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Optical Alignment Procedures

Overview​

The FRAME system requires precise optical alignment to ensure optimal performance. This document provides detailed procedures for aligning the internal optics cube and calibrating the optical path.

Internal Optics Cube Preparation​

Initial Setup Outside the FRAME​

Before installing the internal optics cube in the FRAME, it must be properly aligned using a separate alignment setup.

Components to Align​

The internal optics cube contains three main optical elements that must be aligned:

  1. Transmitted light module with camera and folding mirror
  2. Fluorescence excitation module
  3. Objective mounting system

Alignment Setup​

  1. Create External Test Setup:

    • Remove the internal optics cube from the FRAME
    • Build a test setup on top of the cube that mimics the FRAME's optical distances
    • Mount an objective in the test setup to maintain proper optical spacing

  2. Software Preparation:

    • Start ImSwitch software to enable camera viewing
    • Ensure proper communication with the cube's electronics

Fluorescence Module Alignment​

Köhler Illumination Setup​

  1. Fiber Light Source:

    • Insert a fiber into the fiber holder to act as a point light source
    • This simulates the laser input for fluorescence excitation

  2. Collimating Lens System:

    • The light is collimated by the first lens
    • A second auxiliary lens focuses the light into the back focal plane of the microscope
    • The focus must be positioned at the location of the future objective's back focal plane

  3. Field of View Adjustment:

    • The opening angle/divergence angle at the back focal plane determines the illuminated field of view
    • Adjust this angle to match your sample area requirements

Mechanical Alignment​

  1. Access Adjustment Screws:

    • Use a 2.5mm hex key to access the three adjustment screws on the back of the cube
    • These screws control the position and angle of the optical elements

  2. Laser Centering:

    • Ensure the laser focus is exactly centered where the objective will be positioned
    • The light beam must exit the internal cube parallel to the optical axis
    • This ensures proper Köhler illumination

  3. Objective Installation:

    • Once aligned without objective, install the objective
    • Use a screen to track the beam path outside the objective
    • Safety: Do not look directly into the beam, even with the low-intensity calibration laser

  4. Final Beam Check:

    • The beam must exit the objective centrally and collimated
    • The opening angle should be approximately 0° (parallel beam)
    • The beam bundle should be parallel

FRAME Integration Alignment​

Objective-to-FRAME Alignment​

After internal cube alignment, align the optical axis of the FRAME with the internal cube.

Available Adjustments​

Two adjustment mechanisms are available:

  1. X-axis: Objective can be moved along the X-axis using the slide mechanism
  2. Y-axis: Internal cube can be moved relative to the FRAME using adjustment screws

LED Alignment Tool Setup​

  1. Prepare LED Alignment Tool:

    • Remove the camera from the FRAME
    • Install an LED with C-mount adapter in place of the camera
    • This creates a reference light source for alignment

  2. Pinhole Creation:

    • Use a small pin to create a centered pinhole in front of the LED
    • This creates a point light source for propagation through the optical system

Alignment Procedure​

  1. Initial Positioning:

    • Remove the microscope objective temporarily
    • Turn on the LED alignment tool
    • Observe the point light source projected to the back focal plane

  2. Y-axis Alignment:

    • Adjust the internal cube position along Y-axis using the adjustment screw
    • Move the light point to the center of the back focal plane
    • Use a piece of paper with crosshairs to mark the position

  3. X-axis Alignment (A-axis):

    • Use the electronic controls in ImSwitch to move the objective slide
    • Adjust the A-axis position using the plus/minus controls in the GUI
    • Center the point relative to the back focal plane

  4. Position Storage:

    • Save the A-axis position for both objective slot 1 and slot 2
    • These values are stored permanently in EPROM
    • The system will automatically move to these positions when switching objectives

System Operation​

  • Startup: System homes to physical zero position, then moves to saved position for objective 1
  • Objective Switching: Pressing objective 2 button automatically moves to saved position 2
  • Position Memory: Positions are permanently stored and recalled on each startup

Fluorescence Path Verification​

  1. Test with Laser:

    • Use the actual laser source to test the fluorescence excitation path
    • With objective removed, check focus position at back focal plane
    • The laser spot should be centered like the LED alignment point

  2. Final Verification:

    • Install objectives and verify alignment
    • Test both transmitted and fluorescence illumination paths
    • Confirm proper image quality and illumination uniformity

Final Assembly​

Once all optical elements are properly aligned:

  1. Secure Internal Cube:

    • Tighten the locking knobs to permanently secure the internal cube in the rail
    • Ensure the cube cannot move during operation

  2. System Testing:

    • Perform comprehensive testing of all optical functions
    • Verify image quality across the full field of view
    • Test objective switching and focus mechanisms

Troubleshooting Common Issues​

Poor Illumination Uniformity​

  • Check fiber coupling and position
  • Verify collimating lens alignment
  • Adjust Köhler illumination setup

Image Quality Issues​

  • Verify objective mounting and alignment
  • Check camera sensor alignment
  • Ensure proper back focal distance

Switching Problems​

  • Verify objective position storage
  • Check mechanical alignment of slide system
  • Confirm proper homing sequence

Safety Considerations​

  • Always use appropriate laser safety procedures
  • Never look directly into laser beams
  • Use proper eye protection when working with UV or laser sources
  • Ensure proper ventilation when using cleaning solvents

Maintenance​

  • Regular cleaning of optical surfaces
  • Periodic verification of alignment
  • Check mechanical adjustments for wear
  • Update position calibrations as needed