Nikon microscope 2


Location: Adrian Building EM Facility.
Contact:
Dr Kees Straatman (krs5).

This system is not for general use.

Specifications for Nikon microscope 2

Nikon microscope 2 is a TE300 semi-automatic microscope with manual XY control. The imaging part of the system is automated and controlled by Improvision's Openlab software, running on a Mac (OS X). The system is equipped with an Hamamatsu ORCA-R2 digital camera (pixel size 6.45 x6.45 µm) and an X-cite120 fluorescence illumination system. This system is equipped with a temperature controlled and CO2 fed environmental chamber.

Camera specifications.

List of objectives available.

Switching On

  1. Remove dust cover
  2. Switch on fluorescence bulb
  3. Switch on camera controller
  4. If you want to use the brightfield bulb switch this on
  5. Open Openlab software
  6. Switch on the Ludl box (this controls the filters and shutters)*
  7. Write your name etc in the folder
  8. Check the correct objective is on the microscope before using it

REMEMBER – IF THE OBJECTIVE DOESN”T HAVE THE WORD “OIL” WRITTEN ON IT DON”T USE OIL!!!

* Due to a slight incompatibility issue with the Openlab software and the Ludl Box you must always make sure the Openlab software is fully open before you switch on the Ludl Box. If you don’t the keypad which controls the filters and shutters will not work.

Switching Off

Check to see if anyone is booked on the Microscope after you and if so check they definitely still want to use it. If they do just clean the objective with LENS tissue and leave everything on.

If they don’t …

  1. Shutdown any software which is open
  2. Shutdown computer
  3. Switch off camera controller
  4. Switch off Ludl box
  5. Switch off brightfield bulb
  6. Note down the time you finished and the number of lamp hours in the folder
  7. Switch off fluorescence bulb
  8. Clean the objective with LENS tissue
  9. Replace dust cover making sure it is NOT placed over the bulb houses… else it will melt!

Keypad

This controls the Shutters, the two Filter Wheels housed at the back of the microscope and the Filter Wheel Positions

Three shutters:
Shutter 1 – Fluorescence
Shutter 2 – Brightfield
Shutter 3 – Not used

Two Filter Wheels:
Filter Wheel 1 – No filters, however there is a piece of KG1 glass in Position 1*
Filter Wheel 2 – Contains five different excitation filters

*On Microscope 2 there are two Filter Wheels housed at the back of the Microscope. Each filter wheel can hold a maximum of six filters. Filter Wheel 1 does not contain any filters, however it does have a piece of specialized glass in Position 1. The light from the Fluorescence bulb is very powerful and overtime can damage the filters. Therefore, you should make sure Filter Wheel 1 is in Position 1. This will ensure the light travels through the specialized piece of glass first before it hits the filters in Filter Wheel 2, thus helping to extend their life.

Filter Positions

For Filter wheel 1:
Position 1: KG1 Glass
Positions 2-10: Not in use

For Filter wheel 2:
Position 1: Red (CY5, 630QM50; 162801)
Position 2: Red (Texas Red, 560QM55; XF1413)
Position 3: Red (CY3, 525QM45; XF1403)
Position 4: Green (FITC,  470QM50; XF1411)
Position 5: Blue (Dapi, 365QM35, XF1409)
Position 6 – 10: Not in use

Filter Blocks

Filter blocks are housed in the black box under the objective holder on the right hand side of the microscope. The position of each filter block is numbered and there is a slider underneath to move the filter blocks into position. Unfortunately, we can only have 4 filters in the holder.

Filter blocks available:

Triple bandpass filter for DAPI/FITC/TRITC
Single bandpass filter DAPI
Single bandpass filter FITC
Single bandpass filter TRITC
Single bandpass filter Tex Red
Single bandpass filter Cy5
Nikon filter UV-2A (Ex330-380; DM400; BA420)
Nikon filter B-2A (Ex450-490; DM505; BA520)
Nikon filter G-2A (Ex510-560; DM575; BA590)

Filters can be changed on request. At the moment there is in position:
1: single bandpass filter TRITC
2: single bandpass filter FITC
3: single bandpass filter Cy5
4: triple bandpass filter DAPI/FITC/TRITC

If you have problems with bleed through and want to try the single filter blocks the combinations you need are:

Fluorochrome
Filter Block
Filter wheel 2
DAPI, Hoechst

4

5

FITC, Cy2, Alexa 488

2

4

TRITC, Cy3, Alexa 568

1

3

Alexa 595, Texas Red

3

2

Cy5

Not installed

1

Nikon microscope 2 has 4 filter blocks housed in the black box under the objective holder on the right hand side of the microscope.

Filter block nr 1 allows light between 500nm and 550nm to exite your sample and light above 550 nm to pass through to your detection system. The figure below shows the spectrum from this Omega XF2017 dichroic filter.

XF2017.png

Filter block nr 2 allows light between 450 nm and 500 nm to excite your sample and light above 505 nm to pass through to visualize your signal. The figure below shows the spectrum from the Omega XF2010 dichroic filter.

XF2010.png

Filter block number 3 allows ligh between 330 nm and 390 nm to excite your sample and light with a wave lenght above 400 nm to pass through. The figure below shows the spectrum from the Omega XF2001 dichroic filter.

XF2001.png

Filter block nr 4 is a triple ex/em block; Chroma 61000v2; this is the same filter block as on Nikon microscope 1.

FilterNikon

Bleu = excitation filter
Green = Dichroic mirror
Red = emission filter

The system has also a filter wheel containing 3 excitation filters to select the excitation wavelength at the back of the microscope. The excitation wavelength can be changed by using the keypad:
1. Blank (use to close shutter)
2. FITC (Chroma S484/15x [477nm - 492nm]) Alexa488, GFP etc.
3. TRITC (Chroma S555/25x [543nm - 568nm]) dsREd etc.
4. DAPI (Chroma S395/10x [390nm - 400nm]) Hoechst etc.

Don't use any of the other filter settings as no filters are inserted in the filter wheel and bright light from the X-Cite 120W Metal Halide Bulb will directly enter the microscope read: YOUR EYEs.

This system has no other specific emission filters and the emission light is selected by the emission filter in the Chroma 61000v2 filter block. This increases the change of bleed through from the signal of one fluorochrome in the detection channel of an other fluorochrome. For example you might see the DAPI signal (normally exited with 400nm) when you use the Chroma S484/15x exitation filter using GFP.

Troubleshooting

I can not see anything down the eyepiece / My sample looks very faint

  • Is the fluorescence bulb switched on and the bulb symbol on the display constant?
  • Is the blue dial on the fluorescence bulb unit in the up most position?
  • Is the manual shutter (silver rod on the bottom left hand side of the microscope towards the back) pushed in?
  • Are the neutral density filters (silver rods with black ends on the bottom left hand side of the microscope towards the back) both pushed in?
  • Is the dial (on the right hand side of the microscope) in position A, so 100% of the light is going to the eyepiece?

The colours I am seeing down the microscope don’t look right

  • Are you using the right filter block?
  • Are you using the right filters in filter wheel 2?

The keypad wont work

  • Did you make sure everything else was switched on first and the Openlab software was fully open BEFORE you switched the Ludl box on?
  • Try closing the Openlab software, switching the Ludl box off and waiting a couple of minutes. Now open the Openlab software, wait until it is fully open, and then switch on the Ludl box.

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