- 1. ABSTRACT The following instructions will guide the user to a successful Hoffman Modulation Contrast (HMC) installation. As such, these instructions must necessarily include much detail that may already be familiar to the experienced microscopist. In summary the procedure includes: Focusing low power (10X) objective on specimen.Positioning condenser at its correct working distance and centering condenser lens on optic axis.Aligning the HMC slit aperture(s) in the condenser with the corresponding modulator filter in each HMC objective.Though HMC alignment procedures are simple once mastered, it is recommended that new users carefully follow the accompanying detailed instructions.
2. TABLE OF CONTENTS SECTION IIntroductionSECTION II AInstallation and Alignment of HMC on Inverted MicroscopesSECTION II BInstallation and Alignment of HMC on Upright MicroscopesSECTION IIITechnical Information 1.Working Distance of HMC Condensers2.Cover Slip Correction Objectives3.Filters4.Reflections at the Back Focal Plane5.Kohler Illumination and HMC Condensers6.Troubleshooting Chart 3. SECTION I - Introduction The Hoffman Modulation Contrast system produces an image of high contrast and high resolution. The image has a three-dimensional appearance wherein a rounded object appears dark on one side, bright on the other, with grey in between, against a grey background. Additional features are optical sectioning, directional sensitivity, high resolution and control of contrast and coherence. The Hoffman Modulation Contrast system consists of three components easily added to a compound light microscope (Figures 5A, 5B): 1. An HMC objective containing a special three region filter called a Modulator (Figure 1). The HMC objective (Figure 2) can be used effectively for other modes of illumination, such as brightfield, darkfield, fluorescence and polarized light.Figure 1 - Modulator filter - factory installed in objective.Figure 2 - Typical Hoffman Modulation Contrast objectives.2. An HMC condenser assembly consists of a lens system and one or more rectangular apertures called slits (Figure 3A). They are located on a turret (or slider) just before the condenser lens. This location is similar to the light annulus position in a phase contrast turret. Each slit is in a holder (Figure 3B), which is adjustable so that the slit image, produced at the back focal plane of the objective, can be aligned to the modulator. The adjustments for modulation contrast are as simple and similar to those required for phase contrast.Figure 3A - Model TCT turret condenser assembly. Component at upper left is slit aperture in holder.Figure 3B - Model TCS slider condenser assembly. Shows slit aperture being installed in Spring Clips. This mechanism allows the slit holder to be rotated and moved laterally to permit its alignment to the modulator in the HMC objective. 4. 3. The HMC system usually incorporates the variable contrast feature*. It utilizes a polarizer (P1) (Figure 4), which is rotated to control image contrast. It is placed in the optical path before the slit aperture. Rotating the contrast control polarizer varies the background intensity and the partial coherence of the illumination. As a result of this feature the HMC system is thereby capable of providing optimal contrast and sharp images for a wide range of objects, from relatively flat to round shapes, with small to large refractive index differences relative to their mounting media.Figure 4 - HMC contrast control polarizer (P1).* Systems not equipped with this feature do not utilize a polarizer and always operate at maximum contrast. Nonvariable systems are used on microscopes of relatively low illumination or with polarized light microscopes.Figure 5A - HMC components on an inverted microscopeFigure 5B - HMC components on an upright microscope. 5. SECTION II A - Installation and Alignment of HMC on Inverted Microscopes 1. Before attempting to install the HMC system, review the microscope manufacturers Instruction Book to familiarize yourself with its component parts and their functions. 2. These instructions describe the general procedures to install an HMC condenser on an inverted microscope. An addendum may accompany this booklet with detailed information on your specific microscope. a) Install the condenser mounting flange as shown in Figure 6 using the .050" hex key provided with the system.Figure 6 - Mounting typical flange on Model TCT turret condenser assembly.b) Install the condenser system on the microscope (Figure 7).Figure 7- Model TCT mounted on typical inverted microscope.3. Position the front face of the condenser lens cell at its nominal working distance (Figure 8) from the specimen plane. Working distance (WD) and numerical aperture (na) are marked on the condenser lens cell (Figure 9). 6. Figure 9 - HMC condenser lens cells.Figure 8 - Condenser working distance.4. Rotate the condenser's turret plate (or move slider) to the brightfield position (BF), so that no slit aperture or phase annulus is in the optical path. 5. Install HMC objective(s) onto microscope's nosepiece and select the lowest magnification HMC objective. Focus on the specimen. Once objective is focused at the specimen plane, move the stage so that the specimen is out of the field of view. Note: It is helpful to use a stained specimen during this initial set-up. 6. Using an auxiliary telescope (or microscope's bertrand lens), focus on the modulator (Figure 10) at the back focal plane of the HMC objective. Note: The modulator may be in a different rotational orientation dependent on the microscope's nosepiece thread.Figure 10 - Plan view of modulator.7. If the microscope is equipped with a variable diaphragm near the light source, partially close it until its image is seen through the telescope or bertrand at the periphery of the objectives back focal 7. plane (Figure 11), (see Section III #5 - Kohler Illumination and the HMC Condenser).Figure 11 - Plan view of modulator showing image of variable diaphragm not yet centered.If the diaphragms image is not centered and the microscope has built-in condenser centering screws, use them to center diaphragms image (Figure 12). Once centered, fully reopen the variable diaphragm.Figure 12 - Plan view of modulator showing image of variable diaphragm which has been centered.8. Select the slit aperture that matches the HMC objective (Example: 10X HMC objective/10X slit aperture). Generally, each slit is mounted on a holder which can be individually rotated and moved laterally (Figure 3B). 9. While observing through the telescope or bertrand, focus on the slit image now seen at the objectives back focal plane (Figure 13). There are two parts to the variable contrast slit image. One part always transmits light (nonvariable portion). The transmission of the second part (P2) is determined by the setting of the variable HMC contrast control polarizer (P1) which will be placed at the microscopes light source (Figure 5A). 8. Figure 13 - Plan view of modulator showing slit image superimposed (incorrect alignment).10. Rotate the slit holder so that the long axis of the slits image is parallel to the grey region (Figures 14A, 14B).Figure 14A - Slit image (typical of the unpredictable orientation of the slit image relative to the grey region of the modulator).Figure 14B - Slit image (rotated so that the slit image is parallel to modulators grey region).11. Move the slit holder laterally so that the nonvariable portion of the slit image is registered within the grey region of the modulator (not the P2 section). P2, the transmission of which is controlled by the polarizer, will extend into the clear region of the modulator (Figure 15). It is critical for correct operation of the HMC system that the nonvariable portion of the slit be completely contained within the grey portion of the modulator (see Section III #1 - Working Distance of HMC Condensers).Figure 15 - Plan view of objectives back focal plane showing slit image superimposed on modulator.12. Install the contrast control polarizer, (Figure 5A). Rotate the contrast control polarizer and notice that only the P2 section of the slit image changes intensity (Figure 16). 9. Figure 16 - Plan view of modulator showing minimum and maximum contrast.13. Adjust the contrast control polarizer for maximum contrast (setting it so that no light passes P2 of the slit aperture - Figure 16). 14. If an additional HMC objective and corresponding slit aperture is to be aligned: a. Rotate nosepiece to position the objective in the optical path. b. Rotate HMC condenser turret to the brightfield (BF) position. c. Observing through the telescope or bertrand, focus on the modulator at the back focal plane of the objective. d. Rotate the HMC condenser turret to the HMC slit aperture that matches the HMC objective in use. e. Repeat steps 10 and 11. Follow above for all HMC objectives on the microscope. 15. Replace the telescope with the eyepiece, or disengage the bertrand viewing system. After completing the above installation procedure, your HMC microscope should produce three dimensional appearing images of unstained, rounded objects wherein one side appears dark, the other side bright with grey in between, against an evenly illuminated grey background (Figure 17). 10. Figure 17- Photomicrograph of HMC image.Note: 1. Maximum contrast is attained when the polarizer is adjusted so that P2 of the slit image is completely darkened. 2. If the microscope has an adjustable lamp housing, it may be necessary to adjust the lamp filament to attain maximum and uniform illumination of the HMC image (see Section III #3 - Filters). 3. When viewing objects that are rounded or where there are large refractive index differences between the specimen and the surrounding medium, rotate the contrast control polarizer to attain optimum contrast and image sharpness. 4. Finally, we recommend the use of a light green or light blue