- Proper Alignment and Adjustment of the Light Microscope: Salmon & Canman (1998) Current Protocols in Cell Biology 4.1.1-4.1.16
Salmon & Canman (1998)
The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications (e.g., brightfield, phase-contrast, differential interference contrast, and fluorescence epi-illumination), and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The principles of microscopy and step-by-step alignment and adjustment procedures are described in this unit.
Optical microscopes can be powerful tools in biomedical research and diagnosis if properly aligned and adjusted. This is essential for optimal image quality and accurate quantitative measurements. Video cameras, digital cameras, and electronic image processing can improve visibility of structural detail resolved by the microscope optics in comparison to viewing by eye. However, the quality and accuracy of the image still depends critically on proper microscope alignment and adjustment.
This unit presents protocols for alignment and adjustment of a typical research compound light microscope for transillumination and epi-illumination imaging modes typically used today in biomedical research. The transillumination light modes include bright-field, phase-contrast, and differential interference contrast (DIC). The primary epi-illumination mode is fluorescence microscopy.
The described procedures are for alignment of a research upright microscope (Fig. 4.1.1). The procedures are applicable to inverted microscopes that have similar imaging and illumination light paths to the upright microscope. In either case, the specimen image is produced by an objective lens and the image is projected either to the eye with an eyepiece or to a camera with (and sometimes without) a projection lens. One lamp attached to the back of the microscope provides light for transillumination of the specimen through a condenser lens (Fig. 4.1.2 and Fig. 4.1.3). Another lamp attached to the back of the microscope provides light for epi-illumination of the specimen through the objective using a mirror in a filter cube to bring the illuminating light into the objective light path (Fig. 4.1.4). For both the transillumination and epi-illumination paths, there are field diaphragms for controlling the specimen region illuminated and condenser diaphragms for controlling illumination from the condenser. The lamps, the field diaphragm, and the condenser diaphragm, as well as the condenser and the objective, must be properly focused and centered for the best image formation. In addition, each different mode of image formation has special optical components that also require alignment and adjustment for optimal performance.
The first section in this unit (see discussion of Major Components of the Light Microscope) describes the location and basic functions of the most important features of the upright compound microscope. The second section (see discussion of Basic Imaging and Köhler Illumination Light Paths for Bright-Field and Fluorescence Microscopy) introduces the imaging and illumination light paths of the microscope and describes the principles of Köhler illumination, which is the alignment used typically for both transillumination and epi-illumination in the light microscope. Basic Protocol 1 lists steps in microscope alignment for transmitted-light Köhler illumination. Basic Protocol 2 lists steps in microscope alignment for epifluorescence Köhler illumination. Basic Protocols 3 and 4 provide brief descriptions of the principles of image formation and microscope alignment for phase-contrast and differential interference contrast (DIC) microscopy; these optical modes produce contrast of transparent specimens using transmitted-light illumination. Support Protocol 1 deals with mating cameras to the microscope and Support Protocol 2 deals with calibrating image magnification. Several procedures (see Support Protocols 3, 4, and 5) are given for testing the optical performance of the microscope; these also describe test specimens for microscope performance and their sources. Finally, Support Protocol 6 deals with the care and cleaning of microscope optics. The Commentary refers the reader to references that provide more comprehensive treatments of the basic modes and methods of light microscopy as well as advanced microscope imaging methods like multiwavelength, confocal, or multiphoton imaging, and electronic imaging techniques, including video and digital microscopy.
Major Components of the Light Microscope
Familiarizing oneself with the components of the light microscope is best done using the manual for the microscope as reference. Figure 4.1.1 provides a diagram of an upright research compound light microscope equipped for both transmitted light and epifluorescence microscopy. Locate the following components on the microscope, identify their adjustment screws (if applicable) and movements (e.g., condenser focus), and ascertain that they appear correctly mounted. In this and the following sections, z is a direction along the microscope axis; x and y are perpendicular directions.
Specimen Stage and Focus
A mechanical carrier on the stage holds the specimen slide. Knobs control movement in the x–y direction. Look for the vernier scales that mark the x and y positions. Rotatable stages are typically used for DIC microscopy. The objective is usually fixed and the specimen is focused by moving the stage along the z axis using coarse- and fine-focus knobs on the microscope body. Check the scale on the fine focus. On research microscopes, this is usually is 1 µm/unit.
Objectives and Revolving Nosepiece
Examine the different objectives on the nosepiece. Each objective is usually labeled with the following designations: magnification (e.g., 60×); the degree of optical correction (Apochromat is better than Fluor which is better than an Achromat); the label Plan (if both the center and edges of the field are in focus); numerical aperture (NA, which measures the half-angle of the cone of light from the specimen accepted by the objective; Fig. 4.1.3); immersion medium to be used (e.g., air, oil, water, or glycerin between the objective front lens and the specimen); coverslip thickness; optical tube length (previously 160 mm, currently infinity); and other special features like phase contrast, DIC, or long working distance. Working distance refers to the distance from the front element of the objective to the specimen. Working distance usually decreases with increasing objective magnification and NA. Check that the objectives are mounted in series from the objective of the lowest magnification to the objective of the highest magnification. It is usually best to find the specimen or region of a specimen with a low-magnification objective, and then swing in objectives with higher magnifications and shorter working distances. The high-NA, short-working distance oil-immersion objectives often have the ability to lock up the nosepiece when changing objectives. This prevents running the front element of the objective into the mounting medium on the edges of the coverslip. Check that the nosepiece is down for imaging.
Specimen Slides and Coverslips
Microscope condenser lenses are usually corrected optically for 1-mm-thick glass slides. The objectives are corrected optically for coverslips with a thickness of 0.17 mm; no. 1.5 coverslips are on average this thick. The image quality decreases for thinner (e.g., no. 1) or thicker (e.g., no. 2) coverslips, particularly for non–oil immersion (“dry”) objectives with high NA. For oil-immersion objectives, this problem is not critical when the refractive index of the oil and coverslip (~1.515 to 1.52) match. Coverslips thinner than no. 1.5 are often used with oil immersion, to allow greater range of focus.
In the modern research light microscope, the imaging light leaving the objective and nosepiece is unfocused and the image is projected to “infinity” (Fig. 4.1.2, left). This infinity space above the objective allows insertion of filters without changing the focal position of the image at the intermediate image plane. A positive focusing lens, the tube lens, above the infinity space, is used to bring the imaging light into focus for the eyepieces or camera.
Check for the following possible inserts in the infinity space of the microscope.
This device switches the light between the binocular and the camera port. Check for the percentage of light in each direction. For fluorescence microscopy, it is important to be able to send 100% of the imaging light either to the eye or to the camera.
Eyepiece magnification to the eye is marked on the barrel (e.g., 10×). Note that there are two and maybe three adjustments. The interpupillary distance for the eyes is adjusted by grasping the bases of the eyepiece tubes and moving them closer or further apart. At least one of the eyepieces is adjustable so that the eyes are parfocal.
Check the type of camera adapter and whether it matches the detector. There are several different types of camera adapters. One type uses an eyepiece tube (not the binocular tube) and a projection eyepiece combined with a camera lens to project an image onto the camera detector. This method is common in photographic film cameras (which use ≥35-mm film) and for some video cameras with large-size detectors (e.g., 1-in., equivalent to 2.54-cm). The recent video and cooled slow and progressive scan charge-coupled device (CCD) cameras have small detectors (≤ in., equivalent to 1.7 cm). These cameras require smaller projection magnifications or none—in the latter case the detector is mounted at the intermediate image plane, the focal plane of the objective (Fig. 4.1.2), and no eyepiece or projection lens is used.
Lamp and Housing
The lamp is typically a low-voltage 100-W quartz halogen bulb with a tungsten-filament light source and variable control. Some lamp housings have no adjustments for centering the bulb; the socket is prefixed. Most lamp housings have x–y adjustments for the bulb socket, while an advanced lamp housing also has a mirror in the back for reflecting an image of the lamp back along the microscope axis. This mirror usually has adjustments for x, y, and z positions of the mirror image of the lamp. There is usually a knob on the side of the lamp housing for moving the collector lens back and forth along the z axis to focus the tungsten-filament image onto the condenser diaphragm plane.
Diffuser and Filters
A diffuser and other filters are often inserted in slots in the base of the microscope. The diffuser helps spread the image of the source at the condenser diaphragm plane in order to uniformly fill the condenser aperture. This is important for achieving high resolution. A heat-reflecting filter (e.g., BG 58) blocks infrared light. Video cameras, but not the eye, are often sensitive to this wavelength of light. A green filter is frequently preferred for illuminating living cells. High-efficiency interference filters with a 40-nm bandwidth around 540 nm are often best. Various neutral-density (ND) filters, which are not wavelength selective, are useful for attenuating light to cameras.
This is usually located just above or beneath the mirror that deflects the light up to the condenser lens. It controls the specimen area illuminated by the condenser (Fig. 4.1.2, left).
This is inserted below the condenser diaphragm for polarization and DIC microscopy. Otherwise, it is removed from the light path.
Condenser Focus Knob
This translator moves the condenser along the microscope z axis to focus an image of the field diaphragm on the specimen.
Condenser Centering Screws
There are usually two screws on the condenser carrier which move the condenser in an x–y plane. These screws are used to center the image of the field diaphragm on the z axis.
The condenser diaphragm is located on the bottom of the condenser. It controls the angle (NA) of the condenser cone of illumination of the specimen (Fig. 4.1.3).
Condensers have turrets with inserts for special image-contrast techniques such as phase-contrast and DIC microscopy. Each insert matches certain objectives. In phase-contrast microscopy, the inserts are annuli of different diameters designed to match the phase ring in phase-contrast objectives (Fig. 4.1.5). For DIC, the inserts are typically DIC prisms designed for certain objectives (Fig. 4.1.7). To see these inserts, as well as the opening and closing of the condenser diaphragm, remove the condenser from the condenser carrier by loosening the locking screen and turning it upside down.
The condenser’s main function is to provide bright, uniform illumination of both the specimen field (Fig. 4.1.2, right) and objective aperture (Fig. 4.1.3) for objectives with different NAs. Check the markings on the condenser to see if it is designed for air (dry) or oil immersion with the glass slide. Dry (no oil-immersion) condensers have NA values of ≤0.9 and should not be used with immersion oil. Oil-immersion condensers usually have NA values of 1.0 to 1.4 when using immersion oil.
Lamp and Housing
The light source for the epifluorescence illumination is typically an HBO 50-W or HBO 100-W mercury arc lamp or a xenon lamp of similar wattage. These lamps need to be handled with care because they can blow up if mistreated. Their glass envelopes should be carefully cleaned with 70% ethanol before installation to prevent fingerprints or other materials from inducing hot points that may result in fracturing of the bulbs. Look for the adjustment screws to adjust the x–y position of the lamp. Look also for the rear mirror and its x, y, and z adjustment screws. Identify the focusing knob for the lamp collector lens. Do not turn the lamp on until the bulb has been installed according to the manufacturer’s instructions, the collector lens has been inserted properly, and the lamp housing has been installed on the back of the microscope. These lamps produce intense light, and the mercury bulbs have large peaks in the UV range. Therefore, UV-protective glasses should always be worn when handling and installing these lamps. The light intensity decreases and the probability of explosion increases with the number of hours of operation and lamp starts. Check the timer on the lamp power supply to see that the recommended limits are not exceeded.
Photobleaching is a big problem in fluorescence microscopy. A shutter is used to block the light from the specimen when not taking exposures on a camera or viewing by eye.
A heat-reflecting filter is used to prevent infrared illumination of the specimen and to keep infrared scattered light from reaching the camera. Neutral-density (ND) filters are used to reduce the light intensity of fluorescence illumination by the amount indicated on each filter.
Epi-Illumination Condenser Diaphragm
This diaphragm provides variable adjustment of the illumination intensity. Some microscopes lack this diaphragm.
Epi-Illumination Field Diaphragm
This diaphragm controls the area of the specimen illuminated. Look for the centering screws that control the x–y position of the field diaphragm.
See discussion of Image-Forming Components.
Basic Imaging and Köhler Illumination Light Paths for Bright-Field and Fluorescence Microscopy
The second major step in learning proper alignment of the light microscope is to understand the basic image-forming and Köhler illumination light paths and the functions of the key optical components and diaphragms. These are outlined in Figure 4.1.2, Figure 4.1.3, and Figure 4.1.4.
The upper left section of Figure 4.1.2 shows ray paths for the image-forming light from the objective. The objective, in combination with the tube lens, produces a real, magnified image of the specimen at the intermediate image plane. The eyepiece provides a second stage of magnification to the eye. The magnification to the eye is the product of objective magnification and eyepiece magnification multiplied by any magnification provided by additional lenses in the body tube of the microscope between the objective and the eyepieces.
When a camera detector—film, video, or charge coupled device (CCD)—is mounted at the intermediate image plane, the magnification to the camera is the magnification produced by the objective and body tube. When a camera is mounted above an eyepiece, then the unfocused light leaving the eyepiece must be focused onto the camera detector by a camera lens, which modifies the total magnification to the detector. Special adapters (see Image-Forming Components, Camera Adapters) are also available for projecting the objective image onto a camera detector with magnification, but without the need for an eyepiece.
Transillumination Ray Paths
In the illustration of the standard Köhler method (Fig. 4.1.2), trace the illumination ray paths for a bright-field microscope aligned for Köhler transillumination (right side). A point on the light source is focused by the lamp collector lens onto the front focal plane of the condenser lens, where the condenser diaphragm is located. This light is projected, out of focus, through the specimen by the condenser lens, producing uniform illumination of the specimen. The objective collects the unfocused illumination light and brings an image of the light source into focus at its back focal plane, which is at the back aperture of the objective (Fig. 4.1.3). The light source is again out of focus at the intermediate image plane and at the retina of the eye or the detector of the camera. In between these two points, the light source is in focus at the exit pupil of the eyepiece, at a position ~15 mm above the eyepiece. This position is also called the eyepoint, the position of eye placement above the eyepiece (Fig. 4.1.2, right).
The condenser diaphragm controls the NA (cone angle) of specimen illumination by the condenser lens (Fig. 4.1.3). Opening the diaphragm increases the aperture of illumination, which increases both the light intensity and resolution in bright-field light microscopy (see Support Protocol 3). Note on the right side of Figure 4.1.2 that the image of the condenser diaphragm is in focus where the lamp image is in focus along the microscope axis, at the objective back focal plane and the exit pupil of the eyepiece.
Trace the imaging light rays from the field diaphragm through the microscope (Fig. 4.1.2, left). Note that the field diaphragm controls the specimen area illuminated by the condenser. Note also that the field diaphragm is placed between the lamp collector lens and the condenser lens in a plane where the lamp image is out of focus (compare Fig. 4.1.2, left and right). When the condenser lens has focused the image of the field diaphragm onto the specimen, then the field diaphragm will be in focus with the specimen at the intermediate image plane and at the eye or detector.
Epi-illumination Ray Paths
Figure 4.1.4 shows the optical alignment for an epi-illuminator and Köhler illumination through the objective. As in transillumination (Fig. 4.1.2), the light source is focused by a collector lens onto the condenser diaphragm of a condenser lens in the light path. The condenser lens illuminates a field diaphragm. Another lens collects this light and projects it off a reflective mirror into the objective. The image of the light source is focused at the back focal plane of the objective so that it is out of focus at the specimen plane. In epifluorescence microscopy, filter cubes containing dichroic mirrors are used in combination with excitation and emission filters to efficiently reflect the excitation light into the objective and transmit to the eyepiece or camera only the longer-wavelength fluorescent light emitted from the specimen (see unit 4.2 and Taylor and Salmon, 1989).
Basic Protocol 1: Alignment for Köhler Illumination in Bright-Field, Transmitted Light Microscopy
The following procedures assume that the positions of the quartz-halogen tungsten filament and collector lens are adjustable. Inexpensive, non-research compound microscopes may have the illuminator built into the stand, with no adjustment for the lamp. Such microscopes depend on a ground-glass filter for even illumination. For these microscope stands, skip steps stepRef(‘#cb0401-step-0002’, ‘2’);2, stepRef(‘#cb0401-step-0003’, ‘3’);3, stepRef(‘#cb0401-step-0004’, ‘4’);4, and stepRef(‘#cb0401-step-0008’, ‘8’);8.
Basic Protocol 2: Alignment of the Eyepieces
The binocular usually has adjustments for the inter-eyepiece distance and visual acuity in each eye. The goal is for the image to be in focus for each eye without any eyestrain or discomfort. Only the left eyepiece tube or eyepiece is adjustable on a typical microscope. However, if the microscope has a target reticle in the microscope stand that can be rotated into view, then usually both eyepieces are adjustable.
Basic Protocol 3: Alignment for Köhler Illumination in Epifluorescence Microscopy
The eye is most sensitive to green light. Hence, the following protocol is best done using a filter cube that produces green excitation light (e.g., a filter cube for rhodamine).
Basic Protocol 4: Alignment for Phase-Contrast Microscopy
Phase-contrast microscopy is often used to produce contrast for transparent, non–light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement (Zernike, 1942, 1955, 1958). The last of these references describes the principles of phase contrast and provides an excellent introduction to the wave optics of image formation, resolution, and contrast in the microscope.
The phase-contrast microscope is a bright-field light microscope with the addition of special phase-contrast objectives (Fig. 4.1.5) containing a phase plate or ring and a condenser annulus instead of a diaphragm; the annulus is usually located on a condenser turret because it has to be selected for different objectives. The microscope optics are usually aligned for bright-field specimen illumination by the standard Köhler method. However, there is no condenser diaphragm to adjust. Instead, the phase annulus must be selected and adjusted properly. Modern phase-contrast objectives have a phase plate containing a ring in the back focal plane within the barrel of the objective. This ring absorbs and advances the phase of the light passing through it by ¼ wavelength in comparison to light passing through the rest of the objective aperture. For each phase objective, there is a corresponding annulus in the condenser turret that has about the same relative size as the phase ring in the objective. Light passing through this annulus passes through the phase ring in the objective when the annulus is selected and aligned properly. Light scattered by the specimen mainly passes through the objective aperture outside the phase ring. Light scattered from a thin transparent specimen is ~¼ wavelength retarded from the unscattered illumination light. The additional ¼ wavelength retardation between the scattered and illumination light produced by the objective phase ring makes the scattered and illumination light 180° out of phase. They destructively interfere with each other at the image plane to produce the “dark” contrast of structural detail typical of phase contrast images of biological specimens.
Because the phase annulus and the phase ring reduce the intensity of the background light, a bright illuminator—e.g., a 100-W quartz-halogen illuminator—is necessary at high magnifications (because image intensity decreases as 1/magnification2). For living cells, heat reflection and green illumination filters should be used.
To examine the objective phase ring and matching condenser phase annulus, remove the low- and medium-power phase objectives and the phase condenser from the microscope. View the phase ring (phase plate) within the objective by looking in the back end. The phase ring is located at the position of the objective back focal plane; its diameter is usually ~ 2/3 that of the objective aperture. Note that it is situated within the objective body and is visible because it absorbs light. Locate the annulus for each objective in the condenser turret. The annulus is located at the condenser diaphragm plane, which is situated at the condenser front focal plane. Notice that as the objective NA increases, the diameter of the corresponding annulus in the condenser turret increases. The NA of condenser illumination from an annulus is designed to match that of the phase ring in the corresponding objective.
Replace the condenser on the microscope and illuminate with white light. Hold a piece of tissue paper near the top of the condenser and examine the illumination cone. Notice that it is an annular cone of illumination. Change the annulus from the setting for the low-power objective to the 40× setting. Note that the NA or angle of illumination increases. Points of illumination further from the central axis in the condenser diaphragm plane produce higher-aperture rays passing through the specimen.
Perform alignment using the following steps.
Basic Protocol 5: Alignment for DIC Microscopy
Since its introduction in the late 1960s (e.g., Allen et al., 1969), DIC microscopy has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens—including tissue cells, eggs, and embryos—and does not suffer from the “phase halos” typical of phase-contrast images. See Salmon and Tran (1998) for details of image formation and video-contrast enhancement.
The DIC microscope is a bright-field light microscope with the addition of the following elements (Fig. 4.1.7, middle): a polarizer beneath the condenser; a DIC beam-splitting prism (Nomarski or Wollaston) in the condenser turret; a DIC beam-combining prism (Nomarski) just above the objective; an analyzer above the objective prism in the infinity body tube space; a compensator after the polarizer or before the analyzer in some microscopes (not shown in Fig. 4.1.7); and a rotatable stage. The microscope optics are usually aligned for bright-field specimen illumination by the standard Köhler method.
The polarizer, which produces plane-polarized light (Fig. 4.1.7, left), is typically oriented with its transmission azimuth in an east-west direction facing the front of the microscope. Polarizers with high transmission efficiency are preferred. Polarizers are usually high-quality polaroid material held between thin optical glass flats. Another polarizer is used as an analyzer. The transmission azimuth of the analyzer is oriented north-south at 90° with respect to the polarizer azimuth to produce extinction of the illumination light in the absence of the DIC prisms.
The specimen is held on a rotating stage. Contrast depends on orientation, and frequently specimens must be reoriented to achieve maximum contrast of the structures of interest. Either the stage or the objective and condenser must be centerable.
For the highest sensitivity, the objective and condenser lenses should be selected free of birefringence (Inoué and Spring, 1997). Rectifiers can be used to correct for the rotation of the plane of polarization of light which occurs at the periphery of lens surfaces (Inoué and Spring, 1997).
Figure 4.1.7 outlines the principles of DIC image formation and contrast. The DIC beam-splitting prisms are located at the condenser diaphragm plane, just above the polarizer in the condenser turret. Objectives of different magnification and/or NA usually require condenser prisms of different optical characteristics.
The condenser prism splits the light coming from the polarizer into divergent polarized light wavefronts whose planes of polarization are orthogonal to each other and at 45° with respect to the initial plane of polarization (Fig. 4.1.7, left). The divergent beams are converted by the condenser into two wavefronts, which pass through the specimen separated laterally from each other in the direction of the prism wedge (the shear direction, Fig. 4.1.7, middle) by a tiny amount that is usually less than the resolution limit of the condenser-objective lens combination (Fig. 4.1.7, right). These two wavefronts are recombined just above the objective by a beam-combining prism. Often, each objective has its own prism so that it accurately matches the action of the condenser prism. In some microscopes, there is one beam-combining prism for all objectives and a different condenser prism for each objective. Check which is the case for your microscope.
DIC image contrast depends on the “compensation” or “bias retardation” (spchr(‘Delta’, ‘Math1’, ‘D’)Δ) between the two wavefronts along the microscope axis (Fig. 4.1.7, right). When the objective beam-combining prism is perfectly aligned with the condenser beam-splitting prism and there is no compensation (spchr(‘Delta’, ‘Math1’, ‘D’)Δ = 0), the background light is extinguished and the edges of objects are bright against a black background (Fig. 4.1.7, upper right). When one wavefront is retarded relative to the other by spchr(‘Delta’, ‘Math1’, ‘D’)Δ, this increases the optical path (OP) between the wavefronts (Fig. 4.1.7, middle right) and brightens the background light. One edge of an object becomes brighter than the background while the opposite edge becomes darker (Fig. 4.1.7, upper right). This produces the “shadow cast” appearance of DIC images. Reversing the sign of retardation, reverses the contrast of the specimen edges.
In some microscopes, the objective beam-combining prism (Fig. 4.1.7, middle) is used as a compensator by translating the prism in the direction of prism wedge away from the position of background light extinction. One direction produces positive while the other produces negative retardation (spchr(‘Delta’, ‘Math1’, ‘D’)Δ). In other microscopes, both the objective and condenser prisms are fixed at positions that produce background light extinction, and typically a deSenarmont compensator (a birefringent quarter-wave retarder in combination with a rotatable polarizer or analyzer; Inoué and Spring, 1997) is inserted just above the polarizer or beneath the analyzer.
Bright illumination sources are required for high magnification because of the crossed polarizer and analyzer; at least the 100-W quartz-halogen illuminator is usually needed.
To examine the DIC prisms, remove the objective prisms and the DIC condenser from the microscope. Examine the objective prism. Notice that it is very thin and wide enough to cover the objective aperture. Also notice if there is a screw that can be used to translate the prism back and forth in one direction across the objective aperture. Examine the prisms in the condenser turret. Notice that there are different prisms for low- and high-NA objectives. The highest-resolution (NA) objectives often have special prisms designed only for the optical properties of that objective. In aligning the microscope for DIC, be careful to use the condenser Wollaston prism that matches the objective in use. Reinstall the condenser.
Steps stepRef(‘#cb0401-step-0044’, ‘1’);1 to stepRef(‘#cb0401-step-0049’, ‘6’);6 in the following procedure are for the initial alignment of the microscope. Once this is done, then begin at step stepRef(‘#cb0401-step-0048’, ‘5’);5 for routine use.
Support Protocol 1: Matching Microscope Magnification to Detector Resolution
Support Protocol 2: Calibrating Image Magnification with a Stage Micrometer
Tests for the Optical Performance of the Microscope
Test slides are used to evaluate the performance of the microscope under different conditions.
Diatoms have silica shells shaped like pillboxes. There are pores in the shell arranged in a lattice pattern specific for each diatom species. Figure 4.1.8 shows a low-magnification view of the eight diatoms on the test slide (panel A) plus higher-magnification views of the lattices of three diatoms most useful in testing the resolution performance of microscope optics using phase-contrast or DIC. Number 6 in panel A of Figure 4.1.8, Pleurosigma angulatum, has a triangular pore lattice with spacing of ~0.61 µm between rows (illustrated in Fig. 4.1.8, panel D). Number 5, Surrella gemma, has rows of pores where the rows are separated by ~0.41 µm (illustrated in Fig. 4.1.8, panel C). Number 8 is Amphipleura pellucida, which has horizontal rows of pores separated by ~0.25 µm (illustrated in Fig. 4.1.8, panel B). In transmitted light, the diffraction limit to lateral resolution, r, is given by (Inoué, 1989):
r = spchr(‘lambda’, ‘Math1’, ‘l’)λ/(NAobj + NAcond)
where spchr(‘lambda’, ‘Math1’, ‘l’)λ is the wavelength of light, NAobj is the objective NA, and NAcond is the condenser NA (see Fig. 4.1.3). The lateral resolution, r, is equal to 0.195 µm for the highest objective NA, which is equal to 1.4, with NAcond = NAobj and 546 nm green light. As seen in Figure 4.1.8B, this objective is capable of resolving the rows of pores in the shell of Amphipleura, but not the individual pores, which are slightly <0.19 µm apart.
Use the above equation to calculate the diffraction limit of resolution for your other objectives and use the diatoms to test whether they achieve their theoretical limits.
Cheek cells are a convenient specimen for testing the performance of phase-contrast or DIC microscopes. As seen in the low-magnification view (Fig. 4.1.6) they are large and flat, ≤3 µm thick except near the cell center which contains the nucleus. The upper and lower surfaces have fine ridges which swirl around much like fingerprints. Many of the ridges are <0.2 µm in width and separated by <0.5 to 1.0 µm.
To prepare the cheek cells, gently scrape the inside of your mouth with the tip of a plastic pipet or similar soft tool and spread the cells and saliva on the center of a clean no. 1.5 coverslip. Invert the coverslip quickly onto a clean slide and press down to spread the cell preparation into a thin layer. Seal the edges with a thin layer of nail polish.
Support Protocol 5: Testing Fluorescence Using Red, Green, and Blue Fluorescent Tissue Culture Cell Test Slide
The cells in Figure 4.1.9 are triple labeled: DAPI stained nuclei and chromosomes (blue fluorescence; Fig. 4.1.9A); Alexa 488-phalloidin labeled actin filaments (green fluorescence; Fig. 4.1.9B); and X-Rhodamine immunofluorescently labeled microtubules (red fluorescence; Fig. 4.1.9C). The microtubules and fine actin filamentous arrays are <100 nm in width, but they should appear sharp and in high contrast in the microscope. There should be no “bleed-through” of fluorescence from one fluorescence channel to another if the filters are chosen properly (Taylor and Salmon, 1989).
r = 0.61spchr(‘lambda’, ‘Math1’, ‘l’)λ/NAobj
and the intensity of the image, Iimage, is given approximately by:
Iimage ≈ IexNAobj4/Mp2
where Iex is the excitation light intensity entering the objective and Mp is the projection magnification from the objective to the eye or camera. The rate of photobleaching, Rp, of a specimen depends on:
Rp ≈ IexNAobj2Mobj2
where Mobj2 is the magnification of the objective.
Note from these equations that resolution depends inversely on NAobj (r = 0.24 µm for NAobj = 1.4 and 546 nm green light), image intensity depends on the fourth power of NAobj, while loss of intensity by photobleaching of the fluorophore increases with the square of objective NA (a measure of the excitation light collected by the objective) and magnification (which concentrates the excitation light on the specimen).
Verify these equations by imaging the fluorescent specimen (the microtubules in the spread cells are a good choice) for objectives with different magnifications and numerical apertures. It will quickly be seen why in fluorescence one wants to use the minimum total magnification necessary to resolve the structures of interest in the detector, as well as use the maximum NAobj in order to maximize light intensity for the least amount of excitation light (and the least amount of photobleaching).
Support Protocol 6: Care and Cleaning of Microscope Optics
Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. Below are steps for keeping the microscope clean.
The development of lasers, electronic cameras, digital image analysis, and specific fluorescent molecular probes have recently made light microscopy an enormously powerful tool in the biomedical sciences. There are a number of excellent books and review articles about these applications, as well as the optical principles of light microscope design and image formation. Listing of all these references is beyond the scope of this unit; only a few of these books and reviews are listed, and these can be used to find other useful references.
Spencer (1982), Zernike (1958), and Keller (1995, 1997, 1998) provide excellent introductions to the principles of image formation and contrast in the light microscope for beginners, while Pluta (1988, 1989) provides a more comprehensive treatment. Inoué and Oldenbourg (1995) also review the basic concepts and principles of microscope design and different methods of image formation. The abovementioned references describe the many modes of transmitted-light imaging in the microscope, including bright-field, phase-contrast, and DIC, which are described in this unit, as well as dark-field, polarization, interference contrast, Hoffman modulation, and reflection interference contrast methods. Taylor and Salmon (1989) and unit 4.2 give clear introductions to fluorescence microscopy. The recent edition of Video Microscopy by Inoué and Spring (1997) is a comprehensive introduction to the principles and practical aspects of light microscopy, video, and digital imaging. The Video Microscopy volume of Methods in Cell Biology edited by Sluder and Wolf (1998) also has contributions that cover many basic concepts and practical aspects of light microscopy in the biomedical sciences; it also includes chapters on multiwavelength, multimode digital imaging methods, camera selection, ion ratio imaging, and specimen chambers. Salmon and Tran (1998) review the principles of video-enhanced DIC methods used to image macromolecular complexes invisible in the microscope when viewed by eye. Taylor et al. (1992), Salmon et al. (1998), and Rizzuto et al. (1998) review microscope design for multiwavelength, multimode digital imaging of fluorescent specimens, and four-dimensional microscopy. Advanced quantitative fluorescence methods like ratio imaging, photobleaching, photoactivation, resonance energy transfer, chromophore-assisted laser ablation, and fluorescence lifetime imaging are also reviewed in the Methods in Cell Biology volumes edited by Taylor and Wang (1989), and Sluder and Wolf (1998) as well as in Herman and Jacobson (1990) and Inoué and Spring (1997). Reviews on imaging cells containing expressed protein coupled to green fluorescent protein (GFP) include Heim and Tsien (1996), and Sullivan and Kay (1998). Agard et al. (1989), Carrington et al. (1995), and Rizzuto et al. (1998) review how deconvolution of three-dimensional image stacks can produce super resolution in the light microscope. The recent edition of Handbook of Biological Confocal Microscopy edited by Pawley (1995) is an excellent reference on this important method for imaging fluorescent structures in thick specimens (Smith, 1997). The new technique of multiphoton laser scanning microscopy is described by Denk et al. (1994), while biological applications of optical traps to manipulate organelles and measure molecular forces is reviewed by Svoboda and Block (1994).