step-by-step instructions for building a ......6. take a square mounting plate (#85-737) and slide...

STEP-BY-STEP INSTRUCTIONS FOR

BUILDING A FLUORESCENCE

MICROSCOPE

TECHSPEC®

Optical Cage System

PROPRIETARY - Property of Edmund Optics, Inc. | 2012 Copyright© Edmund Optics, Inc.

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.


OVERVIEW 3

Complete Parts List

Filter Selection

Sub Assemblies:

1. Dichroic

Assembly

2. Objective Arm

3. Source Arm

4. Imaging Arm

Mounting & Alignment


COMPLETE PARTS LIST 4

STOCK # Description QUANTITY 85713 25.4mm Dia. Adapter for 85710 1

85710 Square Tip/Tile Optic Mount 1

Depends on Application 25mm Dia. Dichroic Filter 1

85624 Cage Sphere with (5) 30mm Port and (1) 43mm Port 1

85632 25mm Standard Cage Plate with M6 for Post Mounting 3

38944 0.316” ID, Fiber Optic Adapter SX-6 2

56371/56372

115V AC 50/60HZ, MH-100 Illuminator/220V AC 50/60HZ, MH-100

Illuminator 1

42346 1/4" x 24”, Flexible Fiber Optic Light Guide 1

85588 30mm Cage 25/25.4mm Diameter Thick Lens Mount 1

85737 30mm Square Interface Plate 5

85719 30mm Cage Plate C-Mount Adapter 3

55743 Mitutoyo to C-Mount 10mm Adapter 1

46143 5X Mitutoyo Plan Apo Infinity-Corrected Long WD Objective 1

85488 Cage Support Rod 6mm Diameter x 100mm Length, M3 4


85490 Cage Support Rod 6mm Diameter X 150mm 4

85630 30mm Standard Cage Plate with M6(1 for Post Mounting) 3

86018 Filter Holder Plate 2

85649 Interchangeable Filter Holder 2

Depends on Application Fluorescence Bandpass Filters (see details) 2

54774 MT-1 Accessory Tube Lens 1

58329 Mitutoyo MT-1/MT-2 C-Mount Adapter 1


54633 C-Mount Extension Tube (100mm Length) 1


03625 C-Mount Fine Thread Extension Tube (30mm-50mm Length) 1

65553 25mm Dia. 25mm Efl. Achromatic Lens 1

66014 25mm Dia. 20mm Efl. Aspheric Lens 1

59367 EO-3112C ½” CMOS Color USB Camera 1

59382 5m USB2.0 (mini-B to standard-A) Cable 1

85618 Micrometer Mount Kit 1

58661 Micrometer Head Anvil 13mm 1


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.


1. DICHROIC ASSEMBLY

6


DICHROIC ASSEMBLY 7

STOCK # Description QUANTITY 85713 25.4mm Dia. Adapter for 85710 1

85710 Square Tip/Tile Optic Mount 1

N/A 25mm Dia. Dichroic of choice (see details under filter) 1

85624 Cage Sphere with (5) 30mm Port and (1) 43mm Port 1


85737 30mm Square Interface Plate 1

86018 Filter Holder Plate 2

Parts needed for this step:


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.


2. OBJECTIVE ARM

9


OBJECTIVE ARM 10

STOCK # Description QUANTITY 85737 30mm Square Interface Plate 2

55743 Mitutoyo to C-Mount 10mm Adapter 1

46143 5X Mitutoyo Plan Apo Infinity-Corrected Long WD Objective 1

85719 30mm Cage Plate C-mount Adapter 1

85490 Cage Support Rod 6mm Diameter X 150mm 4

85488 Cage Support Rod 6mm Diameter x 100mm Length 2

85-618 Micrometer Mount Kit 1

58-661 Micrometer Head Anvil 13mm 1



OBJECTIVE ARM 11

4. 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.


OBJECTIVE ARM 12

9. 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 9.

11. 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.


3. SOURCE ARM

13


SOURCE ARM 14

STOCK # Description QUANTITY


38944 0.316” ID, Fiber Optic Adapter SX-6 2

85588 30mm Cage 25/25.4mm Diameter Thick Lens Mount 1



65553 25mm Dia. 25mm Efl. Achromatic Lens 1

66014 25mm Dia. 20mm Efl. Aspheric Lens 1



SOURCE ARM 15

12. 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.


4. IMAGING ARM

16


IMAGING ARM 17

PART # Description QUANTITY 85737 30mm Square Interface Plate 2

85719 30mm Cage Plate C-Mount Adapter 2


54774 MT-1 Accessory Tube Lens 1

58329 Mitutoyo MT-1/MT-2 C-Mount Adapter 1




03625 C-Mount Fine Thread Extension Tube (30mm-50mm Length) 1

59367 EO-3112C ½” CMOS Color USB Camera 1



IMAGING ARM 18

13. 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.


IMAGING ARM 19

16. 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.


MOUNTING & ALIGNMENT

20


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.


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.


START BUILDING NOW! 23

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step-by-step instructions for building a ......6. take a square mounting plate (#85-737) and slide...

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