IncuCyte Training Calibrations and Troubleshooting Distributor Training 3-8-2016 Benjamin D. Bauknecht

IncuCyte Features IncuCyte Components Recap Simply Complex X,Y and Z movement Sits in warm wet environment 24/7 Networked Multi User 3 Tray positions More than one experiment at once IncuCyte Components 3 Motors 2 Circuit boards Expensive camera Sensors, switches and LEDs Server computer MCU RAID 5

How Does the IncuCyte know where to move and quantify data How Does the IncuCyte know where to move and quantify data? Calibrations and Tests (4x, 10x, 20x)

Optics Test (5’, 4’, 6’) Needed materials: Checks the optical pathway Calibration Tray Calibration Slide Checks the optical pathway LED voltages Light Field Dirt Detection Optical Rotation HD Foci Test not calibration Only reads, does not write

Optics Test Order of Operation 2 LED Voltages Moves to open window too check LED voltages and to adjust exposure time Optical Rotation Moves to Window 1 on Cal tray to assess rotation using edge of window. X and Y Light Field Moves back to open window to test Light Field Compares all 16 sections vs the average Dirt Detection Uses Light Field image Runs Algorithm to check for dust or debris that is in the viewing area 1, 3 and 4 Light Field image

Optics Test Order of Operation (Continued) 1 Optics Test Order of Operation (Continued) HD Foci Test Uses calibration Slide to determine optical flatness Focuses on second layer of scratches Cuts image into 108 pieces and determines optical flatness to

Optics Test Results 6 1 2 3 7 4 5 LED Check Exposure Light Field Dirt Detection Best results at 20x Rotation HD Foci Test Filter Details 6 1 2 3 7 4 5

Optics Test Common Failures LED Voltages Change LED Light Field Adjust Phase LED over objective Possible debris Dirt Detection Clean optics path or replace camera if necessary Optical Rotation Drawer alignment HD Foci Flip Calibration Slide Rotate tube lens

Spatial Calibration (F:6’/17’, M:6’/19’, R:6’/21’) Required Material Calibration Tray Maps Drawer Uses “frosted window” on Calibration tray Uses window and edges to find location Done on each window on each tray position (12 total) Kicks the Tires Confirm step tests to see if the movement is repeatable with minimal variation Belt Calibration Tests in each objective to see if system can accurately make incremental movement Calibration not test Writes all values found and saves for reference in Calib.txt

Spatial Calibration Order of Operation Window 1 Finds Z height (Focus) Sweeps to find edge of window in X and Y In X, move to home, move close to edge of square Move back and forth from home ~5 times to get a measure in steps of edge Transition in middle of image Repeats process in Y Repeat in window 2-4

Spatial Calibration Order of Operation (Continued) Confirm Tests repeatability of system to find edge. 20 reps in x, y, z each on Window 1 and Window 2 Only done on one objective Belt Calibration Uses track Does incremental movement to test precise movement Done on all three objectives Y Track Y Track X Track

Spatial Calibration Results Looks at drawer offset Rotation Drawer level Belt Calibration 1-3 4

Spatial Calibration Common Failures Standard Deviation Check belt tension Height in Z too high/low Drawer level is required Still not passing? Was the Zoom off for a long period of time? 2+ weeks Replace belts Gained “memory”

Fluorescence Calibration (5’,6’,5’) Required Materials Calibration Tray Calibration Slide 40µl of Essen Dual Color Calibration Fluid Loaded into Calibration Slide Teaches Zoom on Fluorescence (FLR) intensity Evens out FLR image in GUI Better reporting 40-52 micron channel in calibration slide Must have spatial calibration performed beforehand for best results Objective specific Calibration Fill Fluid Here

Fluorescence Calibration Order of Operation Ambient Light check Looks to see how much light bleeds through in the optics path Channel Depth measurement Records average depth in a 4x4 square on Calibration slide Uses depth to calibrate intensity Multiple exposures Averages for intensity Mean Signal Rate Corners are % of Mean

Fluorescence Calibration Results 1 Ambient Channel Depths Green Channel Summary Red Channel Summary Green Corners Red Corners 3 4 2 5 6

Fluorescence Calibration Common Failures Unable to find channel depth Clean calibration slide Mean Signal Rate/Corners for Green/Red falls below threshold Adjustments required to even out light Up/Down adjustment L/R Adjustment Mean signal and corners

Test and Calibrations Review Optics Checks optical path Light Field HD Foci Dirt Spatial Finds X, Y, Z location Accuracy and Precision Fluorescence Calibrates system to light field Red and Green