STEP-BY-STEP INSTRUCTIONS FOR BUILDING A FLUORESCENCE MICROSCOPE. TECHSPEC Optical Cage System

Transcription

1 STEP-BY-STEP INSTRUCTIONS FOR BUILDING A FLUORESCENCE MICROSCOPE TECHSPEC Optical Cage System

2 INTRODUCTION 2 What is a Digital Fluorescence Microscope? Unlike traditional microscopes, which utilize an eyepiece for performing visual inspection, digital video microscopes utilize cameras to record and capture images, providing higher resolution and higher precision than what is typically seen with the human eye. Since infinity-corrected digital video microscopes can be quite complex, using the modular TECHSPEC Optical Cage System is ideal for simplifying the assembly. In this case another aspect has been added to the design to incorporate a fluorescence aspect to the digital microscope. Fluorescence microscopy is ideal for measuring and analyzing the absorption and excitation of various wavelengths of light. An in-line fluorescence microscopy setup utilizes a dichroic filter to redirect light from an illuminator into the perpendicular optical path. Why TECHSPEC Optical Cage System? Building a customized digital fluorescence microscope with TECHSPEC Optical Cage components allows the user to build a microscope quickly and easily with limitless customization options. Objectives can be changed to modify system magnification, and interchangeable filter holders make swapping filters easy. Custom illumination can be added for bright field, dark field, or in-line illumination options. Additionally, a spectrometer could be added for precise spectral measurements.

3 OVERVIEW 3 Complete Parts List Filter Selection Sub Assemblies: 1. Dichroic Assembly 2. Objective Arm 3. Source Arm 4. Imaging Arm Mounting & Alignment

4 COMPLETE PARTS LIST 4 STOCK # Description QUANTITY mm Dia. Adapter for Square Tip/Tile Optic Mount 1 Depends on Application 25mm Dia. Dichroic Filter Cage Sphere with (5) 30mm Port and (1) 43mm Port mm Standard Cage Plate with M6 for Post Mounting ID, Fiber Optic Adapter SX / V AC 50/60HZ, MH-100 Illuminator/220V AC 50/60HZ, MH-100 Illuminator /4" x 24, Flexible Fiber Optic Light Guide mm Cage 25/25.4mm Diameter Thick Lens Mount mm Square Interface Plate mm Cage Plate C-Mount Adapter Mitutoyo to C-Mount 10mm Adapter X Mitutoyo Plan Apo Infinity-Corrected Long WD Objective Cage Support Rod 6mm Diameter x 100mm Length, M Cage Support Rod 6mm Diameter x 50mm Length, M Cage Support Rod 6mm Diameter X 150mm mm Standard Cage Plate with M6(1 for Post Mounting) Filter Holder Plate Interchangeable Filter Holder 2 Depends on Application Fluorescence Bandpass Filters (see details) MT-1 Accessory Tube Lens Mitutoyo MT-1/MT-2 C-Mount Adapter Cage Support Rod 6mm Diameter x 300mm Length, M C-Mount Extension Tube (100mm Length) C-Mount Extension Tube (50mm Length) C-Mount Fine Thread Extension Tube (30mm-50mm Length) mm Dia. 25mm Efl. Achromatic Lens mm Dia. 20mm Efl. Aspheric Lens EO-3112C ½ CMOS Color USB Camera m USB2.0 (mini-b to standard-a) Cable Micrometer Mount Kit Micrometer Head Anvil 13mm 1

5 OPTICAL FILTERS 5 A basic fluorescence microscope requires 3 filters. Each application will use a different combination depending on the system requirements: Excitation Filter: placed within the illumination path of a fluorescence microscope. It filters out all wavelengths of the light source except for the excitation range of the fluorophore or specimen under inspection. Dichroic Filter: placed between the excitation filter and emission filter at a 45 angle. It reflects the excitation signal towards the fluorophore under inspection and transmits the emission signal towards the detector. Emission Filter: placed within the imaging path of a fluorescence microscope. It filters out the entire excitation range of the fluorophore under inspection and transmits the emission range of the fluorophore.

6 6 1. DICHROIC ASSEMBLY

7 DICHROIC ASSEMBLY 7 Parts needed for this step: STOCK # Description QUANTITY mm Dia. Adapter for Square Tip/Tile Optic Mount 1 N/A 25mm Dia. Dichroic of choice (see details under filter) Cage Sphere with (5) 30mm Port and (1) 43mm Port mm Standard Cage Plate with M6 for Post Mounting mm Square Interface Plate Filter Holder Plate 2

8 DICHROIC ASSEMBLY 8 1. Carefully insert the 25mm Dia. Dichroic Filter into the 25.4mm Dia. Adapter (#85-713). Lock into place with the M3 set screws. Insert the adapter into the Square Tip/Tilt Optic Mount (#85-710) and secure. 2. Attach the 25mm Standard Cage Plate (#85-632) to the Cage Sphere (#85-624) opposite the 43mm port with M3 SHCS. Slide the base of the tip/tilt assembly from step 1 into the sphere and secure within the plate. (Alignment will be done later). 3. Attach two Filter Holder Plates (#86-018) with M3 SHCS to two adjacent sides of the sphere. Keep the the openings of the plates facing the 43mm port. Attach a Square Mounting Plate (#85-737) to a side opposite of a filter holder plate.

9 9 2. OBJECTIVE ARM

10 OBJECTIVE ARM 10 Parts needed for this step: STOCK # Description QUANTITY mm Square Interface Plate Mitutoyo to C-Mount 10mm Adapter X Mitutoyo Plan Apo Infinity-Corrected Long WD Objective mm Cage Plate C-mount Adapter Cage Support Rod 6mm Diameter X 150mm Cage Support Rod 6mm Diameter x 100mm Length Micrometer Mount Kit Micrometer Head Anvil 13mm 1

11 OBJECTIVE ARM Thread the C-Mount Adapter (#85-719), Mitutoyo Adapter (#55-743), and Mitutoyo Objective (#46-143) together. Then secure the C-Mount Adapter part of the assembly within the square plate with M3 set screws. 5. Insert the four 150mm long rods (#85-490) into the outer rod holes of the square plate and secure with M3 set screws. 6. Take a Square Mounting Plate (#85-737) and slide it onto the 150mm long rods. Lock the plate onto the rods approximately 20mm from the end of the objective. 7. Insert two100mm long rods (#85-488) into two adjacent inner rod holes of the square plate from step 6. Keep the ends flush with the plate and secure with M3 set screws. 8. Slide the 2 springs from the micrometer assembly onto the 100mm long rods.

12 OBJECTIVE ARM Attach the stainless steel plate (not shown) to a Square Mounting Plate (#85-737) with M3 SHCS. Slide the square plate onto the rods with the steel plate between the100mm long rods and facing away from the springs. Keep the plate loose on the rods. 10. Mount the micrometer (#58-661) in the micrometer holder. Slide the micrometer assembly onto the two 150mm rods. The micrometer should be between the two 100mm long rods Be sure the micrometer is in the half extended position and the shaft contacts the stainless steel plate from step Push the micrometer assembly and loose square plate towards the secured square plate compressing the springs to approximately ¾ their uncompressed length. Lock the micrometer assembly on the rods with M3 set screws keeping the springs under tension. The loose square plate should now translate when the micrometer is adjusted. This loose square plate will serve as the microscope stage.

13 13 3. SOURCE ARM

14 SOURCE ARM 14 Parts needed for this step: STOCK # Description QUANTITY mm Standard Cage Plate with M6 for Post Mounting ID, Fiber Optic Adapter SX mm Cage 25/25.4mm Diameter Thick Lens Mount Cage Support Rod 6mm Diameter x 100mm Length, M mm Standard Cage Plate with M6 for Post Mounting mm Dia. 25mm Efl. Achromatic Lens mm Dia. 20mm Efl. Aspheric Lens 1

15 SOURCE ARM Insert the 25mm Dia. x 25mm FL Achromatic Lens (#65-553) into a 25mm Cage Mounting Plate. Secure with M3 set screws. 13. Insert the 25mm Dia. x 20mm FL Plastic Aspheric Lens (#66-014) into another 25mm Cage Mounting Plate. Secure with M3 set screws. 14. Place one of the Fiber Adapters (#38-944) into the 25mm Thick Lens Mount (#85-588) and secure with retaining ring. Insert the assembly into a 30mm Cage Mounting Plate (#85-630). Secure with M3 set screws. 15. Insert two 100mm long rods onto the filter holder next to the microscope objective. Secure with M3 set screws. 16. Slide the plates from steps 12, 13, and 14 onto the rods in order of assembly: Achromatic Lens, Aspheric Lens, then Fiber Adapter. Make sure the more convex surfaces of the lenses facing the fiber adapter. 17. Temporarily secure the plates on the rods for later alignment. Note: there is an extra plate shown in these images between the achromat and filter holder. This is optional for mounting purposes.

16 16 4. IMAGING ARM

17 IMAGING ARM 17 Parts needed for this step: PART # Description QUANTITY mm Square Interface Plate mm Cage Plate C-Mount Adapter Cage Support Rod 6mm Diameter x 50mm Length, M MT-1 Accessory Tube Lens Mitutoyo MT-1/MT-2 C-Mount Adapter Cage Support Rod 6mm Diameter x 300mm Length, M C-Mount Extension Tube (100mm Length) C-Mount Extension Tube (50mm Length) C-Mount Fine Thread Extension Tube (30mm-50mm Length) EO-3112C ½ CMOS Color USB Camera 1

18 IMAGING ARM Secure the MT-1 Tube Lens (#54-774) within the Tube Lens C-Mount Adapter Assembly (#58-329). 14. Thread a Cage C-Mount Adapter (#85-719) onto the female threading of the tube lens assembly. Insert the adapter into the 30mm aperture of one of the Square Plates (#85-737) and secure in place with M3 set screws. 15. Thread the C-Mount Fine Thread Extension Tube, 100mm C-mount Extension Tube, and 50mm C-Mount Extension Tube (#03-625, #54-632, #54-633) onto the male threading of the tube lens assembly.

19 IMAGING ARM Thread the remaining C-Mount Adapter (#85-719) onto the end of the tube assembly. Slide a Square Plate (#85-737) onto the C-mount adapter. 17. Thread the C-Mount Camera (#59-367) onto the C-Mount Adapter after the square plate. 18. Insert all four 300mm Long Rods (#85-492) through the rod holes in the Square Plates. Keep the ends flush with the plate connected to the tube lens. Secure rods with M3 set screws. 19. Use two 50mm Long Rods (#85-486) to connect the Square Plate attached to tube lens to the Filter Holder Plate opposite the objective on the sphere. Secure rods with M3 set screws.

20 20 MOUNTING & ALIGNMENT

21 MOUNTING 21 Adding two standard cage plates to the microscope assembly make it easy to mount this microscope either horizontally or vertically: 1. Add a plate to the source arm between the achromatic lens and the filter holder. The second can be placed along the 50mm rods between the filter holder and tube lens. (make sure the M6 or ¼-20 mounting holes are facing the same direction. 2. Attach mounting posts to the two new plates and adjust the placement of the plates to match the breadboard or bench plate spacing.

22 ALIGNMENT 22 Rotate the tip/tilt mount from step 1 so the dichroic filter reflects the light source toward the camera (instead of toward the objective). Adjust the location of the achromatic lens and aspheric lens until the fiber optic light guide is in focus. Lock the location of the plates on the rods. Rotate the tip/tilt mount again so the dichroic filter reflects the source through the objective onto the sample. Adjust the angle until the focused spot is in the center of the sample area. Using the micrometer, find the smallest focused spot. Once the microscope is assembled and aligned, the excitation and emission filters can be secured within two Interchangeable Filter Holders (#85-649). The Filter Holders slide into the filter holder plates for inspection.

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