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.

TABLE OF CONTENTS

SECTION I Introduction

SECTION II A Installation and Alignment of HMC on Inverted Microscopes

SECTION II B Installation and Alignment of HMC on Upright Microscopes

SECTION III Technical Information

1. Working Distance of HMC Condensers

2. Cover Slip Correction Objectives

3. Filters

4. Reflections at the Back Focal Plane

5. Kohler Illumination and HMC Condensers

6. Troubleshooting Chart

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.

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 microscope

Figure 5B - HMC components on an upright microscope.

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).

Figure 8 - Condenser working distance.

Figure 9 - HMC condenser lens cells.

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

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).

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 objective’s 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).

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).

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 filter to optimize the image contrast.

If you have any questions, call:

MODULATION OPTICS INC. Customer Support Department

(516) 484-8883

SECTION II B - Installation and Alignment of HMC on Upright Microscopes

1. Before attempting to install the HMC system, review the microscope manufacturer's 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 upright microscope. An addendum may accompany this booklet with detailed information on your specific microscope.

3. Install condenser onto microscopes condenser carrier.

4. Rotate the turret plate of the HMC condenser system to the brightfield (BF) position so that no slit aperture or phase ring is in the optical path. Note: Make sure the aperture diaphragm located at the brightfield position is fully open.

5. Install HMC objective(s) onto microscope's nosepiece and select the lowest magnification HMC objective. Focus on the specimen. Note: It is helpful to use a stained specimen during this initial setup.

6. Attaining the correct condenser setting: (See Technical Information Section III #1 - Working Distance and III #5 - Kohler Illumination and HMC Condensers).

a. Partially close the microscope's field aperture. Raise or lower the condenser as required to obtain a focused image of the field aperture on the specimen plane.

b. Use the microscopes built-in condenser centering screws to center the condenser lens on the optic axis by centering the field apertures image (Figure 18). Fully reopen the field aperture once it is centered.

Figure 18 - Centering field aperture image.

c. Position the front face of the condenser lens at its nominal working distance from the specimen plane (Figure 19). The nominal

working distance (WD) is marked on the HMC condenser lens cell (Figure 20).

Figure 19 - Condenser working distance

Figure 20 - HMC condenser systems with different lens cells.

7. Move the stage so that the specimen is out of the field of view.

8. Using an auxiliary telescope or bertrand lens, focus on the modulator (Figure 21) at the back focal plane of the HMC objective. Note: The modulator may be in a different rotational orientation dependent on the microscopes nosepiece thread.

Figure 21 - Plan view of modulator.

9. Select the slit aperture that matches the HMC objective (Example: 10X HMC objective/10X slit aperture). The slit is either on a single insert in the condenser housing or in a turret plate (Figure 22). Generally, each slit is mounted in a holder. This is installed in a spring clip which can be individually rotated and moved laterally.

Figure 22 - HE Condenser

10. Observing through the auxiliary telescope or bertrand lens, focus on the slit image now seen at the objectives back focal plane (Figure 23). 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 microscope's light source (Figure 5B).

Figure 23 - Plan view of modulator showing slit image superimposed (incorrect alignment).

11. Rotate the slit holder so that the long axis of the slit’s image is parallel to the grey region (Figure 24A, 24 B).

Figure 24A - Slit image (typical of the unpredictable

orientation of the slit image relative to the grey region of the modulator).

Figure 24B - Slit image (rotated so that the slit image is

parallel to modulator's grey region).

12. 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 25). 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 25 - Plan view of objective's back focal plane showing slit image superimposed on modulator.

13. Install the contrast control polarizer (Figure 5B). Rotate the contrast control polarizer and notice that only the P2 section of the slit image changes intensity (Figure 26).

Figure 26 - Plan view of modulator showing minimum and maximum contrast.

14. Adjust the contrast control polarizer for maximum contrast (setting it so that no light passes P2 of the slit aperture - see Figure 26).

15. 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 11 & 12.

Follow above for all HMC objectives on the microscope.

16. 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 27).

Figure 27- Photomicrograph of HMC image.

Note:

1. Maximum contrast is attained when the polarizer is adjusted so that the P2 section of the slit image is completely darkened.

2. If the microscope has an adjustable lamp housing, it may 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 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 to optimize the image contrast.

If you have any questions, call:

MODULATION OPTICS INC. Customer Support Department

(516) 484-8883

SECTION III - TECHNICAL INFORMATION

1. WORKING DISTANCE OF HMC CONDENSERS

The working distance specification of your HMC condenser system is a nominal value. Working distance is defined as the distance between the front face of the condenser lens housing and the specimen plane. This dimension is adequate for initial system set-up, but may require fine-tuning in your application. The precise setting must accommodate the fact that the refractive index and thickness of the media between the object and the condenser affect its working distance. Media is herein defined as the material through which the light passes, be it in air, glass, plastic, liquid or combination of these.

In modulation contrast, the width of the slit image should be just slightly smaller than the width of the grey region of the modulator. In phase contrast, the light annulus image match to the phase ring in the objective is generally not critical. However, the light ring image should be completely contained within the objectives phase ring.

If, in your particular application, the slit or annulus image size is modified by the refractive index and/or the media, adjust the height of the condenser to correct the situation.

On inverted microscopes, chambers are often used. Here, the media is the chamber wall thickness, plus the height of any liquid present, plus the air space both inside and outside. On upright microscopes, the total media thickness is usually a 1 mm glass slide and the air between it and the condenser.

2. COVER SLIP CORRECTION OBJECTIVES

Standard objectives are designed to work best with 0.17mm thick cover glass. For some applications the cover glass is thicker (For example, tissue culture chamber walls). These situations require use of special objectives: those designed for a specific coverglass thickness or those which can be adjusted to the application. The latter are often called cover slip correction objectives and are adjusted as follows:

a. Set the correction ring to the thickness of the coverglass.

b. Bring the specimen into sharp focus by means of the microscope's focusing system.

c. Turn the correction ring slightly to the left. As a result, the image will now be slightly out of focus. Use the microscope's fine focus knob I make it as sharp as possible. If the result is better than the

original image, repeat the procedure until the highest image quality is attained. If the image quality is found inferior, turn the correction collar in the opposite direction and re-focus the microscope.

d. Repeat this procedure until the image shows the highest quality

e. NOTE: The correction ring is not intended as a parfocality adjustment device. Doing so often results in reduced image quality.

3. FILTERS

Light Green or Blue Filters

The use of a light green or blue filter is recommended when viewing phase (non-stained) specimens. This type of filter can enhance image sharpness and reduce any residual chromatic aberration to assure viewing comfort during long periods of observation.

Frosted Filters

When HMC condensers are used on some microscopes, it is possible that the filament may be imaged on the specimen plane. In addition, on some microscopes where filament position is non-adjustable or the microscope does not have a provision for moving an auxiliary lens in/out of the optic path, the specimen plane may not appear fully illuminated. In such cases, the difficulty may be overcome by inserting a lightly frosted filter in the optic path.

Heat Filters

As part of the variable contrast feature, HMC uses a plastic polarizing material over a part of the slit aperture. This material is damaged when subject to intense heat. To avoid this potential problem it is necessary to use the heat absorbing filter supplied with your HMC system.

4. REFLECTIONS AT THE BACK FOCAL PLANE OF HMC OBJECTIVES

There are many glass surfaces in the optical train of a microscope. Reflections may appear at the objectives back focal plane. Although these reflections could contribute to stray light at the image plane, in practice it usually has no significant effect and the contrast system maintains a high degree of contrast.

5. KOHLER ILLUMINATION AND HMC CONDENSERS

HMC Condensers are designed to accomplish the primary goals of Kohler Illumination, namely, the efficient production of an evenly illuminated field of view at the required numerical aperture. An HMC

condenser, correctly positioned at its nominal working distance, provides these attributes.

It should be noted that when special purpose condensers are used, for example HMC systems, the image of the field diaphragm on the specimen plane may no longer be an appropriate indicator of Kohler Illumination. HMC condenser systems are clearly labelled with their nominal working distance enabling them to be simply and correctly positioned.

6. TROUBLESHOOTING CHART

PROBLEM SOLUTION

1. Low contrast Contrast control polarizer not in optical path.

Contrast control polarizer needs to be rotated.

Incorrect alignment of slit.

Video Systems:

a. Lamp intensity too low.

b. Monitors contrast and/or brightness incorrect.

2. Uneven illumination in image field.

Slit image not registered correctly.

Filament needs adjustment.

Incorrect slit.

3. Slit image does not register in grey region of modulator.

Wrong slit in optical path.

Condenser not at proper working distance.

Condenser not on optic axis.

Objective not focused on object.

4. Slit image does not fill most of grey region of modulator.

Same as #3.

Notes