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Modulation contrast setup


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  • 1. Modulation Contrast Modulation Contrast User Manual Document no. 6-81-500UM, Issue 1, ECN822, 22nd August 2011 Research Instruments Ltd Bickland Industrial Park, Falmouth, Cornwall TR11 4TA, U.K. Tel: +44 (0) 1326 372 753 Fax: +44 (0) 1326 378 783 E-mail: website:
  • 2. Modulation Contrast
  • 3. Modulation Contrast Contents Introduction���������������������������������������������������������������������������������������������������� 2 Summary�������������������������������������������������������������������������������������������������������� 3 Overview�������������������������������������������������������������������������������������������������� 3 Components��������������������������������������������������������������������������������������������� 3 Set-up Summary��������������������������������������������������������������������������������������� 3 Tools required������������������������������������������������������������������������������������������� 3 Assembly�������������������������������������������������������������������������������������������������������� 4 Condenser assembly (all systems)������������������������������������������������������������ 4 Fit condenser to microscope�������������������������������������������������������������������� 4 Fit objectives�������������������������������������������������������������������������������������������� 4 Fit IMC slider (Leica IMC systems)������������������������������������������������������������ 4 Adjust height (Modulation Optics condensers)���������������������������������������� 5 Adjust height (Nikon, Olympus, Leica and Zeiss condensers)������������������� 5 Alignment������������������������������������������������������������������������������������������������������� 5 Centre condenser (all systems)���������������������������������������������������������������� 6 Align slit apertures (all systems)��������������������������������������������������������������� 7 Align slit apertures (Modulation Optics systems)������������������������������������� 8 Align slit apertures (Leica S40 on DMIx000B)������������������������������������������ 9 Align slit apertures (Nikon NAMC and Olympus RC condensers)����������� 10 Correction collars����������������������������������������������������������������������������������� 10 Notes on Use������������������������������������������������������������������������������������������������ 11 Support��������������������������������������������������������������������������������������������������������� 12 1
  • 4. Modulation Contrast Introduction Please read this manual carefully to ensure that the modulation contrast system will work correctly and give the expected image quality. Please also read the manuals supplied with the original equipment. This manual is designed to summarise modulation contrast installation and to provide specific details relevant to IVF use. The information in this manual is current at the time of publication. Our commitment to product improvement requires that we reserve the right to change equipment, procedures and specifications at any time. In no event does Research Instruments assume the liability for any technical or editorial errors, or for any damages arising therefrom. © This manual is protected by copyright, all rights reserved, and no part hereof may be photocopied or reproduced in any form without the prior written consent of Research Instruments. “Hoffman Modulation Contrast” and “HMC” are trademarks of Modulation Optics Inc. “Relief Contrast” is a trademark of Olympus. “Nikon Advanced Modulation Contrast” is a trademark of Nikon. “Integrated Modulation Contrast” is a trademark of Leica Microsystems. 2
  • 5. Modulation Contrast Summary Overview Modulation contrast is a method for increasing the contrast of transparent specimens, giving them a 3D appearance. The 3D effect is artificial, and does not indicate actual geometry. Hoffman Modulation Contrast (HMC) systems are manufactured by Modulation Optics Inc. Systems using the modulation contrast technique are also manufactured by the major microscope manufacturers under different names, for example Olympus Relief Contrast, Nikon Advanced Modulation Contrast (NAMC), Leica Integrated Modulation Contrast (IMC). Components Field aperture Polariser Slit aperture Condenser lens Objective Modulator (inside objective) Set-up Summary • Assemble condenser and fit to microscope • Adjust condenser height • Centre condenser • Align slit apertures • Align correction collars Tools required A basic toolkit as supplied by RI is sufficient to install most systems. A centring telescope suitable for the microscope model will make alignment easier for microscopes not fitted with Bertrand lenses (see page 7). 3
  • 6. Modulation Contrast Assembly Condenser assembly (all systems) Follow the instructions supplied with the specific system. In general, all components provided must be used. The condenser lens may be supplied separate to the condenser turret, and is usually screwed in place. Fit condenser to microscope Follow the instructions supplied with the condenser or microscope. Typically, the condenser unit fits into a mount attached to the illumination pillar and is fixed with a locking screw. Fit objectives Modulation contrast requires special objectives that are matched to the condenser. Suitable objectives are supplied with the modulation contrast system. Fit the objectives into the nosepiece in order of increasing magnification. Fit IMC slider (Leica IMC systems) The microscope stand must be fitted with the port for the slider in the left hand side underneath the eyepieces. This port cannot be retrofitted, so contact your Leica distributor for advice if necessary. 4
  • 7. Modulation Contrast Alignment Adjust height (Modulation Optics condensers) Use the height card provided to set the condenser at the correct height. The correct height is the working distance figure marked on the side of the condenser. The example shown is 45mm. Do not use the Köhler method as described below. Adjust height (Nikon, Olympus, Leica and Zeiss condensers) Follow the instructions supplied with the condenser or microscope. Typically, systems from microscope manufacturers are set up using the Köhler method as follows. Focus on the top surface of a Petri dish. Select a brightfield position on the condenser turret. Close the field aperture diaphragm on the microscope until the bright area becomes smaller than the field of view. 5
  • 8. Modulation Contrast Alignment Adjust the height of the condenser until the edge of the diaphragm comes into sharp focus. Centre condenser (all systems) Leica S40 systems are factory centred, and have no adjustment. Other systems typically have two centring screws. Adjust the screws to move the aperture to the centre of the field of view (1) and (2). Increase the size of the aperture until it nearly fills the field of view (3), and adjust the centring if necessary. Finally, open the aperture fully. 1 2 6 3
  • 9. Modulation Contrast Alignment Align slit apertures (all systems) Each slit aperture must be aligned to the modulator in the corresponding objective. Select the lowest magnification modulation contrast objective (usually 20x), and focus on the top surface of a Petri dish. Select the matching position on the condenser turret. There are three options for viewing the slit apertures. In order of preference: 1. If the microscope has a “B” (Bertrand lens) position. On Olympus microscopes this may be labelled as “CT”. This is usually on a control below the eyepieces, and has an adjustable focus control. Select the B position and adjust the focus control to bring the slit apertures into focus. (See next page for slit aperture images.) Note: do not adjust the microscope focus. B Focus B Example: Nikon Ti 2. Use a centring telescope. These are available from the microscope manufacturer or can be supplied by RI or your distributor. Remove one of the eyepieces and fit the centring telescope instead. Turn the top of the telescope to adjust the focus. 3. Remove one of the eyepieces, and look straight down the tube. The slit aperture will be visible, but the image will be smaller than with methods 1 and 2. Higher magnification objectives give smaller images. 60x objectives may be difficult to set up by this method. 7
  • 10. Modulation Contrast Alignment Align slit apertures (Modulation Optics systems) Remove the oval cover plate and align the slit aperture using your fingers. Rotate and slide the slit aperture until it is aligned to the modulator in the objective, which appears fixed. Note: the modulator will not necessarily appear at the bottom right as shown below. The overall orientation is not important, what is important is that the slit aperture is aligned to the modulator. 1 2 Replace the oval cover plate. 8 3 Correct alignment
  • 11. Modulation Contrast Alignment Align slit apertures (Leica S40 on DMIx000B) The rotational alignment is set at the factory and should not need further adjustment. Pull fully out for B position Adjust B focus (3mm hex key) Adjust modulator position The modulator should be adjusted by rotating the knob shown above until the modulator is aligned to the slit aperture. 1 2 3 Correct alignment 9
  • 12. Modulation Contrast Alignment Align slit apertures (Nikon NAMC condensers) The slit aperture appearance is the same as in the Modulation Optics systems. However, the adjustment is carried out using a hex key instead of moving the slit aperture directly. Centring adjustment Centring adjustment Rotation adjustment Correction collars Plan Fluorite objectives are usually fitted with coverslip correction collars. These must be in the correct position to obtain the best image quality. Correction collar 10
  • 13. Modulation Contrast Alignment Start by turning the collar to align the scale to the nominal thickness of the Petri dish (typically 1.0mm). Note that some objectives have a fixed cap which provides some correction, so the correction collar should be set such that the sum of the two is 1.0mm. For example, if the cap shows a correction of 1.1mm, then the collar scale should be set to -0.1. Fine adjustment may be needed each time a new sample dish is used. Bring the sample into the field of view. Turn the correction collar slightly and refocus. If the image appears better then try a further slight adjustment. If the image is worse then try a small adjustment in the other direction. The focus must be adjusted each time the correction collar is moved. Notes: Correction collars are not intended to as parfocality adjustments. Using the collar in this way may lead to poor image quality. The correction collar is easy to move by mistake when changing objectives. Notes on Use For the best image quality, systems must be set up following the instructions exactly. Misalignments have a greater effect on the image quality with higher magnification objectives. IMSI systems using 60x objectives are particularly sensitive to small errors in alignment. Rotate the polariser on the top of the condenser to adjust the amount of contrast. Avoid mixing parts from different systems, even if they appear the same. Modulators and slit apertures are carefully designed to match a particular condenser. 11
  • 14. Modulation Contrast Support If you have any questions that have not been covered by this manual then contact your distributor or RI’s Service Team. Research Instruments Ltd Bickland Industrial Park, Falmouth, Cornwall TR11 4TA U.K. email: Tel: +44 1326 372 753 Note: contact us for advice before returning any goods. Returned goods will not be accepted without prior agreement. 12