Microscope bugs

M4

M2

Microscope 2

Power measurement

15/02/24

02/06/20 (european "normal" expression: d/m/y)

Measurements before fiber were done right before the lens and after the polarization beam splitter.

Fixing MM2 errors

Easy fixes

Booster clip needs to be reset

If you get this message, click on ok and in the Micro-Manager menu "Configuration", load a new configuration: the "booster to normal" configuration. After loading, close Micro-Manager. Start the Toptica software (the red dot), connect to the Booster, click on reset clip and quit the software. Start Micro-Manager 2 again and load the normal M2 configuration.

COMport error

Micro-Manager 2 often gives COMport errors on M2. Just try to "reload the hardware configuration" from the MM2 Configuration menu. In most cases it works! If the error changes (different COMport number), try again. If the error is the same, there might be an actual error with the device.

BFP slider not working

It might happen, that the BFP slider sometimes freezes and does not move the lens in position. This is because the device that controls it is entering some error state and cannot move anymore. To solve this:

• Close MicroManager
• Open ELLO software (look for it using the search function in start menu)
• connect to COM12 (top left)
• When the slider control menu opens, click "Home"
• The issue should now be solved. 

You can now reopen MicroManager and resume your imaging session.

Camera presets

# Labels

# Configuration presets
# Group: Camera
# Preset: Conventional
ConfigGroup,Camera,Conventional,Evolve512,Gain,1
ConfigGroup,Camera,Conventional,Evolve512,MultiplierGain,1
ConfigGroup,Camera,Conventional,Evolve512,PMode,Frame Transfer Parallel Clocking
ConfigGroup,Camera,Conventional,Evolve512,Port,Normal
ConfigGroup,Camera,Conventional,Evolve512,ReadoutRate,5MHz 16bit

# Preset: EM100 10MHz
ConfigGroup,Camera,EM100 10MHz,Evolve512,Gain,1
ConfigGroup,Camera,EM100 10MHz,Evolve512,MultiplierGain,100
ConfigGroup,Camera,EM100 10MHz,Evolve512,PMode,Frame Transfer Parallel Clocking
ConfigGroup,Camera,EM100 10MHz,Evolve512,Port,Multiplication Gain
ConfigGroup,Camera,EM100 10MHz,Evolve512,ReadoutRate,10MHz 16bit

Ports

PI Settings

Installation

Once only:

• map analog input voltage to µm for external sensor:  0…10V = -1…+1µm (permanent memory):
  Sensor Mech. Correction 1: 0x02000200 = 1
  Sensor Mech. Correction 2: 0x02000200 = +/- 0,01
• read out soft limits of the piezo (needed to go back to full range):
  Soft Voltage Low Limit: 0x0c000000 à SPA? 1 0x0c000000
  Soft Voltage High Limit: 0x0c000001 à SPA? 1 0x0c000001

• The password is "advanced"

External/Internal sensor

Switch to external sensor:

1. closed loop, internal sensor: move to focus. External sensor needs to have signal then
2. get piezo voltage (VOL? 1) -> =Vx
3. switch off servo (piezo voltage stays at previous value)
4. write matrix and limits for external sensor into volatile memory:
   Pos Sensor 1: 0x07000500 = „0“ ; Anpassung der Sensormatrix
   Pos Sensor 2: 0x07000500 = „1“
   Soft Voltage Low Limit: 0x0c000000 = „[Vx – 5V]“ ; limits for voltage, in case signal gets lost. or use values smaller 5V
   Soft Voltage High Limit: 0x0c000001 = „[Vx + 5V]“
5. move to 0 µm (=5V).
6. switch on servo

Switch back to internal sensor:

• change matrix and write back soft limits into volatile memory

Prozedur: 1. Scannen des kompletten Bereiches des P-726 (100µm) in closed loop (über den internen Sensor geregelt). Dabei den externe Sensor überwachen bis dieser ein Messsignal hat. 2. Auf „Messposition“ fahren. 3. Piezospannung abfragen (VOL? 1) 4. Regler ausschalten. (die Piezospannung bleibt auf dem letzten Wert) 5. Matrix + Limits auf externen Sensor in den flüchtigen Speicher schreiben: Pos Sensor 1: 0x07000500 = „0“  ; Anpassung der Sensormatrix Pos Sensor 2: 0x07000500 = „1“

Soft Voltage Low Limit: 0x0c000000 = „[gemessene Spannung – 5V]“  ; maximal Begrenzung der Spannung für den Fehlerfall z.B. außerhalb des Sensorsignals Soft Voltage High Limit: 0x0c000001 = „[gemessene Spannung + 5V]“ 6. Sollwertvorgabe analoger Eingang: 5V = 0µm 7. Regler einschalten. (=Autofocus ein)

Achtung: Beim Umschalten auf internen Sensor, müssen die Soft Limits und die Sensor Matrix wieder auf den ursprünglichen Wert gesetzt werden.

Camera

1. Group: Camera
2. Preset: EM200 20MHz

ConfigGroup,Camera,EM200 20MHz,Evolve512,ClearMode,Pre-Sequence ConfigGroup,Camera,EM200 20MHz,Evolve512,Gain,1 ConfigGroup,Camera,EM200 20MHz,Evolve512,MultiplierGain,200 ConfigGroup,Camera,EM200 20MHz,Evolve512,PMode,Frame Transfer ConfigGroup,Camera,EM200 20MHz,Evolve512,Port,Multiplication Gain ConfigGroup,Camera,EM200 20MHz,Evolve512,ReadoutRate,20MHz 16bit

1. Preset: Conventional

ConfigGroup,Camera,Conventional,Evolve512,Gain,1 ConfigGroup,Camera,Conventional,Evolve512,MultiplierGain,1 ConfigGroup,Camera,Conventional,Evolve512,PMode,Frame Transfer ConfigGroup,Camera,Conventional,Evolve512,Port,Normal ConfigGroup,Camera,Conventional,Evolve512,ReadoutRate,5MHz 16bit ConfigGroup,Camera,Conventional,Evolve512,ClearMode,Pre-Sequence

1. Preset: EM200 10MHz

ConfigGroup,Camera,EM200 10MHz,Evolve512,Gain,1 ConfigGroup,Camera,EM200 10MHz,Evolve512,MultiplierGain,200 ConfigGroup,Camera,EM200 10MHz,Evolve512,PMode,Frame Transfer ConfigGroup,Camera,EM200 10MHz,Evolve512,Port,Multiplication Gain ConfigGroup,Camera,EM200 10MHz,Evolve512,ReadoutRate,10MHz 16bit ConfigGroup,Camera,EM200 10MHz,Evolve512,ClearMode,Pre-Sequence

1. Preset: EM100 10MHz

ConfigGroup,Camera,EM100 10MHz,Evolve512,Gain,1 ConfigGroup,Camera,EM100 10MHz,Evolve512,MultiplierGain,100 ConfigGroup,Camera,EM100 10MHz,Evolve512,PMode,Frame Transfer ConfigGroup,Camera,EM100 10MHz,Evolve512,Port,Multiplication Gain ConfigGroup,Camera,EM100 10MHz,Evolve512,ReadoutRate,10MHz 16bit ConfigGroup,Camera,EM100 10MHz,Evolve512,ClearMode,Pre-Sequence

Microscope 4

PI Linear actuators Objective height

1. Start PIMicorMove
2. Connect to controller E/873 and axes
3. Click Servo (box on the right, might be out of the window) to activate closed loop
4. Put in new position or step size and take step
5. If there is an error, we have to initialize the axes

Intializing axes (necessary after rebooting controller):

1. Open the command window with the small button >_ under the E-873 tab
2. Send the command RON 1 0 (this sets the mode for referencing for axis 1 to 0).
3. Do the same for axes 2 and 3 with RON 2 0 and RON 3 0
4. Set the reference position for axis 1 to 0: POS 1 0
5. Do the same for axes 2 and 3 with POS 2 0 and POS 3 0
6. Now the movement should work.

To do

• Camera water cooling (Done)
• Servo in detection beam path
  • Decide cylindrical lens for 3D (now, once the f=1.5m lens arrives)
    • 1m
    • 2m
    • 1.5 m (ordered)
  • Mount cylindrical lens to servo (now)
  • Bertrand lens (now)
    • install after cylindrical lens
• Mount new dichroic
  • make sure the holder fits the microscope, talk to Joran (Done)

• Clear excitation beam path
• New excitation beam path
  • Mount new Ploessl lens(es)
  • Put in new dichroic
  • Align Ploessl lens
• Excitation lasers
  • check status of multi-mode laser box, talk to Anindita (Done)
  • sketch out how to arrange single mode + multi mode ( + high power lasers) (now)
  • Do the math for the single mode collimation lens (now)
  • Install any laser
  • Install EM-CCD
  • Install filters for EM-CCD
• Ensure mechanical stability
• How to run both cameras in the same Micro Manager instance (now)
• Build housing
  • conventional emission beam path including camera
  • excitation beam path
  • Em-CCD camera
• Fitting, talk to Jonas
• Integrate hood including LED, talk to Joran (now)
• Monitor temperature and laser power, talk to Joran (now)

Old roadmap

• Milestones
  • Water lens + 3D for biology
  • Dual color ratiometric
  • Automated 3D + dual color ratiometric

• Emission
  • Multi-mode
  • Smaract stage for TIRF

• Detection
  • Cylindrical lens
  • BFP lens
  • Filters
  • Housing

• Software
  • Micro Manager Integration
  • Dual camera support in Micro Manager
  • Hardware integration
  • FPGA-Box (connection)
  • SMAP fitting

• Dual color
  • Boxing

• Objective lenses
  • Objective stage angle (using water lens)
  • Test PSF for all objective lenses (including M3)

• Hardware
  • Buy missing filters
  • Smaract controllers
  • Focus lock for water lens
  • Check PI parameters of PI
  • Hood + LED
  • Sensors
    • Temperature
    • Laser Power

Align 3 axis PI controllers

Important: before moving the actuators, check that the plate is perfectly sitting on them (actuator tips in the small rails underneath the plate) !!

• To use the actuators in Pi MikroMove, you need to zero all actuator positions: after connecting to the controller, you reach the step "Start up axes". For each axe, select it and click on "Advanced...". There you can zero the position.
• Moving actuators towards negative values moves objective lens up

• Tilting stage around X-axis (as seen from the camera):
  • Move actuator 1 by 0,012800000 mm (or multiple n) -- (harmonized units: 117 = 3x39)
  • Move actuator 2 by 0,003900000 mm (or multiple n) -- (harmonized units: 039)
  • Keep actuator 3 in position
  • Refocus using z-piezo

• Tilting stage around Y-axis (as seen from the camera)
  • Move actuator 1 by 0,002200000 mm (or multiple n) -- (harmonized units: 039)
  • Move actuator 2 by 0,012200000 mm (or multiple n) -- (harmonized units: 234 = 6x39)
  • Keep actuator 3 in position
  • Refocus using z-piezo

Sample holder for slides (old one)

• When putting on the sample holder for slides, the objective has to be driven up by 250 µm. I.e., all three actuators have to go to -0.250 mm.

Changing the lens to NA 1.7 from M3

• When changing to NA 1.7 from M3, the objective holder stage has to go up by 260 µm. I.e., the actuators need to be driven by -0.260 mm

FAQ

24 h microscopy hotline

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+49 157 56 74 58 86

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Software

IMAQ error on labview when using activation

• Check if during your multi-dimensional acquisition in Micro-manager you selected "Image stack file". It should be "Separate image files".

Hardware

It looks like there is no electrical power on the set-up

For instance camera not working (no ventilation) or no light on the focus-lock laser.

• Sometimes the fuse switches jump on the set-up (microscope 1, fuse F8), look for on "off" and turn it "on".
• It may happen that the power is off but the fuse on, then turn it off and on again.

The joystick of the Smaract is not working

• When the Smaract sensors are in closed-loop, the joystick doesn't work --> Go the the menu, by pressing two buttons corresponding to X and Y --> choose Control Modes option by pressing one of the knobs --> choose "simple" mode for X and Y --> joystick becomes functional again

Laser box is not working (lasers are not triggered)

• switch off everything (including permanent socket - this can be done by switching the fuses off and on), then switch on everything
• if that does not work disconnect the power and USB on the National instruments card on top.

Back focal plane lens is not moving anymore

• A screw can be loose on the arms of the mechanical slider.

When nothing else works

First aid when something doesn't work

• Have you tried turning it off and on again? Restarting Micromanager, Labview or the whole set-up might help.

Sometimes you even have to unplug and replug the permanent power socket.

Ports