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Nanolive 3D Cell Explorer-fluo User Manual

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Summary of Contents for Nanolive 3D Cell Explorer-fluo

  • Page 1 fluo 3D Cell Explorer- User Manual...

  • Page 2: Table Of Contents

    Contents Safety Notes fluo 3D Cell Explorer- • CoolLED illumination system • Maintenance & Care fluo 3D Cell Explorer- • CoolLED’s illumination system • Introduction Instrument Description & Main Features • Typical Field of Applications • fluo 3D Cell Explorer- terminology •...

  • Page 3: Safety Notes

    3D Cell Explorer- fluo fluo Thank you for purchasing the Nanolive 3D Cell Explorer- . Before using your 3D Cell Explorer- and the CoolLED illumination system, carefully read the safety notes to ensure safe handling and usage of the device at all times. Failure to do so may result in personal injury or damage to other items.
  • Page 4 Safety Notes fluo 3D Cell Explorer- fluo 4. Contaminations 6. The 3D Cell Explorer- conforms to the requirements of the Safety Standards as follows: The user has the responsibility to avoid disastrous contaminations by biological samples. Nevertheless, contamination might occur •...

  • Page 5: Coolled Illumination System

    Safety Notes CoolLED illumination system • Safety precautions must always be exercised when using 7. Safety Notes for the CoolLED illumination system electrical equipment to prevent operator shock, fire hazard or • UV light may be emitted from this product depending on the equipment damage.

  • Page 6: Maintenance & Care

    Maintenance & Care fluo 3D Cell Explorer- fluo Maintenance and Care for the 3D Cell Explorer- fluo 1. Carefully, open the shipment box with the top face up to avoid damaging the 3D Cell Explorer- fluo 2. Handle the 3D Cell Explorer- from the side or the bottom plate;...

  • Page 7: Coolled's Illumination System

    Maintenance & Care CoolLED illumination system Maintenance & Care for the CoolLED illumination system 1. Carefully unpack the components from the shipping cartons. 2. The CoolLED illumination system requires little or no maintenance throughout its life. There are no field service- able parts so there is no need to remove the covers.

  • Page 8: Introduction

    Introduction Instrument description and main features Plug & Play systems • 2 imaging modalities: - Physical structural imaging (based on refractive index, RI) - Chemical fluorescence imaging • 3 Fluorescence Channels: 2 configurations available: - DAPI (Excitation 392 nm / Emission 432 nm), FitC (Excitation 474 nm / Emission 515 nm), TritC (Excitation 554 nm / Emission 595 nm) - FitC (Excitation 474 nm / Emission 515 nm), TritC (Excitation 554 nm / Emission 595 nm), Cy5 (Excitation 635 nm / Emission 730nm)

  • Page 9: Typical Field Of Applications

    Introduction Typical Fields of Applications fluo The typical fields of applications for the 3D Cell Explorer- • Merging of fluorescence microscopy and 3D cell tomography • Calibration of the refractive index map with markers (Chemical dyes, fluorescent antibodies, protein labeling with GFP, etc.) •...

  • Page 10: Cell Explorer- Fluo Terminology

    Introduction fluo 3D Cell Explorer- terminology fluo fluo fluo 3D Cell Explorer- – front view 3D Cell Explorer- – side view 3D Cell Explorer- – rear view Scanning head Peripheral mirror Rotating arm Cell container interface Hi-grade stage for XY knob for sample screening sample screening Z knob for...

  • Page 11: Coolled Illumination System Terminology

    Introduction CoolLED illumination system terminology CoolLED illumination system – front view CoolLED illumination system – side view 1 Liquid Light Guide (LLG) Not needed BNC plugs Global Trigger CoolLED illumination system – rear view CoolLED illumination system – Top/Side View Excitation filters slots go back to contents go back to contents...

  • Page 12: Technical Specifications Sheet

    4. Technical Specification Sheet (TSS) fluo 3D Cell Explorer- & CoolLED illumination system fluo Specifications CoolLED illumination system / Specification 3D Cell Explorer- Epifluorescence Holotomography 380 x 170 x 445 77 x 186 x 162 Dimensions (width x depth x height in mm) Weight (in kg) Permissible ambient temperature, and can be 5-35°C...

  • Page 13: Computer Technical Specifications

    5. Computer Technical Specifications fluo 3D Cell Explorer- fluo The 3D Cell Explorer- is provided without a computer. Therefore, before you acquire a computer, please be aware of the minimum requirements listed below which are absolutely necessary for correct operation of the instrument.* CPU: 1.5 GHz or faster multi-core CPU CPU Memory:...

  • Page 14: Start-Up

    Start-up Components fluo The 3D Cell Explorer- package contains the following: fluo the 3D Cell Explorer- WEEE certificate 1 USB 2.0 cable protective cap for the MO and 1 USB 3.0 cable protective cap for the LLG connection to the microscope 1 power supply (10) 1 BNC cable.
  • Page 15 Start-up Components The CoolLED Illumination System packaging contains the following: (11) LED Light Source (12) DC Power Supply (13) IEC Power Cable (14) 1 USB 2.0 cable (15) liquid light guide (LLG) protected with red vinyl caps (use the red vinyl caps to protect both end of the LLG when not in use) (16) 3 holders equipped with excitation filters.
  • Page 16 3D Cell Explorer- while it is removed out of its box. fluo In case of damages, Nanolive reserves the rights to examine the 3D Cell Explorer- and verify how the damage occurred. All warranty claims will be cancelled in case of fluo mishandling the 3D Cell Explorer- by ignoring the instructions.
  • Page 17 Start-up Installation and setup 2.     C arefully remove the protective accessories • Properly dispose of the original packaging, or keep it for storage or return of the instrument to the manufacturer. • Remove the disposable plastic foil and red vinyl protective cap. Disposable plastic foil Red vinyl cap For safety reasons, please proceed to install the CoolLED illumination system first.
  • Page 18 Start-up Installation and setup 3. Install the CoolLED Illumination System on a stable, flat, horizontal and firm workbench • Lift the CoolLED Illumination System carefully out of the box together with its accessories. go back to contents go back to contents à...
  • Page 19 Start-up Installation and setup 4. Install the excitation filters on the CoolLED Illumination System • Place the little drawers containing the excitation filters in the dedicated slots. • Mind the order and the direction. Channel 2 Channel 1 Channel 3 go back to contents go back to contents à...
  • Page 20 Start-up Installation and setup 5. Install the LLG on the CoolLED Illumination System • Remove the red cap from the LLG from one end (Figure 1) Protective cap LLG header Figure 1 • Insert the LLG as far as it can go into the LED Light Source as shown below (Figure 2). ✔...
  • Page 21 Start-up Installation and setup 6. The CoolLED illumination system with the inserted LLG should look like the image below. fluo Place the light source close to the 3D Cell Explorer- in order to avoid any strain on the LLG or too short radius turns. Figure 3 200 mm 200 mm...
  • Page 22 Start-up Installation and setup fluo 7. Connect the LLG to the 3D Cell Explorer- • Remove the red cap from the LLG from the other end (Figure 5) Protective cap LLG header Figure 5 fluo • Insert the LLG as far as it can go into the injection head on the 3D Cell Explorer- as shown below (Figure 6).
  • Page 23 Start-up Installation and setup • Use an Allen key n°1.5 to secure the LLG in place. • Bring the 3 screws first in mechanical contact without effort one after the other. Gently apply some force to keep the LLG in place one after the other.
  • Page 24 Start-up Installation and setup 8. Handling note on the LLG (Figure 8) fluo • The LLG is carrying the light from the CoolLed illumination system to the 3D Cell Explorer- . It is fragile and should be handled with care. •...
  • Page 25 Start-up Installation and setup 9. Diverse connections of your CoolLed illumination system • Plug the power connector from the DC power supply to the CoolLED illumination system as shown below. Ensure that the DC power supply is the one supplied with the product. Using a non-CoolLED power supply may damage the illumination system and will invalidate the warranty.
  • Page 26 IEC connector into the DC power supply and switch the power on at the socket. Camera / data transfer USB 3.0 Computer USB 2.0 3D Cell Explorer Nanolive SA SN: YY MM DD 00 00 X X XX XX Power supply & power cable BNC cable Power supply &...
  • Page 27 Connect the Nanolive power cable to the Nanolive power supply. Then, plug the Nanolive power supply into the power port. • Plug the Nanolive USB 3.0 cable into the USB 3.0 port on the side of the 3D Cell Explorer (the camera), tightly screw the locks and connect it to the USB 3.0 slot on your computer.
  • Page 28 Updated drivers: you need to have the latest Nvidia Graphics Card drivers, USB 3.0 drivers and fully updated Windows. fluo • Go to www.nanolive.ch/register, fill out the form to register the 3D Cell Explorer- and download the software. fluo •...

  • Page 29: Handling

    Handling fluo Correct handling of the 3D Cell Explorer- -fluo The following section’s purpose is to explain how to avoid any damages to the 3D Cell Explorer during moving, carrying, transportation and repacking. -fluo If you want to move, carry, transport or repack the 3D Cell Explorer , please make sure to: -fluo 1.
  • Page 30 Handling fluo Correct handling of the 3D Cell Explorer- Instructions on how to secure the sample stage fluo Before moving the 3D Cell Explorer- , please ensure that the sample stage is blocked in top position by using the z-screw to avoid damaging the Microscope Objective as shown in the picture below: ✖...
  • Page 31 Handling fluo Correct handling of the 3D Cell Explorer- Instructions on how to protect the Microscope Objective (MO) with the red vinyl protective cap Place the protective cap on the MO through the sample stage aperture ✔ ✖ Push it until it leans on the knurled part of the MO go back to contents go back to contents à...
  • Page 32 Handling fluo Correct handling of the 3D Cell Explorer- Instructions on how to protect the LLG header and how to properly take care of the LLG • Use the protective cap for both ends when the LLG is not connected to the devices (Figure 15 & 16) •...
  • Page 33 Handling fluo Correct handling of the 3D Cell Explorer- fluo Instructions on how to carry the 3D Cell Explorer- correctly Once all cables are unplugged and the sample stage is secured and in top position, hold/carry your device as shown below. ✔...

  • Page 34: Sample Preparation

    Sample Preparation Sample Preparation Manual: fluo The 3D Cell Explorer- allows measuring the inside of a living cell offering the researcher the possibility to acquire high resolution 3D images of a cell within seconds: • cells fixed on glass coverslips or FluoroDish™ •...

  • Page 35: Cleaning Procedure For Optical Elements

    Cleaning Procedure for optical elements First, inspect the optical elements to determine the location of the con- taminants. This allows you to anticipate the cleaning (typically by a swiping movement) so that the contaminant is removed from the surface of the optical element as soon as possible (avoid dragging it around).
  • Page 36 Cleaning Procedure for optical elements Figure 19 Figure 20 go back to contents go back to contents à à...

  • Page 37: Software Steve

    10. Software STEVE Important Note: Fluorescent filter configuration Fluorescence filter configuration Before you start your acquisitions, set up STEVE with the correct fluorescence filter configuration as shown below. fluo The 3D Cell Explorer- is delivered with the options FITC (green), TRITC (orange) and DAPI (blue) or Cy5 (red). The configuration may be changed from the Microscope —...
  • Page 38 Please also refer to our video on our homepage Getting started with STEVE STEVE is Nanolive’s software for exploring the data acquired using the 3D Cell Explorer. For STAIN PICKER each recorded frame, the 3D Cell Explorer measures the Refractive Index of your cell in three : New stain - choose a name (not required) and a color for the stain.
  • Page 39 10. Software STEVE Interface Fluorescent 2D View Panel Viewer 3D View The 2D view shows a X-Y slice of the sample's Refractive Index overlaid with The Panel Viewer represents a 2D space of Refractive Index and Index Gradient. The 3D view shows the stained data in three dimensions. user-defined digital stains and fluochromes resulting in a multi-channel image.
  • Page 40 10. Software STEVE Preparation for a measurement Note! fluo In order to start your measurements, verify that all the cables are properly plugged in, the 3D Cell Explorer- and the CoolLED illumination system are turned on and connected to your computer and STEVE is running. fluo Operate and control the 3D Cell Explorer- and the CoolLED illumination system easily with STEVE if you wish to do the following:...
  • Page 41 10. Software STEVE Selecting the mounting medium‘s refractive index How to select the mounting medium’s refractive index Before either a single-shot or a time-lapse acquisition is started, a dialog will appear in STEVE (Figure 23) prompting you to select the mounting medium refractive index (e.g.
  • Page 42 10. Software STEVE Single-frame image acquisition How to do a single-frame image acquisition A single frame image acquisition is an instantaneous measurement that results in a unique image of the object. To perform a single shot, click on the 3D button or on the Single Shot from the Microscope menu. The mounting medium refractive index selection dialog will appear (see section "How to select the mounting medium’s refractive index"...
  • Page 43 10. Software STEVE Acquiring fluorescence images Multi-channel image A multi-channel image combines a series of monochrome images into one image. The multi-channel image generally shows a sample that has been stained with numerous different fluorochromes. The multi-channel image is a combination of the single fluorescence images. You can have the individual fluorescence images displayed separately (showing, e.g., the plasma membrane or mitochondria) or also as a superimposition of all of the fluorescence images (overview) (Figure 27).
  • Page 44 10. Software STEVE Acquiring fluorescence images How to create multi-channel images To perform a single shot, click on the 3D button or on the Single Shot from the Microscope menu. A selection dialog will appear to choose the acquisition type which can be individually set up (Figure 28). Once a refractive index and the fluorescence channels are selected, a new dialogue will appear to adjust the fluorescence channel parameters (Figure 29).
  • Page 45 10. Software STEVE Acquiring fluorescence images To be able to acquire good fluorescence images, follow our guidelines hereafter. At the time the dialogue appears you are able to adjust the fluorescence channel parameters, in order to acquire the best image possible (Figure 30). Live-image of the selected channel Tab for setting-up each of the selected channels Tab for activating the white-light mode and perform bright-field of the sample...
  • Page 46 10. Software STEVE Acquiring fluorescence images For the best image quality, keep the intensity as low as possible in order to avoid bleaching while you search for a suitable fluorescent cell. You will need to maximize the gain to be able to see something. At the same time, the exposure time should be typically below 200 ms, to have enough frame refreshing rate and screen comfortably the sample (Figure 31).
  • Page 47 10. Software STEVE Acquiring fluorescence images 3. To acquire an image without background, adjust the intensity parameters. The intensity should stay low (<20-30) (again to minimize photobleaching). Now, reduce the gain until the background disappears. The maximum gain to be used can be up to 40%. Increase the exposure time if you need to see more details of the image but try to keep it as low as possible (100 ms or lower = very good;...
  • Page 48 10. Software STEVE Displaying multi-channel images When you visualize a multi-channel image, the overlay parameter must be set to 0 (Figure 33). The fluorescence control area in the lower right part of your software window provides access to all of the fluorescent channels (Figure 34). •...
  • Page 49 10. Software STEVE Time-lapse acquisitions How to do a time-lapse acquisition In a time-lapse acquisition, multiple 3D reconstructions in a range of time are attained. To perform a time lapse, click on the 4D button or on Time Lapse in the Microscope menu. The mounting medium refractive index selection dialog will appear (see section "How to select the mounting medium’s refractive index"...
  • Page 50 10. Software STEVE Fluorescence time-lapse acquisition Acquiring fluorescence time-lapses To perform a time lapse, click on the 4D button or on Time Lapse in the Microscope menu. A selection dialog will appear to specify the speed and the duration of the acquisition, which can be individually set up (Figure 36). Once the settings for the time lapse, the refractive index and the fluo- rescence are selected, a new dialogue will appear to adjust the fluorescence channel parameters (Figure 37).
  • Page 51 10. Software STEVE Time Line Area -fluo While the 3D Cell Explorer acquires multiple 3D reconstructions to create a time-lapse, you will be able to follow the exact chosen fluorescent and refractive index parameters for each frame in the lower part of the STEVE interface (Figure 38). Figure 38 Time Line Area go back to contents à...
  • Page 52 10. Software STEVE Digitally staining How to digitally stain cells with STEVE 1. Choose a meaningful slice on the 2D visualization panel (left side). • Dragging the mouse up to down (left click pushed) on the 2D visualization panel or by moving the Slices slider. 2.
  • Page 53 10. Software STEVE Digitally staining 7. How to manipulate your stain: • Change the opacity (Opacity slider or drag up-down with the right click pushed). • Change the edge softness (Edge softness slider or drag up-down with the right click pushed on a stain edge). •...
  • Page 54 10. Software STEVE Data Saving How to save your data The data can be saved by clicking on the Save icon (Figure 39). The holograms are kept by default within the .vol file. Figure 39 Saving Window go back to contents à...
  • Page 55 10. Software STEVE Data Saving STEVE allows to save data as a 3D or a 4D .vol file (readable only with STEVE). Alternatively, it is possible to export the data in other file formats by opening the export dialog shown in the Figure 40. Figure 40 Export data setting The data can be exported by choosing the following parameters: •...
  • Page 56 10. Software STEVE Data Annotation How to work with data annotation For each measurement you can visualize and modify the measurement properties, called also data annotation. In the File Menu under “Properties” open the annotation dialog (Figure 41). All the visible properties, marked with “Eye icon” will be added to the current measurement after the button “Apply”...
  • Page 57 10. Software STEVE Software Updates How to update the software Steve will automatically update when a new version is available. The user will be alerted to close Steve and the Steve Maintenance Tool will start. In the Steve Maintenance Tool choose “Update components” and click the “Next” button. Follow the messages provided by the update dialog. go back to contents à...

  • Page 58: Troubleshooting

    11. Troubleshooting Calibration Calibration failed err.1.1 If applicable, reduce the quantity of liquid above your sample (-> Check our Sample Preparation Protocol in Section 5). Check that no opaque object can intersect the laser beam while the arm is rotating. If using a dish, check that you are imaging close to its center such that the laser beam cannot intersect the wall of the dish instead of the top.
  • Page 59 11. Troubleshooting Connection failure err.3.3 STEVE cannot establish communication with the 3D Cell Explorer. The port to which the USB 3.0 cable (coming from the camera) was connected to was recognized as USB 2.0. This cable must be connected to a USB 3.0 port. If you have correctly connected the USB 2.0 and USB 3.0 cables and the error persists, please verify that your USB 3.0 adapter drivers are properly installed or contact your system administrator.

  • Page 60: Compliance

    WEEE All qualifying products that are subject to the WEEE Directive and supplied by Nanolive are compliant with the WEEE marking requirements. Such products are marked with the "crossed out wheelie bin" WEEE symbol and in accordance with European Standard EN 50419.

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