Confocal Microscope
Switch on/off
1. Switch on 4 plug sockets on wall.
2. Switch on discharge switch for laser 1 on small black box.
3. Turn on laser 2 by turning key to on, and pressing ignition button on box under bench
(microscope side of bench).
4. Turn mercury lamp on and press ignition for fluorescence (if not using eye pieces, you don’t
need this). Make sure you note down the numbers on the counter at the beginning and at
the end of the session.
5. Turn on other 2 boxes next to mercury lamp (for bright field, next to mercury lamp, and
bottom one for stage of microscope).
6. Switch on microscope (switch at back RHS), then switch on hardware computer (cream
machine under bench), then software computer (black machine on shelf). Password is the
same as on screen name, and program is the rainbow icon (EZ-C3-391).
7. To turn off, turn off key on laser 2,( or red button and key), switch discharge switch to off
on laser 1,but do not switch off at plugs until the fan stops as this cools the laser cool down.
The fan will switch off automatically when cooled. Switch off other boxes and computers.
Need to leave Tungsten on for at least 30mins per session or you will ruin the bulb. Note
down numbers on counter on sheet.
Getting ready to view
1. Insert plate for either plates or slides using the 3 screws. Adjust by pressing the 2 buttons
down.
2. Pull out slide at back top for bright field, and push in for confocal.
3. Slides should be used coverslip down.
Checking pathway of condenser and bright field light (see diagram)
Field Diaphragm
Focusing knobs
Contrast diaphragm
Polarizer
Centring screws
Phase ring needs to be
turned to “A” to do this!
1.
2.
3.
4.
5.
Focus your sample.
Close field diaphragm.
Use focusing knobs until you see a sharp hexagon through the eye pieces.
Use centring screws to centre the hexagon.
Open the field diaphragm until you can’t see the hexagon.
Phase contrast
1. After centring bright field, check phase ring is set as on objective.
2. To check alignment of phase ring, select Bertrand lens under eye pieces. Use focus wheel
(white small wheel on side). You will see light and dark circles. To align these, use 2
screwdrivers in the 2 screws on the phase ring.
DIC
1. Polariser needs to be in light beam (see diagram above). Move out at other times. Moving
ring alters where the shadow is. Phase ring needs to be on DICN2. Slider needs to be clipped
under the 60X objective.
Fluorescence
1. ND4, ND8 filter (right arm) and back slider (marked F) all need to be pulled out otherwise
you will get a dark shadow on image.
PFS
1. This is for time lapse experiment. Turn “on” using the buttons on the front of the
microscope. When not flashing it means it is in focus. Use separate wheel now to focus. D
slider at top needs to be pushed in.
Buttons
1. On sides of microscope – light intensity, changing objectives etc.
2. L100 – confocal, there is no R100, L80 – will turn lasers off!!!
3. Black knob when upright = 1X, if turned = 1.5X.
Joystick
1. Moves the stage. Twist to change setting of the red light.
Confocal
Needs to be on L100. Program is EZ-C3-391. Confocal button is 5th from right (on screen?).
Turn gain down to decrease saturation. Offset should be in middle (127).
Laser depends on the dye on your cells. Detector is filter for detection.
The higher the required resolution, the more pixels you need. However,1024x1024 is 4 times
slower than 512x512.
5. Pinhole changes depth of section. Small = low light but good image for a thin slice of tissue.
Average = if fuzzy channel, can average several images. Frame Lamda = averaging, improves
signal to noise ration what results in a nicer image.
1.
2.
3.
4.
Time Lapse Experiment
1. You can set up how often you want to scan and mark where you have added reagent.
XY
1. You can zoom or crop. Zoom produces higher resolution therefor better image. Crop just
reduces the size of your image. Click on the cube (top right of screen) to use
2. Reset sets screen back to the live scan.
3. Can angle back by curved cursor in the middle of the zoom/crop square.
Spectral
1. This can be useful if several dyes spectrums overlap considerably or if there is a lot of auto
fluorescence. On top of confocal port (on L) is a black switch to select spectral imaging. Ours
has 32 grating (apparently very good ).
Saving Images
1. Save images as ics/ids files as this will save images and all of the data associated with it.
Nikon C1Si Confocal Microscope
Location: Robert Kilpatrick Clinical Sciences Building, room 229
Contact: Sonja Khemiri (scj6; ext.5837) and Jonathan Barber (jrb15, ext.3135)
Specifications for Nikon C1Si confocal laser scanning
microscope
The Nikon C1Si CLSM is built on a Nikon eclipse Ti microscope stand equipped with 4 lasers
resulting in the laser lines:
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405 nm
457 nm
488 nm
514 nm
561 nm
640nm.
The system is filter block based and has 4 detectors (PMTs) which can be used as 3 fluorescence and
one transmitted light detector or in a configuration with 4 fluorescence detectors. Besides the standard
PMTs this system has also a spectral detector unit with an array of 32 PMTs. This array makes for fast
spectral imaging with a resolution of 1.5 nm, 5 nm or 10 nm and is the fastest microscope based
spectral imager in our facility.
Objectives on the microscope:
- CFI Plan Fluor 10x (NA = 0.3) phase contrast (dry!)
- CFI Super Plan Fluor 20x (NA = 0.45) extra long working distance (dry!)
- CFI Super Plan Fluor 40x (NA = 0.6) extra long working distance (dry!)
- CFI Plan Apochromat VC 60x (NA = 1.4) oil
Training:
If you wish to use the microscope you must firstly contact one of the technicians responsible for the
system and provide a brief explanation of the work and techniques that you wish to use. Individual
accounts will be established for all new users which will be required before you are able to make
bookings.
Users are not to alter either the software or microscope set-up without prior permission.
To book, follow link below:
https://www2.le.ac.uk/colleges/medbiopsych/facilities-and-services/cbs/lite/aif/bookequipment/nikon-c1si-clsm-booking-calendar
To get user ID: fill in and sign a user form, together with your PI. User forms can be found at
http://www2.le.ac.uk/colleges/medbiopsych/facilities-and-services/cbs/lite/aif/pdf-1/Userform.pdf
Rules for use of the microscope