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"The Microscope Group Archives" This paper is reproduced with permission from Humana Press, Division of Springer (L) Cell Biophys. 1981 Jun; 3(2), pp.167-173 |
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AbstractA simple, zero-cost, reversible modification of a bright field microscope permits visualization of phase gradients in cells by transmitted illumination, yielding a Normaski-like effect. This modification, based on schlieren optics, is simultaneously compatible with high-aperture epi-illumination flourescence excitation. For many objectives that are intended for use in flourescence work, but are unavailable in phase contrast versions, the modification provides a simple means for locating cells in culture with good image contrast and resolution. Introduction Examination of cells by epi-illumination (i.e. through the objective) flourescence excitation is greatly facilitated by high-aperture liquid-immersion microscope objectives. Unfortunately, most such high aperture objectives intended for epi-flourescence are compatible only with transmitted illumination in bright field mode; they are often not commercially available with any means for phase-sensitive observation, such as phase contrast, to visualize unstained cells. This paper discusses a simple modification of ordinary bright field objectives that allows imaging of phase gradients in microscopic samples. This modification can be performed reversibly in a few minutes on either high- or low-aperture and magnification objectives, and at virtually zero cost with only two pieces of black masking tape. The modification is completely compatible with simultaneous use of the objective for epi-illumination flourescence or bright field illumination. Similar microscope systems were originally described by Abbe (1) in 1873 for creating relief contrast in microsocope images, and by Saylor (2) in 1935 for accurately measuring the refractive index of crystals immersed in fluids of closely matched refractive indices. Although the optical system described here is only slightly different from Saylor's, its potential application for visualizing biological cells with high contrast through bright field/epi flourescence objectives is not well known among cell biologists and deserves attention. The physical principle is based on schlieren optics (3), whereby a ray of light passing through a sample with a phase gradient is bent toward or away from an opaque barrier positioned behind the objective. Previous versions of schlieren microscopes (4-6), other than Saylor's (2), require extra lenses and significant alteration of the conventional microscope's sample mounting system. Modulation contrast microscopy (7), which is also based on the schlieren principle, is somewhat similar to the system discussed here, except that modulation contrast requires installation of a somewhat more sophisticated (and expensive) plate of graded opacity behind the objective. All forms of schlieren microscope optics produce image intensity variations sensitive to phase gradients in the sample. The resulting "three-dimensional" effect qualitatively resembles that of the much more expensive Normaski differential contrast (DIC) system, which is particularly popular for viewing biological cells. Method Figure 1 shows a simple schlieren modification of a bright field microscope with Köhler illumination. An opaque edge stop (e.g. a piece of black masking tape) is positioned in the front focal plane of the condenser so that it blocks about 4/5 of the condenser diameter (the exact fraction is not critical). Another opaque edge stop (again, black masking tape is sufficient) is inserted at the back focal plane of the objective. The two edge stops must be oriented with their edges parallel so that a narrow band of illumination light can propogate unimpeded between the edge stops. |
 Figure 1 |
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Fig.1. The optical path of rays through the schlieren microscope system for three idealized samples of different phase retardation profiles: zero, positive, and negative phase gradients. Typical rays are shown schematically emanating from two points in the condenser front focal plane: one near the opaque edge stop and the other somewhat farther from the edge. (Actual transmitted illumination is a continuum). For the positive-phase gradient sample, both rays are shown to be transmitted past the objective edge stop; for the zero gradient, one ray is transmitted; for the negative gradient neither ray is transmitted. The view of the objective back focal plane (seen directly with the ocular removed) shows the objective edge stop on the right-hand side of each circular field of view and the inverted image of the condenser edge stop on the left-hand side. For an actual sample, typically with a zero-average phase gradient, the condenser edge stop should be laterally positioned to give a back focal plane view similar to the zero phase gradient case shown here. The relative positioning of these opaque edge stops can be easily checked by looking down through the microscope eyepiece tube with the ocular removed; both the edge stop in the objective's back focal plane and an image of the edge stop in the condenser's front focal plane should appear as in Fig.1. A flat sample with zero phase gradient will not bend the illumination light and the narrow band of transmitted illuminated light will have a width 'Wo' as it passes the objective edge stop. A sample with a constant non-zero phase gradient normal to the edge stops will bend the illumination light so that the transmitted band will have an altered width W+ (greater than Wo) or W- (less than Wo) depending on the sign of the gradient. The width of the transmitted band determines the brightness of the sample image. Images of samples with variegated phase retardation will appear bright where the phase gradient is "positive", dim where the phase gradient is "negative", and intermediate elsewhere. Contrast in the field of view is increased by decreasing the width Wo of the band of transmitted illumination through the eyepiece tube. Figure 2 shows living cells on glass coverslips photographed with schlieren illumination as described. |
 Figure 2A, 2B |
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Fig.2A, cells in a primary culture of dissociated rat muscle tissue (8), viewed by schlieren optics with a 10x, NA 0.25 Leitz achromat objective and a NA 0.60 Leitz condenser. Fig.2B, phase contrast of the same field with a 10x, NA 0.25 Leitz achromat objective and a NA 0.60 Leitz phase condenser. |
 Figure 2C |
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Fig.2C, same cell type as A and B viewed by schlieren optics with a 50x, NA 1.00 water-immersion Leitz achromat objective especially intended for epi-flourescence and a NA 0.90 Leitz condenser. |
 Figure 2D |
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Fig.2D, human cheek epthelial cells viewed by schlieren optics with a 100x, NA 1.25 oil-immersion Leitz achromat objective and a NA 0.90 Leitz condenser. Note: All bars are 50 um. |
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In practice, the focal planes of the objective and condenser are not always easily accessible for insertion of the black masking tape edge stops. Fortunately, exact longitudinal (i.e. along the optical axis) positioning of the condenser edge stop is not necessary. This edge stop can be mounted in a plane as close to the front focal plane as practicable consistent with easy accessability for moving it laterally and rotationally as needed. The logitudinal positioning of the objective's back focal plane edge stop can be somewhat more critical, especially in high magnification objectives. Many achromat objectives are nearly hollow, thus enabling the edge stop to be mounted on the end of a thin-walled hollow tube inserted from the back of the objective. High magnification objectives in which rear access to the back focal plane is blocked by a lens cannot be modified easily for schlieren optics. The positioning of the masking tape barrier in low magnification objectives (less than 20x) is less critical; if an insert tube is not feasible, the masking tape can be placed at the back surface of the objective. Non-ideal longitudinal positioning of the edge stops causes parallax between the edge stops as viewed down the eyepiece tube and a smooth gradation of background intensity from one side of the field of view to the other. However, the schlieren effect is often preserved over most of the field of view. The two opaque edge stops in the schlieren technique are analogous to the condenser slit and the graded density modulator plate in the modulation contrast technique (7). Both techniques are sensitive to a preferred directon of phase gradients; both are free of halos characteristic of phase contrast; and both show little confusion from slightly out-of-focus phase gradients ("optical sectioning"; see Fig.3). |
 Figure 3 |
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Fig.3; "Optical sectioning" on a human cheek epithelial cell viewed by schlieren optics with a 100x, NA 1.25 oil-immersion Leitz achromat objective and a NA 0.90 Leitz condenser. Both A and B show the same filed of view, but their planes of focus differ by about 5 um. The nucleus is prominennt in A whereas cytoplasmic granules are prominent in B. The bar is 25 um. The schlieren technique has the advantage that it can be installed non-professionally, requiring no specially manufactured modulator plate and that the transmitting portion of the objective aperture is entirely unaltered. The Schlieren modification discussed here is completely compatible with simultaneous use of the system for bright-field and epi-illumination. Removing the condenser edge stop restores the view to brightfield. The lateral offset of the edge stops from symmetry around the optical axis serves to leave most of the objective's aperture clear. [In Saylor's schlieren system (2), the objective obscures approximately half of the aperture.] This offset is of particular advantage in the epi-illumination flourescence where high objective apertures are desirable. |
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Acknowledgement This research was supported by NIH grant #NS14565 References 1. Abbe, E. (1873) Arch.Mikroskop.Anotomie 9, 469
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