Light Sheet Microscope Z1 (SPIM) tutorial

1T.Laroche V: 3.0 20180410

Light Sheet Microscope Z1

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Before using the Light Sheet Microscope Z1 ,please reserve the microscope on the reservation system =

https://sv-bbs.epfl.ch/ptbiop/

Light Sheet Microscope Z1 (SPIM)

• Reservation System.• System Start up (page 4-8)• Setup Pathway (page 9-16)• Setup Hardware (page 17-24)• Dichroic Filter Change (25-26)• Preparing > 2mm small sample (mounting) (page 27-30)• Preparing > 4mm large sample (mounting) (page 31-32)• Preparing < 4mm very large sample ( mounting) (page 33-44)• How to acquiring an image (page 45-51)• System Shut down (page 52)

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System Start Up

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Front cavitydoorFront

systemdoor

Upper system cavitydoor

Upper rearsystem

door

System Start Up

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1) Turn on SYSTEM

2) Turn on PC

3) Turn on INCUBATION

4) Turn on the Computer

System Start Up

• Once you have entered your login on the microscope PC, you need also to entered your login on the PC workstation,or SWAPP.

• PLEASE NEVER TURN OFF THE WORKSTATION, JUST DO A LOG OFF

• Select Start System.

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System Start Up

• Once you have entered your login, you will see the ZEN icons.• To start it double click it.

• Select Start System

• During the system initialisation, a displaycan appear like: Please remove sample, Press OK to continue the initialisation.

• Remove your sample from the XYZ Rotationholder (can some damage duringthe initialisation). Press OK

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System Start Up

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• An Error Log window can appear on the left corner at the bottom of the screen.

• This Error Log, appears when the cell chamber is not connected to the temperature /CO2 controller. Connect the cell chamber to the microscope, or if you use a cell chamber without temperature controller, select Clear Errors or Cancel.

Setup

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• Once Zen is initiailised, select the objectives you want to used.• Select Maintain / Objectives/Illumination Optic/• Select the objective icon.• Select Objective.• Choose the appropriates illumination objective.

• LSMFM 5x/0.1.• LSMFM 10/0.2.• LSMFM 10/0.2 clearing.

• The Illumination Optic are the 2 objectives for the light sheet illumination.

Setup Pathway

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• Select Maintain / Objectives/Detection Optic/• Select the objective icon.• Select Objective.• Choose in the liste, the appropriates detection objective.

• EC Plan-Neofluar 5x/0.16.• W Plan-APOCHROMAT 20X/1,0 UV-VIS.• W Plan-APOCHROMAT 40X/1,0 VIS-IR.• LSFM 20X/10 Corr clearing.

• The Detection Optic is the objective that collects the image (emission).

Setup Pathway

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• Select Light Path.• Select the Laser Icon.• Select the appopriate laser wavelenght.• Use 4-5% laser intensity for each laser that you selected

(you can select 2 lasers in the same time).

Setup Pathway

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• Select Mirror, here you choose your appropriate,dichroic filter like:

DAPI/510/GFP = For Dapi and GFP like fluorophores.DAPI/510/RFP = For Dapi and RFP like fluorophores.GFP/560/RFP = For GFP and RFP like fluorophores.GFP/560/LP-RFP = For GFP and RFP like fluorophores.GFP/560/F_RED = For GFP and Far red like fluorophores.

Setup Pathway

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To acquire GFP and Cy5 =Laser 488 and 638 @ 4%Dichroic GFP/560/F_RED Camera 1 /BP 505-545 = GFPCamera 2 / LP 660 = Cy5

To acquire GFP and tdTomatoes =Laser 488 and 561 @ 4%Dichroic GFP/560/LP-RFP Camera 1 /BP 505-545 = GFPCamera 2 /LP 585 = tdTomatoes

Setup Pathway

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To acquire DAPI and GFP =Laser 405 and 488 @ 4%Dichroic DAPI/510/GFP Camera 1 /BP 420-470 = DAPICamera 2 /BP 525-545 = GFP

To acquire DAPI and RFP =Laser 405 and 561 @ 4%Dichroic DAPI/510/RFP Camera 1 /BP 420-470 = DAPICamera 2 / BP 575-615 = RFP

To acquire GFP and RFP =Laser 488 and 561 @ 4%Dichroic GFP/560/RFP Camera 1 /BP 505-545 = GFPCamera 2 / BP 575-615 = RFP

Setup Pathway

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• You can build a Multitrack set-up, for a sequential acquisition, in case you have 3 or 4 fluorophores, or if you suffer from cross-talk.

• Here an example show you a configuration with only one track.

• If you have 3 or 4 fluorophores add a new track.

Setup Pathway

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• HeConfiguration for the acquisition of 4 fluotophores with two tracks.:

Track1 DAPI+RFP Track2 GFP+Cy5

• As a starting point, Select Channels and use an Exposure Time of 100 ms, for each Track.

Setup Hardware• Choose a lens matching the refractive index of the mounting medium.• Typically the EC Plan-Neofluar 5x/0.16; the W Plan-APOCHROMAT 20X/1.0 UV-VIS and the W Plan-

APOCHROMAT 40X/1.0 lenses are used for specimens mounted in a media with a refractive index close to n=1.33 (e.g. PBS/water).

• Typically the LSFM 20x/1.0 Corr clearing lens is used with the cleared fixed specimens mounted in a medium with a refractive index close to n = 1.45 e.g. Cubic or Histodense. It is also possible to use the EC Plan-Neofluar 5x/0.16 for cleared specimen if an adaptor ring is added at the bottom of the lens.

• Mount the Detection Lens first (placed at the back of the microscope cavity), then mount the 2 Illuminations lenses (on the side of the microscope cavity).

• Different lens configuration.

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Condition n Detection Lens Illumination Lens RingWater/PBS 1.33 EC Plan-Neofluar 5x/0.16 LSMFM 5x/0.1 Adapter Ring 1.33Water/PBS 1.33 W Plan-APOCHROMAT 20X/1,0 UV-VIS LSMFM 10/0.2 Adapter Ring 1.33Water/PBS 1.33 W Plan-APOCHROMAT 40X/1,0 VIS-IR LSMFM 10/0.2 Adapter Ring 1.33

Cubic/ Histodense 1.45 LSFM 20X/10 Corr clearing LSMFM 10/0.2 clearing No RingCubic/ Histodense 1.45 EC Plan-Neofluar 5x/0.16 LSMFM 5x/0.1 Adapter Ring 1.45

Setup Hardware

• Under the table of the pc workstation, there is a container with 3 drawers.• The top drawer contains the lenses.• The middle drawer contains capillary tube, piston, holders, O-ring, and coverslide.• The bottom drawer contains the samples chambers.

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Set LSFM 20x 1.0 Corr clearing Lens

• To acquire images with the LSFM 20x 1.0 Corr clearing (1) detection lens you need to use:• The 2 LSMFM 10x/0.2 (2) clearing illuminations lens.• The 20x Clearing Sample Chamber (3).• The Fixed Filterwheel. (4) See page 24-25

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1.

2. 2.

3.

4.

Set EC Plan-Neofluar 5x/0.16 Lens

• To acquire images with the EC Plan-Neofluar 5X/0.16 detection lens you need to use:(1)• The 2 LSMFM 5x/0.1 illuminations lens.(2)• The 5x Clearing Sample Chamber(3) if you use clearing medium or.• The 5x 1.33n Sample Chamber(4) if you use PBS medium.

• You need to fix the 1.45 n Ring (5)on the back side of the 5x Lens, if you use clearing medium and the Motorised Filterwheel (7).

• You need to fix the 1.33 n Ring (6) on the back side of the 5X lens,and the Motorised Filterwheel (7) if you use the PBS medium . See page 24

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1.

2. 2.

3. 4.

5. 6.7.

Set LSFM 20x, 40x or 60x 1,0 W Lens

• To acquire images with the LSFM 20x,40x or 60x 1,0 W Lens (1)detection lens you need to use:• The 2 LSMFM 10x/0.2 illuminations lens (2).• The 20x 1.33n Sample Chamber (3).• Fix the 1.33 n Ring on the back side of the Lens (4) .• The Motorised Filterwheel (5). See page 24-25

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1. 1. 1.

2. 2.

3.

4.5.

Set LSFM 20x 1,0 W Lens

• View of the LSFM 20x 1.0W Lens + the Ring

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Setup Hardware

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• Open the Front Cavity Door.

• Insert and fixed carefully the Detection Lens in theback of the microscope cavity.

• Insert and fix carefully the two Illumination Lensplaced at the 2 sides of the microscope cavity.

Setup Hardware

• Introduce carefully the Sample Chamber, and screw it correctly(1).

(1)

• If, as the photo shows, you use the Sample Chamber 20x 1,33n, it is also necessary to fix the 2 electricals connections, and the 2 liquids connections, to the microscope, otherwise if you use another Sample Chamber, skip this step.

• Close the microscope Front Cover.

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(2)

(3)

(2)

Setup Hardware, Filter Change

• Depending on the lens used, it will be necessary to change the dichroic wheel behind the lens.

• Open the Upper Rear System Door, which is on the top of the microscope.

• Unscrew the two screws that close the lid, and remove the lid of the cavity where the dichroic filter is located.

• Push the lock which releases the turret containing the filter.

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Setup Hardware, Filter Change

• Pull the filter turret, and put it in the first slot of the top of piece of furniture .

• The motorized filter is used for the following purposes: LSFM 5x, 20x, 40x or 60x 1.0 Water Lens.

• The fixed filter is used specifically for the following purpose: LSFM 20x 1.0 Corr Clearing only.

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Preparing a small sample

• Preparing a sample embedding container with a capillary.• We supply 4 different diameter capillary glass, depending on the size of the sample,• # 4 ref: 701910 (Brand) = 2.15 mm diameter / 100 ml volume • # 3 ref: 701908 (Brand) = 1.5 mm diameter / 50 ml volume • # 2 ref: 701904 (Brand) = 1 mm diameter / 20 ml volume• # 1 ref: 701902 (Brand) = 0.68 mm diameter / 10 ml volume

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• Introduce the Plunger into the Capillary tube, until the plunger is at the bottom of the capillary. Use the plunger as a small pump.

• Transfer the sample into final 1% agarose low temp (Roth, n° 6351.1),diluted with water or PBS, in a Eppendorf tube at 38 C°.

• Quickly, pump the sample with agarose until the blue mark and visible on the plunger.• Remove the agarose that is around the capillary ( clean it).• Put the capillary at 4C° for 5min.

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Preparing a small sample

• Introduce the two Sleevesto the Sample Holder(each one have a coded color) that corresponds to thecolor of the capillary, ( e.g. blue).

• Introduce the Sample Holder to the Sample Holder Disc.

• Introduce the Clamp Screwto the Sample Holder.

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Preparing a small sample

• Introduce the capillary into the Sample Holder untilthe Blue Mark is visible to the end of the holder.

• Screew correctly the Clamp Screw to prevent the capillary from moving.

• Open the Upper System Cavity Door.

• Insert the Sample Holder into the Light Sheet microscope.(the White mark on the disc holder must correspond to thewhite mark which on motorised stage).

• Close the Upper system Cavity Door.

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Preparing a small sample

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Preparing a > 4mm large sample

• A) Use a 1ml Syringe (Braun #9161406V)• B) Cut away the tip of the syringe• C) Pump with the Plunger 0,1-0,2 ml of 1% Low melt agarose.• D) Insert the sample (>4mm large) into the syringe.• E) Pump 0,1-0,2 ml of 1% Low melt agarose.

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• There are two components to secure the syringe: the Sample Holder for the syringe, and the Ring Adapter.

• Insert the syringe into the ring adapter.

• Insert the syringe into the Sample Holder Disc, rotate the syringe to clip it into the sample holder.

• Introduce the Sample Holder Disc into the microscope (the white mark on the disc holder must correspond to the white mark on motorized stage).

• Close the cap.

Preparing a > 4mm large sample

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• Custom made holders are available to mount a specimen that is significantly thicker than the syringe.

• The disadvantage of these tools is that it is necessary to stick the specimen to the tool, and therefore a place of the specimen becomes unusable.

• The cyanolite-based adhesive is used.

Preparing a <4mm very large sample.

• The tip of the Syringe (1) has been cut, a Magnet 4mm diameter (2) is inserted into the syringe, the magnet should extend beyond the tip of the syringe.

• A small plastic Cap of 5 x 5 mm square shape (3) , where one end has a screw thread, in which a flat head screw can be attached. The Iron Screw (4) is maintained by the Magnet(5).

• Samples of 1cm cube size can be fixed on the Cap with glue based on cyanolit.

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(1)

(2)

(4)

(5)

(3)

Preparing a < 4mm very large sample with a Cap.

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• In this example, the Screw is longer (1.5 cm) than the first example, which makes it possible to bring the material closer to the lens; because the outer diameter of the syringe is 6mm, and the screw is only 2mm in diameter, the probability that the syringe comes in contact with the cell chamber and weaker than in the first example.

• At the same time, the risk that the syringe will come into contact with the medium which is in the cell chamber, and is less than the first example.

Preparing a < 4mm very large sample with a Cap 1.5 cm long.

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• In this example, the syringe is cut half (1) to the length, the Magnet (2) is fixed to the end of the syringe, a piece of LEGO ID:395726 (Black Antenna) (3) whose bass to a cavity allows to fix an 4mm diameter iron washer (4), The other end of the antenna will be used to fix a sample with cynolit-based glue (4).

• The Antenna is very long (3.5 cm) and very thin (3mm), in this example, it possible to bring the material very close to the lens because the thickness of the screw is thinner than the syringe, thereby It does not come in contact with the top of the cell chamber.

• Furthermore, it makes it possible to move the sample in the y-axis without the syringe coming into contact with the edge of the cell chamber.

• The disadvantage of the antenna and that it is not possible to stick too large and large samples because there is a risk that the point of adhesive is not sufficient to maintain the weight of the sample.

• A sample of 5x5 mm is good.

(2)(1)

(3)

(4)

(5)

Preparing a < 4mm very large sample with a antenna.

• In this example, the antenna LEGO (1) is even longer (5.5 cm) and very thin is now possible to bring the material very close to the lens because the thickness of the screw is thinner than the syringe, thereby It does not come in contact with the top of the cell chamber.

• Furthermore, it is possible to move the sample along the y-axis by avoiding a contact between the syringe and the edge of the cell chamber.

• It is not possible to stick extended samples because there is a risk that the weight of the sample is too heavy.

• Typical sample size: 5 mm x 5 mm x zz mm

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Preparing a<4mm very large sample with a antenna 5.5 cm long.

(1)

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• In this example, this antenna (levier de vitesse) LEGO ID:73737 which is mobile (1), it possible to give an angle of the sample with respect to the objective

• The disadvantage of this antenna is that it is not possible to fix too heavy and too big samples.

• Typical sample size: 3 mm x 3 mm x zz mm.

(1)

Preparing a < 4mm very large sample with a mobile antenna.

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Preparing a < 4mm very large sample with a iron rod.

• This custom built holder was desigend to mount cover-slips.

• It is made up of a Plastic Cover (A) (6,15 cm long and 0,7 cm in diameter),

• An Iron Plunger (B) (7,35 cm long and 0,5 cm in diameter) with 2xO ring;

• An Iron Rod (C) (3.5 cm long and 0,25 cm in diameter) and one of its end a Tray (D) of 7mm long, 4mm wide and 1mm thick

• The holder is very stable, in the sense that the rod can be fixed to the plunger with high precision in the XY axes, which allows to keep the samples stably in front of the acquisition objective. The plunger has at its end a cavity containing a Magnet (E) , the iron rod will easily inserted into the plunger cavity.

A

B

D C

E

E

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• A 24mm x 5 mm cover-side is cut with a diamond pencil, this cover-slide is held with a Magnet (F) on the flat end of the iron rod.

• The sample can be glued, with cynolit based glue on one side of the cover-slip.

Preparing a < 4mm very large sample with a iron rod.

F

• For all mounting methods where the sample is bigger than the syringe, you must first assemble the syringe (with the magnet )in the sample holder. Be sure that the holder and position as high as possible.

• Then insert the sample attached to one of the supports developed by the BIOP trough the main front door of the microscope,

• Be sure that the support comes to fix on the magnets of the syringe, • Introduce the cell chamber, and fix it.• Fill the cell chamber with the desired media.• Close the main door.• Bring down the holder/sample into the cell chamber, allowing the sample to plunge into the medium.

• Please follow step by step the following steps which are described in the following page.

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Preparing a < 4mm very large sample.

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• There are two components to secure the syringe: the Sample Holder Disc for the syringe, and the Ring Adapter.

• Insert the Ring Adapter on the syringe.

• Insert the syringe into the Sample Holder Disc, rotate the syringe to clip it into the Sample Holder Disc.

• Introduce the Sample Holder Disc into Motorized Stage(the white mark on the disc holder must correspond to the white mark which on Motorized Stage).

• Close the cap.

Preparing a < 4mm very large sample.

• Deposit the sample delicately on a sheet of Parafilm (1) with a tweezer, remove the Histodenz or Cubic as much is possible from the sample; e.g. by using Kimtech paper.

• Place a drop of Cyanolit-based (2) glue at the end of the holder.

• Paste the sample, and maintain a slight pressure for 15 seconds.

• Now the sample is ready to use.

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Preparing a <4mm very large sample.How to fix the sample on the holder (BIOP).

(1)

(2)

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• Open the main front door.• Then insert the sample fixed on the antenna or another of the supports

developed by the BIOP.

• Be sure that the support comes to fix on the Magnetsof the syringe.

• Bring, with the Joystick or software; The highest possible Holder + Sample.

• Introduce the Cell Chamber, and fix it.• Fill the cell chamber with the desired media.• Close the main door.• Bring down the holder/sample into the cell chamber, allowing the sample to

plunge into the medium.

Preparing a < 4mm very large sample.How to fix the samble on the holder (BIOP).

• Select Locate Capillary.

• Into the Specimen Navigator, use the Y-axis to lower the capillary/syringue until it is seen.

• Push a bit the capillary plunger to remove the sample out from the capillary tube (embedded by agarose) until you see the sample.

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Acquire images

• Select Locate Sample

• Into the Specimen Navigator

• Use the X-axis to move the specimen in left or right position

• Use the Y-axis to move the specimen from the top to the bottom position.

• Use the Z-axis to focus the specimen

• Use the icon to rotate the specimen

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Acquire images

• Set the laser / dichoic filter and the ccd.• For the different possible configurations,

please reefer to the page 12.

• For example a configuration for the GFP, (laser 488 @ 4% and exposure of 100msec).

• Select Continuous.• If the grey level is to low, select Auto under

the Display.• Now you can use the joystick to move/focus

the sample.

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Acquire images

• Under Acquisition Mode there are several options available.

• The Zoom, within the zoom range from 0.36 to 0.7 the image quality toward the edge can decrease.

• The Single Side illumination is displayed by default from the Left Direction.

• When Dual Side when Experiment is chosen the sample is illuminated by the light sheet from left and by the light sheet from the right sequentially, producing two image for each plane.

• If Online Dual Side Fusion is selected, the image are fused successively after being acquired

• The Pivot Scan is used to move the light sheet in the focal plane of the detection optics in a wavelike motion, this result is a reduction of shadows wich might otherwise be cast by optically dense structures within the sample.

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Acquire images

• If Dual Side when Experiment is selected the light sheet should be aligned, first adjusts the Right laser correctly, then the Left, otherwise adjust it automatically, by selecting Auto-Adjust, and follow the wizard.

• If you want to do the alignment yourself, proceed as follows:

• Select the Right laser, select Continuous, focus in the desired region, look at the image and change the Rightlaser offset, this value in general, is not very far from 0.

• If the adjustment is correct, the image should be the most sharp and intense possible; if not, change this value by moving away from 0.

• Once this value has been found, it is necessary to do the same operation but with the Left laser, often the value found on the Right laser will be positive, and the value of the Left laser will be the same (or close) value but expressed in negative.

• Be aware when you change the Zoom during the acquisition session, the light sheet alignment needs to be done again.

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Acquire images

• Advanced button informs you about the thickness in the center and in the border of the created light sheet in microns within the field of view. To manually change the ticknessof the center thickness use the slider.

• To return to the ideal light sheet thicknessratio, use the Zoom slider in the AquisitionMode tool window.

• The ratio or thickness in the center of the light sheet and at its border should be ideally1:2 for the use frame.

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Acquire images

• Quick steps to a Z-Stack

• Select Z-Stack in Multidimensionalacquisition field to acquire an imageseries in Z.

• Select Continuous to start a live image acquisition using the currently active imaging parameters for defining the First Slice and Last Slice within the Z-Stack control panel.

• Pressing the Optimal button sets Iinterval to a value of half the opticalslice thickness.

• Click Start Experiment to beginrecording the Z-stack.

• Save your data to the SWAP underyour Folder name

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Acquire images Z-Stack

System Shut down

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1) Remove the sample and the Holder from the microscope. 2) Remove the Cell Chamber.3) Do a shutdown through the PC.

4) Turn OFF SYSTEM.

5) Turn OFF PC.

6) Turn OFF the INCUBATION.

7) After stopping the microscope and the pc, please wash the cell camber, the syringe and the tube correctly with water.

Wipe and dry the small cell chamber window correctly with KIMTECH paper.