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Tilt

User’s Guide

Assembling Mizar

Cube Chambers

Everything you will need to get started:

Mizar Cubes, Mizar Cube holder w/ helper insert, 22x40mm #1.5 coverslips,
silicon vacuum grease

How to assemble Mizar Cube imaging chambers

Spread a thin layer of vacuum grease on the inside of a petri dish, or similar flat surface.

Using a gloved hand to avoid fingerprints, firmly press a Mizar Cube into the thin
layer of grease; inspect the bottom of the Mizar Cube for complete coverage with
the grease.

Содержание Tilt

Страница 1: ...Tilt User s Guide 1 www mizarimaging com User s Guide v1 0 Tilt ...

Страница 2: ...tents Warnings and Safety 3 Definitions of Safety Symbols 3 Warnings 4 Safety Features 5 How theTilt Works 6 Getting to KnowYourTilt 7 Getting to KnowYourTilt 8 AdjustmentTips 9 How to Image 10 Advanced Alignment and Installation 12 Installation 12 Aligning Tilt Optical Components 13 Tips for Mounting Samples 17 Recommended Specimen Chambers 18 Assembling Mizar Cube Chambers 19 Troubleshooting 21 ...

Страница 3: ...review the following safety information before using the Tilt Definitions of Safety Symbols SYMBOLS USED IN THIS MANUAL Warning Notice Instructions containing a warning notice detail the hazards and safe operation of the Tilt Ignoring these safety instructions can result in serious injury or property damage SYMBOLS USED ON THE TILT Laser Hazard Label This symbol is displayed near the laser output ...

Страница 4: ...nd Use of the Tilt for any other purpose including the use of unsupported microscope stands and laser products can result in serious injury and property damage Complete information on microscope stand compatibility and supported laser products can be obtained from Mizar Imaging 3 Installation Installation of the Tilt must only be performed by a trained service professional Improper setup may impai...

Страница 5: ...bove the sample and is intended to protect the user from stray or reflected laser light II LightTrap The light trap is placed opposite of the optical assembly aperture and pre vents further travel of any laser light that has passed through the sample field III Locking Screws The locking screws are located on each of the Tilt sled arms and prevent removal of the sled optical assembly from the stage...

Страница 6: ...ld be too narrow to cover the field of view of the objective resulting in poor illumination of the sample To solve this problem we use a photomask containing four slits before the beam enters the cylindrical lens This method Golub et al 2015 has the effect of creating an interfer ence pattern at the focal point of the cylindrical lens This mathematically and in practice elongates the sheet to 300 ...

Страница 7: ...p and down above the objective Adjust the Green knob to position the light sheet vertically at the focal point of your objective 2 LATERAL ADJUSTMENT BLUE Adjusting the Blue knob will slide the Tilt sled forward and back Fig B thereby changing the lateral position of the light sheet Use the Blue knob to center the focused light sheet over your objective 3 REAR HEIGHT ADJUSTMENT PINK Turning the Pi...

Страница 8: ...8 www mizarimaging com Tilt User s Guide Getting to Know Your Tilt ...

Страница 9: ...he optic assembly or further away depending on which way you are turning the knob 5 While continuing to turn the Blue Focusing knob place your other hand on the Green Angle Adjustment knob and begin to fine tune the lightsheet Try to get the narrowest lightsheet you can directly above the objective lens 6 Once you have developed a sense for how the adjustments of both the Blue and Green work focus...

Страница 10: ...mple chamber and place sample chamber onto the sample holder platform For detailed tips on mounting samples seeTips for Mounting Samples on page 17 See also Recommended Specimen Chambers on page 18 3 Coarse Fine Focus the Lightsheet 3 1 Locate specimen and focal plane of interest With the laser off use any type of transmitted light to find your sample Focus on the ap proximate focal plane of inter...

Страница 11: ... the Tilt to check angle of the your lightsheet Be sure to zero the inclinometer on the stage prior to measuring If your lightsheet is outside normal operating range 3 3 0 5 use the Pink Rear Height Adjustment knob to raise or lower the back part of the Tilt Re adjust the Green knob so that your sample is illuminated 4 Ready to Image 4 1 Adjust laser power and camera settings Once you have complet...

Страница 12: ...ting microscope stage 1 4 Install theTilt optical assembly Place the Tilt optical assembly on top of the three adjustable posts above the Green and Pink knobs on your Tilt stage Attach a single mode fiber from your laser launch to the rear of the Tilt optical assembly To prevent removal of the optical assembly from the stage install the provided screws through the holes in each the top and bottom ...

Страница 13: ...s initial installation and only on rare instances will it become necessary to perform a realignment If you feel yourTilt optical components require realigning call our trained scientists at 1 508 939 7500 TO BE CARRIED OUT ONLY BY INSTALLER OR CERTIFIED ENGINEER Inside the Tilt enclosure there are two mirrors used for aligning the collimated laser with the lens assembly Each has two screws for adj...

Страница 14: ...a tively use a laser launch set to 2mW 2 Remove the collimating lens by loosening the three set screws and pulling straight back 3 Set the lens on a flat surface and ensure it is pointed away from nearby people 4 Turn on the laser and aim it at the alignment template Adjust the beam by turning the threaded head of the collimating lens until the beam fills the target 5 Move the template along the b...

Страница 15: ...ns is properly matched with the fiber 3 Turn on the laser You should see an ellipse projected onto the alignment target 4 Using the adjustment knobs on each of the mirrors center the ellipse both vertically and horizontally within the target making sure to fill the target ellipse as much as possible and that the ellipse is brightest in the center Refer to the tips and figure on the next page for d...

Страница 16: ...speaking Mirror 2 will be used to set the final vertical and lateral positions and Mirror 1 is used to fill Mirror 2 Issues with ellipse width height too narrow short rotation twisting or clipping result from poor alignment of Mirror 1 Adjust knobs 1 and 2 until the ellipse almost fills the template centering the ellipse on the target with knobs 3 and 4 on Mirror 2 as you go If the beam is not ent...

Страница 17: ...ration of the lightsheet Here are several tips for minimizing obstructions Keep the sides of your sample chamber clear of fingerprints oil grease etc Make sure there is adequate medium in your sample chamber The meniscus can have a lensing effect and distort the incoming lightsheet If you use agarose or similar mounting gel make sure you fill the entire well either with the gel or a medium that ma...

Страница 18: ...lass is ideal for lightsheet imaging ibidi μ Slide 2 4 8 Well chambers The ibidi μ Slide 2 4 and 8 well chambered coverslips come pre sterilized and are a good disposable option for general use and cultured cells Many scientists already comfortable with culturing cells on chambered slides prefer this option as only minimal changes in workflow and sample prep are required for successful lightsheet ...

Страница 19: ...t 22x40mm 1 5 coverslips silicon vacuum grease How to assemble Mizar Cube imaging chambers 1 Spread a thin layer of vacuum grease on the inside of a petri dish or similar flat surface 2 Using a gloved hand to avoid fingerprints firmly press a Mizar Cube into the thin layer of grease inspect the bottom of the Mizar Cube for complete coverage with the grease ...

Страница 20: ...ay a 22x40mm coverslip on top 4 Align the Mizar Cube squarely with the coverslip and press firmly to seal the Mizar Cube 5 The assembled Mizar Cube chamber is now ready to loaded with your sample Alterna tively a sample can be immobilized on a 22x40mm coverslip prior to sealing the Mizar Cube chamber around it and filling with media ...

Страница 21: ... of the Tilt using an inclinometer Use the Pink adjustment knob to lower the angle back to 3 3 Centering the lightsheet in your field of view is more critical at low magnifi cation as the entire 300μm waist will be in use Make sure the Tilt angle is optimal at 3 3 and use the Blue knob to better center the lightsheet to your image Review pages 7 11 for more tips on adjusting the lightsheet Dim Ima...

Страница 22: ...Tilt User s Guide 22 www mizarimaging com 7 MBL Street Lillie 220 Woods Hole MA 02543 1 508 939 7500 info mizarimaging com www mizarimaging com ...

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