot emerge from the top lens without oil and will be totally reflected back into the condenser. Light emitted from the illumination source is reflected at the mirrored glass surfaces within the interior of the condensers and exits the top of the condensers at much higher angles of inclination than the critical angle (approximately 41 degrees) at which total reflection occurs for passage of light from glass to air. In the situation of the oiled paraboloid condenser (Figure 5(b) and the condensers in Table 2) where the refractive index of the condenser glass, immersion oil, and glass slide are equal, light emitted from the condenser passes through the specimen unrefracted by glass-air interfaces.
Hollow Light Cone Numerical Aperture

Use this tutorial to visualize how the hollow cone of light changes with numerical aperture in reflecting darkfield condensers.
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Reflecting high numerical aperture condensers listed in Table 2 cover a wide range of designs used to produce the oblique hollow cone of light necessary for high-magnification darkfield microscopy. The paraboloid darkfield condenser has been discussed in detail above. Another very useful design is the cardioid condenser that is illustrated in Figure 1. This condenser design utilizes a mirrored hemisphere in the center of the condenser that serves as both a light stop and a reflector to direct light onto a second reflecting surface shaped to resemble a cardioid of revolution, from which the condenser derives its name. The combination of spherical and cardioid reflecting surfaces produces a condenser that is free from coma and both spherical and chromatic aberration. There are several technical drawbacks to using a condenser of such high numerical aperture. The cardioid condenser is very sensitive to alignment and must be carefully positioned to take advantage of the very sharp cone of illumination, making it the most difficult darkfield condenser to use. In addition, the condenser produces a significant amount of glare, even from the most minute dust particles, and the short focal length may result in poor illumination on objects that exceed a few microns in size or thickness. When choosing microscope slides for quantitative high-magnification darkfield microscopy, make certain to select slides made from a glass mixture that is free of fluorescent impurities.

High numerical aperture reflecting condensers (Figures 1, 5, 6 and Table 2) with darkfield illumination provide the method of choice for observing and photographing collections of very small particles or colloidal suspensions, even when the particle diameter is significantly lower than the limit of resolution for the objective. This is due to light diffracted by the particles, which passes through the objective and becomes visible as bright diffraction disks. Each particle is visible as a minute diffraction disk, provided the lateral distance between adjacent particles is greater than the limit of resolving power of the objective. As illumination intensity is increased, the optical difference between minute diffracting particles and their background increases. Simultaneously, even smaller particles (detectable solely by their ability to scatter light) now diffract enough light to become visible and suspended particles can be seen even when their diameters are smaller than 40 nanometers, which is about one-fifth the 200 nanometer resolution limit with oil immersion objectives of the highest numerical aperture. In biological applications, the movements of living bacterial flagella that average about 20 nanometers in diameter (too small to be seen in brightfield or DIC illumination) can be observed and photographed using high numerical aperture darkfield condensers.

Careful attention should be paid to the details of oiling a high numerical aperture condenser to the bottom of the specimen slide. It is very difficult to avoid introduction of tiny air bubbles into the area between the condenser top lens and the bottom of the microscope slide, and this technique should be practiced to perfection. Air bubbles will cause image flare and distortion, leading to a loss of contrast and overall image degradation. Problems are also encountered when using microscope slides that are either too thick or too thin. Many darkfield condensers contain the range of usable slide thickness inscribed directly on the condenser mount. If the slide is too thick, it is often difficult to focus the condenser without resorting to a higher viscosity immersion oil. On the other hand, slides that are too thin have a tendency to break the oil bond between the condenser and the slide. It is a good idea to purchase precision microscope slides of the correct thickness to avoid any of the problems mentioned above.
Darkfield Condenser Adjustment

Explore how alignment and configuration of a darkfield condenser affects image quality.
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A unique situation arises when specimens immersed in aqueous medium are being imaged using a high numerical aperture darkfield condenser. Under these conditions the refractive index of the aqueous solution limits the angle of inclination under which light can pass from the glass microscope slide (n = 1.515) into the water (n = 1.336) surrounding the specimen. The maximum numerical aperture of light passing from glass to water is given by the following equation:

NA (illumination) = 1.555 × sin(i) = 1.336 × sin(90°)

and because sin(90°) = 1

NA (illumination) = 1.336

Even though reflecting darkfield condensers designed for oil immersion are listed with upper limits of numerical aperture as high as 1.50 (see Table 2), light contributing to the illumination of specimens in aqueous media must have a numerical aperture no greater than 1.336, reducing the effective upper limit of darkfield illumination. In the case of specimens immersed in liquids of higher refractive index, the effective upper limit of the numerical aperture of darkfield illumination can approach a maximum of 1.50, although this is difficult to achieve in practice.

High numerical aperture condensers, whether intended for use dry or with oil, must be accurately centered in the optical path of the microscope to realize optimum performance. To achieve this, many darkfield condensers are built with a small circle engraved onto the upper surface to aid in centering the condenser. Centering is performed with a low power (10x-20x) objective by imaging the engraved circle and using the condenser centering screws to ensure the circle (and condenser) are correctly centered in the optical path. For more detailed information about microscope alignment for darkfield illumination, consult our section on darkfield microscope configuration elsewhere in the microscopy primer.

In general, objects imaged under proper conditions of darkfield illumination are quite spectacular to see (e.g. try a drop of fresh blood in darkfield). Often specimens containing very low inherent contrast in brightfield microscopy shine brilliantly in darkfield. Such illumination is best for revealing outlines, edges, boundaries, and refractive index gradients. Unfortunately, darkfield illumination is less useful in revealing internal details.

Other types of specimens, including many that are stained, also respond well to illumination under darkfield conditions. Figure 7 illustrates darkfield photomicrographs of three types of specimen, all of which produce good contrast in both brightfield and darkfield illumination. Details in the body of the deer tick (Ixodes demmini) shown in Figure 7(a) can be washed out in brightfield, unless the condenser aperture is stopped down to maximize contrast. However, in darkfield, most of the specimen detail in the tick becomes visible and can be easily captured on film. The heavily stained helminth trematode (Echinostoma revolutum, Figure 7(b)) also reveals considerably more detail when illuminated under darkfield conditions, as does the silkworm trachea and spiracle illustrated in Figure 7(c). In addition to the examples presented above, a number of other specimens can also be viewed and photographed under both brightfield and darkfield illumination to achieve the desired effects.

During the first half of the Twentieth Century, darkfield microscopy had a very strong following and much effort was expended in optimizing darkfield condensers and illuminators. This intense interest slowly began to fade with the emergence of more advanced contrasting-enhancing techniques such as phase contrast, differential interference contrast, and Hoffman modulation contrast. Recently, a renewed interest in transmitted darkfield microscopy has arisen due to its advantages when used in combination with fluorescence microscopy.

Darkfield microscopy is still an excellent tool for both biological and medical investigations. It can be effectively used at high magnifications to photograph living bacteria, or at low magnifications to view and photograph cells, tissues, and whole mounts. Marine biologists continue to use darkfield illumination at low powers to observe and record data about fresh and salt water organisms such as algae and plankton.

dark field fluorescence microscopy

dark field fluorescence microscopy Excludes Scleroderma in Raynaud’s

 

he absence of a systemic sclerosis (SSc) nailfold pattern in patients with Raynaud’s phenomenon or suspected connective tissue disease is of high clinical value as a biomarker to rule out SSc, according to a large cohort study from the U.K.

For identifying patients who met the 2013 American College of Rheumatology/European League Against Rheumatism or the Very Early Diagnosis of Systemic Sclerosis (VEDOSS) criteria for SSc, the study found that a nailfold capillaroscopy pattern had a negative predictive value of 90% (95% CI 86 to 93), according to Maya H. Buch, MBChB, PhD, and colleagues from the University of Leeds, writing in BMC Musculoskeletal Disorders.

That pattern also had a sensitivity of 71% (95% CI 61 to 80), a specificity of 95% (95% CI 91 to 97), and a positive predictive value of 84% (95% CI 74 to 91).

“We were very impressed with nailfold capillaroscopy’s utility in negative prediction,” Buch said in an interview with MedPage Today. “The most valuable result here is the low likelihood of scleroderma in patients with Raynaud’s phenomenon who do not have any scleroderma-specific features on nailfold capillaroscopy. In practice, this means we can more confidently reassure such a patient and discharge care back to the patient’s general practitioner.”

The researchers noted that to the best of their knowledge, this is the first study to demonstrate that the absence of any SSc pattern on nailfold capillaroscopy maintains its known negative predictive value, including for patients with secondary Raynaud’s phenomenon, who are considered at increased risk of SSc. “This study is only one of two to include a large unselected cohort of patients with Raynaud’s phenomenon — mirroring clinical practice in which rheumatology departments frequently receive referrals of patients with Raynaud’s from GPs,” Buch said.

Primary Raynaud’s phenomenon is associated with normal microcirculation architecture, whereas microangiopathies are associated with secondary Raynaud’s, she explained. The SSc nailfold capillaroscopy pattern correlates with disease duration and severity, and also predicts future vascular and visceral organ damage. Nailfold capillaroscopy also detects vascular problems in glaucoma.

Although an SSc nailfold capillaroscopy pattern is sometimes present in other connective tissue diseases, “nailfold capillaroscopy could be performed to provide reassurance to the rheumatologist in the assessment of both [primary and secondary] Raynaud’s phenomenon,” the researchers wrote.

Buch and colleagues studied 347 patients referred for nailfold capillaroscopy to a tertiary-care center from January 2009 to October 2013. The mean age of the cohort was 47 years and 83% were female. Clinical review showed that 54 patients (16%) did not have true Raynaud’s phenomenon, 69 (20%) had primary Raynaud’s, 172 (50%) had secondary Raynaud’s, and 52 (15%) had SSc.

At referral, 46 patients (89%) met either VEDOSS or the 2013 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) criteria for SSc. Of the patients with secondary Raynaud’s, 71 (41%) were being managed for connective tissue disease or inflammatory arthritis, while 101 (59%) had an antibody and/or a red-flag feature for SSc.

A nailfold capillaroscopy pattern for SSc was detected in 80 patients (23%) — 43 with early, 31 with active, and six with late-pattern vasculopathy. This pattern was observed in 37 patients (71%) diagnosed with SSc, 30 (17%) with secondary Raynaud’s, nine (13%) with primary Raynaud’s, and four (7%) without Raynaud’s.

Considering only those patients with non-SSc connective tissue disease or inflammatory arthritis, 16 of 71 patients (23%) had an SSc pattern. This was detected in two of five patients with SLE, eight of 42 with undifferentiated connective tissue disease, four of six with mixed connective tissue disease, one of three with Sjogren’s syndrome, and one of 14 with inflammatory arthritis.

Interestingly, the team said, participants meeting ACR/EULAR criteria were more likely to have an SSc nailfold capillaroscopy pattern than those meeting the VEDOSS criteria were: 84% versus 42%, respectively. “This may be related to the earlier stage of disease in those meeting VEDOSS with less time for detectable vasculopathic changes at the nailfold to develop,” the researchers wrote. “These findings are important as the earlier detection and management of SSc may lead to reduced morbidity and earlier detection of its complications.”

Among the study limitations were the lack of formal measurements to determine enlarged capillaries and the use of two different nailfold capillaroscopy methods, which might have introduced bias. As in clinical practice, the examiners were not blinded to the clinical diagnosis, possibly introducing investigator bias. In addition, the retrospective analysis may have missed important data, particularly the presence of telangiectasia.

The authors cited the need for larger, more defined prospective studies of a heterogeneous group of Raynaud’s patients. Buch noted that the current study is part of a larger Leeds program to identify biomarkers for accurately identifying patients at risk for scleroderma or those with scleroderma at risk for poorer outcomes.

dark field fluorescence microscopy

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