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Optics11 Life PAVONE, User Manual

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Optics11 Life PAVONE User Manual Download Page 46

 

46

  optics11life.com 

3.5

 

Camera settings and Microscopy 

To have a good image of your sample or probe, camera settings need to be adjusted by the user in the 

“Camera” 

option  in  the  main  window  (see 

Figure  39

).  One  can  select  parameters  to  be  adjusted 

automatically by ticking ‘Automatic’ or 

use  a one-time  automatic adjustment  from the main software 

window 

“Auto Setup Camera”. The maximum ‘Frame rate’ of the camera is 47 fps and it is dependent on 

the  exposure  time  e.g. exposure  time of 100  ms  (0.1 s)  limits the frame  rate to 10 fps  (1/0.1=10). To 
achieve higher frame rates, set shorter exposure time. From the main window, one can select to turn on 
3 different LED rings: Brightfield, Phase one and Phase two. 

“LED intensity” 

sliding bar in the main window 

allows to adjust the intensity of LED rings.  
For saving the images, 

“

File format

”

 

of the “Camera”

 window (see 

Figure 39

) allows setting image saving 

options 

when using “

S

ave image“ button from the main window

 

(‘Front panel’)

 or automatic saving of 

images or videos 

“D

uring experiments

”

. Images taken during the experiment will be saved in the same 

location  as  the  text  files  of  indentation.  Moreover,  the  objective  type  (4x,  10x,  20x  and  40x)  can  be 
selected in the Objective menu. If another objective is used, um/pixel value can be calibrated using XY 
stage movement in the camera view.  
  
 

Summary of Contents for PAVONE

Page 1: ...PAVONE USER MANUAL ...

Page 2: ...RESS VU University Campus W N Building room O 236 De Boelelaan 1081 1081 HV Amsterdam The Netherlands SHIPPING ADDRESS De Boelelaan 1081 1081 HV Amsterdam The Netherlands COMPANY INFORMATION Optics11 B V KvK CC 52469417 VAT NL850459734B01 Amsterdam NL Please see our website for more information about our products www optics11life com ...

Page 3: ...ety 5 Introduction 6 1 Installation 7 2 Preparing the setup for measurement 9 3 Using the Pavone 25 4 Additional system calibration and settings 48 5 Optimizing instrument stability and sample mounting 54 6 The OP1550 interferometer 58 ...

Page 4: ...ics11life com REVISION HISTORY Version 1 0 New document V1 March 4 2019 Ernst Breel Version 1 5 Update for V1 5 2 February 18 2021 Nelda Antonovaite Version 1 6 Update for V1 6 0 October 20 2021 Ali Paknahad ...

Page 5: ...instrument before changing the fuse or before switching from 220V to 110V or vice versa Do not open the box as this might result in serious injuries Water damage to the Pavone components is not covered by the warranty Please make sure that no liquids are spilled on the piezo objective or other components PC of the Pavone is provided by the external supplier which includes operating system Optics11...

Page 6: ...ribes the preparation of the Pavone and probes for experiments How to perform the measurements and operate the instrument is described in detail in Chapter 3 Chapter 4 describes how to calibrate camera and microplates Chapter 5 describes the optimization of sample preparation and measurement conditions Chapter 6 describes how to operate the OP1550 interferometer Figure 1 The Pavone in action measu...

Page 7: ...ment body which also includes a CCD Camera with the mount b Objective 2 Power supply unit 3 Interferometer OP1550 4 PC which also includes a Keyboard and mouse b USB splitter c USB camera for remote training 5 Optics11 Life probes Figure 2 Pavone main components For the best user experience it is recommended to first power on all separate components before running the software 1 2 Connecting compo...

Page 8: ...smit the outgoing optical signal to the acquisition electronics in the Pavone and the USB B cable to the PC allowing for communication with the Pavone through the PC Pavone is connected to a PC with two USB B cables The Camera being a flexible component connects to the PC through a separate micro USB cable Please note that all USB cables and connections should be of the USB 3 0 type only connect t...

Page 9: ...are by double clicking the Pavone software icon on the desktop The software is loading all devices while checking the hardware connection status which should all be Idle see Figure 5 after a maximum waiting time of 60 seconds The software will then proceed to the main user interface Figure 5 Hardware status check After starting the instrument the system will prompt homing of the XY sample stages t...

Page 10: ...mple Young s moduli in the table below These are estimated for a tip radius of 25 µm indentation depth of 2 µm The full range is given considering minimum cantilever bending of 0 01 µm and a maximum of 30 µm The optimal range is given for cantilever bending of 0 05 µm and a maximum of 20 µm Table 1 Young s modulus and advised cantilever stiffness range tip radius of 25 µm and indentation depth of ...

Page 11: ...ack right position When handling a probe take special care to avoid any contact with the cantilever as the glass cantilever at the end of the glass ferrule is extremely fragile and almost any unintended contact will cause permanent damage Therefore when handling the probe always hold it by the plastic adapter keep the optical fiber away from the cantilever and avoid touching the glass part Given t...

Page 12: ...s Figure 8 Main Pavone components Y stage X stage 100 µm piezo probe stage objective stage plate 1 plate 2 probe Cantilever with tip 1 Piezo signal in V Non linearized cantilever signal in V 10 11 X stage Y stage 2 3 4 5 6 8 7 9 Figure 9 Main software window ...

Page 13: ...e plate 1 A4 current probe position within wellplate Piezo µm current position of the piezo together with the bar 4 Microscopy Bright field turns on bright field LED Phase one turns on phase contrast LED suited for x20 objective Phase two turns on phase contrast LED suited for x40 objective LED intensity adjusts LED intensity 5 Temperature control ON OFF turn on and off the temperature control Set...

Page 14: ...ion when the surface is detected 9 Find surface X µm move to point X coordinate Y µm move to point Y coordinate Move to point executes the function Speed µm s the speed at which XY stages move when running Move to point function Coord list allows to set up a list of coordinates to be measured 10 Live signals In blue piezo position signal in Volts In green non linearized cantilever position in Volt...

Page 15: ...ip is hovering above the center section of the objective see Figure 10 images on the right 5 Turn on Brightfield and Phase 2 and press Auto cam adjust in the software If the image of the camera is dark increase LED intensity or go to Camera settings You should see an image or shadow of the probe If necessary adjust the height of the objective stage so that the probe is in the focal plane check the...

Page 16: ...ounting Caution When using high magnification objectives please ensure that the objective is not touching the probe Caution Please ensure to untighten the black knob at the top of the condenser before adjusting the probe position and tighten again after adjustment Figure 10 From left to right knob to release manual stages circled in red manual micromanipulators for probe adjustment circled in red ...

Page 17: ... microplates from Greiner LINK 96 and 6 Softwell plates from Matrigen LINK Follow these steps 1 First mount the microplate by pushing it down until all sides touch the bottom of the plate holder and the plate feels firm in the X direction there are springs on the right side that keep the plate firm If the microplate can move in the Y direction push it to the top Choose the well that will act as th...

Page 18: ...e top left corner without hitting the edge of the well see Figure 13 left image If the probe position with respect to the well is not correct calibrate the wellplate see Section 4 1 4 Prewet the probe by pipetting the medium the same solution as used in the calibration well over the front face of the probe until you can clearly observe a droplet hanging underneath the probe see Figure 13 right ima...

Page 19: ...meaningful measurement results Probe parameters that need to be set in the program are the cantilever spring constant k N m and probe tip radius R µm These parameters can be entered in the Calibration meniu see Figure 14 The numbers can be found on the side of the probe packaging box and are unique for each probe They are calibrated in the air by indenting on a scale1 If you accidentally forget to...

Page 20: ...so that the probe can adjust to the new environment When performing experiments with temperature control the calibration should be accomplished at the same temperature as the measurement temperature This is because probe is sensitive to temperature variations 6 Click the Scan OP1550 Wavelength button once The interferometer screen will now show a progress bar The live signal window on the screen s...

Page 21: ...length rapidly from 1565nm to 1525nm and creates an interference pattern As the photodiode s output voltage depends on the intensity of the interference pattern an automated offset and gain setting will be set which optimizes the photodiode output for the 0 10V range When changing the medium or probe the new wavelength scan needs to be performed in order to optimize the laser settings 2 2 Chavan D...

Page 22: ...eans that the cantilever is not in contact and you should either increase the threshold value in the main window and repeat the find surface step or manually bring down the probe to contact in small 1 µm steps Figure 16 Manual finding of the surface position by pressing down the probe stage at 1 µm steps Caution For the softest probes of k 0 025N m the threshold value most likely will have to be i...

Page 23: ...when probe is in contact with the hard surface left and when it is out of contact right c Successful top and failed bottom calibration messages d Calibration curve e Signal movement in time on demodulation circle after it is calibrated f There is no indentation on a hard surface after calibration red line is flat a b c d e f ...

Page 24: ... probe is in contact with a stiff surface 12 When calibration is completed New factor is given By pressing Use new factor the software automatically saves the new Calibration factor in the probe configuration menu see Figure 17 c The calibration factor should be 1 33 times lower in the medium than it is in the air which is given on the box of the probe e g if the number on the box is 3 2 then the ...

Page 25: ...ion can be verified by performing an indentation directly after the calibration Load or make an experiment file Config experiment using the default displacement mode settings and changing to a calibration distance to displace the probe again on the stiff substrate When Run experiment check the demodulation circle in the interferometer window The white signal should be on top of the red circle duri...

Page 26: ...of the contact point approach distance where the probe is out of contact with the sample is needed as shown in Figure 20 d to fit the Hertz model red line from which contact point is given as one of the fitting parameters Caution The maximum distance that piezo can extend is 100 µm thus the distance to the sample s surface after the Find Surface procedure cannot be higher than that Adhesion mode i...

Page 27: ... of contact part of the curve needed for contact point determination b Find Surface procedure when Z above surface is too low due not ideal probe and sample stiffness combination resulting in in contact measurement shown in e c Z above surface is too low due to the adhesive sample resulting in in contact measurements shown in e f Out of contact In contact Starts in contact Contact Adhesion a d b e...

Page 28: ... volume of material and thus has approximately the same surface position fill in 1000 µm lower value in Transit µm e g 22 000 µm or move up by 1000 µm and press Set transit height 4 If samples in different wells have different positions of the surface then you will need to find the one with the highest surface position and set transit height based on that well Otherwise you can also set transit he...

Page 29: ... Read Section 4 1 about wellplate calibration 3 3 2 Move to point The X Y stage can also be moved using the camera after clicking anywhere on the camera feed window the green dot will appear and its coordinates will be recorded in X µm and Y µm see Figure 22 Then by clicking Move to point in the Stage location tab the selected location will be brought under the center of the probe The find probe p...

Page 30: ...l and a red circle will indicate the selected well Plates can be changed in Wellplates meniu in the main software window If Move Absolute is selected see Figure 24 b probe and objective stages can be moved to the absolute coordinates One can either fill them in manually by typing in the coordinates or by checking Transit Height and Focal Plane boxes which will use corresponding coordinates selecte...

Page 31: ...tings in the main software window one can select the speed at which the probe moves down Speed µm s distance to which the probe is retracted after the surface is found Z above surf µm and Adhesion mode settings can be used when the sample is sticky see Section 3 1 for more details see Figure 25 Caution make sure that the signal is on the demodulation circle after moving the probe from air to mediu...

Page 32: ...e or absolute to allow the probe to stabilize to the environment and temperature of the medium see Figure 26 Figure 26 Settings of wait step 3 4 4 Clean When measuring biomaterials some residue might get stuck to the probe tip especially when samples are adhesive Also the residue might deposit on the gold coating of the cantilever from the salts in the medium which would decrease the signal to noi...

Page 33: ...on Single indentations Single indentations can be configured in the Indentation tab The first subtab General allows to select the location of the data the folder name and write comments The second tab Profile allows setting an indentation profile in one of the modes displacement load or indentation The third tab is dedicated to setting up oscillatory frequency sweep also called dynamic mechanical ...

Page 34: ...rtz model fitting During all three modes of operation the instrument is recording piezo movement blue and cantilever bending green shown in Figure 28 a Indentation depth is calculated by subtracting cantilever bending from piezo movement while in contact with the sample The load is calculated by multiplying cantilever bending with the spring constant of the cantilever The contact point with the sa...

Page 35: ...tion depth depends on the distance from the sample surface and the ratio of cantilever and sample stiffness during piezo movement while in contact with the sample Since the piezo is not receiving any feedback signal from the sample cantilever bending it is working in an open loop with the sample meaning one has no control over indentation speed depth or load The displacement µm and time s variable...

Page 36: ...t optics11life com Indentation depth control I mode Operating in I mode gives you the possibility to set an exact indentation depth and indentation speed and also indentation depth can be kept constant over time for a stress relaxation experiment see Figure 29 c Before setting the indentation profile simply click Indentation control in the Mode Selection part of the indentation configuration windo...

Page 37: ...the corresponding profile menu Oscillation parameters like frequency periods amplitude and relaxation time are freely adjustable to provide a flexible measurement design see Figure 30Figure 31 DMA data analysis in DataViewer The minimum initial relaxation time should be 5s However if the sample shows a long relaxation duration the time should be chosen so that equilibrium is reached before DMA sta...

Page 38: ...le Min initial relaxation time 5s Min relaxation time 2s Min amplitude 2x noise level Lowest frequency possible 0 01 Hz Highest frequency possible 20 Hz closed loop 75Hz open loop Min oscillation time 1s Figure 30 Configuration of DMA profile Dynamic Mechanical Analysis DMA uses a cyclic motion with frequency 𝑓 while controlling indentation depth or load with an amplitude of ℎ0 or 𝐹0 respectively ...

Page 39: ...mplitude of oscillations the depth should be considered due to the curvature of the sphere as it is assumed that the contact area does not change during oscillations so that ℎ0 ℎ 0 25 The performance of the DMA can be evaluated in the DataViewer by comparing the amplitude which was measured with the amplitude which was set in the experiment and by looking at the R squared values of cosine fits Fig...

Page 40: ...probe at the top left corner of the region that needs to be mapped Once the parameters have been set the scan is initiated by pressing the Start scan button Before continuing to a new point including the first indentation of a matrix scan the probe moves up to a safe transportation height and subsequently moves to the indentation location After reaching this location the find surface procedure is ...

Page 41: ...ee Figure 34 Simply press Add and then select a location on the camera image and the XYZO coordinates will be filled in the list Continue with all the objects locations of interest When finished press Save and the Coordlist json file will be created To run indentation measurements on selected coordinates go to Config exp and add Run Coordinate List Caution when running indentations on the list of ...

Page 42: ...Z position of stages and Objective position see Figure 35 At first all the stages are moved to the selected coordinate followed by an Automatic find surface if it is ticked and then a single indentation is performed Coordinates can be also manually modified by directly opening the json file with Notepad Data will be saved in a separate folder Coordinates ...

Page 43: ...l The temperature setpoint from 15 to 50 C during the experiment can be set in Environment control menu see Figure 36 One should wait 30 min to let the system and the sample plate to warm up to the desired temperature Figure 36 Setting up the temperature set point in the Environment control menu ...

Page 44: ...nder a specific name by changing the name in the experiment path and pressing Save 1 Move to well 2 Move to transit and focal height or Move Absolute or Move Relative 3 Wait 4 Find Surface 5 Matrix scan Coordinate list Indentation 6 Go to step 1 and repeat the sequence for n times n represent the number of selected wellplates more details are described in the following section Figure 37 Configure ...

Page 45: ...e the slot of the wellplate then wellplate type e g 6well 12well 24well and etc and then click on the wells that the programmed experiment needs to be performed at By selecting each well the color of that specific well will turn black see Figure 38 Note that you can use more than one Goto function to program your experiments with different configurations for different wells or wellplates The impor...

Page 46: ...rame rates set shorter exposure time From the main window one can select to turn on 3 different LED rings Brightfield Phase one and Phase two LED intensity sliding bar in the main window allows to adjust the intensity of LED rings For saving the images File format of the Camera window see Figure 39 allows setting image saving options when using Save image button from the main window Front panel or...

Page 47: ...processing Also the DataViewer program can be used to quickly load all data and make small adjustments in the fitting where necessary For each matrix scan a tab separated text file is saved with a matrix of the Effective Young s Modulus per coordinate Additionally per indentation a separate text file is saved that contains all information for this indentation When reanalyzing the data in DataViewe...

Page 48: ...owing the header lines and the start of the five data columns 4 ADDITIONAL SYSTEM CALIBRATION AND SETTINGS 4 1 Wellplate calibration adjusting the safety areas Each wellplate has safety areas which are determined by the dimensions of the well plate see Figure 41 and calibrated by the user This guide will show how to add well plates and how to adjust safety areas ...

Page 49: ... probe in Z direction to see that the probe can enter safely the well The step size can be set in Travel µm You can move XY stages by clicking on the blue arrows on the bottom side of the current window see Figure 42 d First move the stage to a place where the probe can safely enter the well in the Top left position Then by choosing small steps sizes Travel µm slowly move the Z stage down with the...

Page 50: ...50 optics11life com Figure 42 Different stages of wellplate calibration process a b c d ...

Page 51: ... strong so that you can easily readjust them 2 Place any type of well plate and focus on a small object such as dust You do not need to mount the probe 3 Press with the cursor on the small object and note the coordinate Y of the green cursor 4 Move the stage in Y direction across the field of view so that you can still see the small object 5 Press with the cursor on the small object and note the c...

Page 52: ...to bottom choose a point with a green cursor Y coordinate 106676 7 Move up the Y stage and press on the same object Y coordinate 106677 2 Rotate the camera clockwise Move stage down again Y coordinate 106674 6 Continue until the Y coordinate is aligned ...

Page 53: ...tended to be changed by the user unless they are specially trained to do it by one of the application specialists of Optics11 Life see Figure 45 The default maintenance settings are saved in the desktop folder settings Figure 45 Example of maintenance settings ...

Page 54: ...nical disturbances such as ground vibrations from other labs other equipment or appliances ensure that the Pavone is mounted on a stable bench Preferably this bench is not shared with other vibrating machines such as vortexes pumps or large fans Optionally the Pavone can be bolted to the top of a third party stabilization platform such as an air table Make sure that the air table is inflated when ...

Page 55: ...r bursting the bubble Note that you should avoid directly touching the cantilever the cantilever is very fragile and can break easily when touched Holding the tissue at a safe distance will also allow you to dry the probe without touching the cantilever see Figure 46 Suitable probe cantilever stiffness To perform successful indentation experiments it is necessary to have a probe cantilever with a ...

Page 56: ...umed to be an infinite half space thus tip size should be chosen so that this assumption is true 4 When performing indentation mapping the step size should be at least two times the contact radius a to avoid oversampling Regarding the tip size you should balance between the spatial resolution of the mapping which depends on the heterogeneity of the sample and the scale of the measurement in terms ...

Page 57: ...t is important to immobilize the sample to eliminate lateral and height drift during and between indentations Indenting drifting or floating samples results in unreliable indentation measurements such as high noise or irregular curves Especially when performing automated matrix scans make sure the sample is immobilized correctly For more information about how to mount the sample please read the se...

Page 58: ...ursor over the button or field and press the center button enter In case a field is selected the field will turn blue and the up and down buttons can be used to alter the value the cursor highlights The interface is built up out of two menus The first menu Measurement Wavelength scan Demodulation and Settings can be accessed using the buttons on the top of the screen The second menu Laser ADC Outp...

Page 59: ...ference signal i e peak to peak signal of fringes is optimal Wavelength scan can be performed by pressing the Scan wavelength button at the bottom of the screen in the Wavelength scan menu or through the software in the Initialize window A good wavelength scan results in a sinusoidal curve as shown in Figure 49 b The Demodulation tab displays the linearized signal of interference fringes presented...

Page 60: ...e minimum laser power is 6 31mW and the maximum one is 25mW Caution Please consider increasing laser power to a maximum one when measuring in the medium as it will increase your signal to noise ratio see the video https youtu be coKxhkDsS6Q However for the wavelength scan in the air please decrease the laser power as there might be too much signal which would result in a saturated signal flat line...

Page 61: ...it is recommended to keep them at the default settings The Modulation menu contains the controls to induce a small modulation on top of the laser output signal by quickly and continuously modulating the laser wavelength This modulation is required for the Pavone to function properly and should always be turned on when performing indentations The modulation is active when the mode is set to On the ...

Page 62: ...ons Do not use live calibration if the samples are sticky as it will cause a reset of scaling factors to the over bended position In general better to use a stiffer probe for very sticky samples or contact us for ideas on how to decrease the stickiness Please see the videos on how demodulation signal looks with and without live calibration https youtu be 1VL6cXOqMsE live calibration on https youtu...

Page 63: ...63 For troubleshooting support or questions while working with the Pavone please contact Optics11 Life at Tel 31 20 5987917 E mail support optics11life com Office hours are between 9h and 18h CET ...

Page 64: ......

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