Instruction Manual Leica DM-RA(X) Microscope

Rules:

  • The microscopes can only be used after an introduction by
    Jan Stap (M3-106.1, tel: 64771) or Ron Hoebe (M3-106.1, tel 64743)
  • Always make a reservation using:  http://agenda.cellularimaging.nl
  • Please clean up after using the microscope
  • Clean the lenses with a soft tissue with ethanol
  • Switch of the fluorescence lamp if the microscope will not be used within 15 minutes

 

Checklist

1) Microscope

Switch on:

  • “Power on” at the right site of the microscope
  • Transmitted light (next to the “power on” switch) if needed
  • Fluorescence lamp (left side of the microscope), please leave/turn off if not needed

EL6000       IMG_2926       IMG_2922

Fluorescence Filter Cubes

Operation buttons of the fluorescence cubes are located on the right side of the microscope stand.

Name

Fluorophores

Excitation

Excitation filter

Dichroic mirror

Suppression filter

 Microscope

A DAPI, Hoechst UV BP 340-380 400 LP 425 RA, RXA
L5 FITC, GFP Blue BP 460-500 505 BP 512-542 RA, RXA
TX2 Texas-Red, PI Green BP 540-580 595 BP 608-682 RA, RXA
Y3 CY3 Green BP 510-560 565 BP 573-647 RA
N3 CY3 Green BP 538-550 565 BP 580-620 RXA
Y5 CY5 Red BP 590-650 660 BP 663-737 RA
HD RA, RXA
C1 DAPI / CY3 UV/Green RA
C2 FITC / CY3 Blue/Green RA

Focusing

  • Motorised, use button on the right side of the microscope above wheel to change step-size, S0 = fine — S3 =coarse (see indicator on display)
  • Fast movement of stage with the two buttons on the right side of the microscope behind the wheel. You can set or delete the upper-level with the button at the front-right of the stand

IMG_2917       IMG_2919

Objectives

DMRA: Changing objectives is manual, do not mix Dry and Oil.

DMRXA: Changing objectives is automated. Use the two button on the left side of the stand behind the focus wheel. To switch between Dry and Oil you have to press both buttons on the front-right of the stand first.

DMRA DMRXA
N PLAN 2.5x / 0.12
N PLAN Fluotar 10x / 0.25
N PLAN Fluotar 40x / 0.65 PH2PL Fluotar 25x / 0.75 Oil
PLAN APO 40x / 1.25 Oil
PLAN APO 100x / 1.40 Oil PH3
N PLAN 5x / 0.12 PH0
PL Fluotar 10x / 0.3
PL Fluotar 20x / 0.5
PL Fluotar 40x / 0.65 PH2PLAN APO 25x / 0.75 Imm
PLAN APO 40x / 1.25 Oil
PLAN APO 100x / 1.40 Oil PH3

Phas Contrast: Manually select the correct Phase Contrast Ring as stated on the objective. Choose HD for Bright Field. (There is no Phase Contrast on the DMRXA)

2) Taking pictures with the CCD Camera

Starting up

  • Always switch on the microscope first before switching on the computer.
  • The camera is cooled 25 degrees below room temperature. For this reason the camera is always on. You can see in the sofware is the camera is cooling. If not click start cooling.
  • Start “Image pro plus”
  • Choose in the menu LCAM (or CMO) the item “Acquire Fluorescence Image”

Software

Settings

  • Select red, green and/or blue by selecting/unselecting the checkbox. Any combination is possible.
  • Select a fluorescence filter-cube and exposure-time for every chosen color
  • Select an area in the center of the field of view. The largest area is 2048×2048 for the DM-RA and 1800×1800 for the DM-RXA. The pixel-size of the CCD Camera is 7.4 µm square. For a 100x objective this means 74 nm square for the pixel in the acquired image.
  • If needed select binning of pixels. Binning combines neighboring pixels together resulting in brighter pixels with a lower magnification.
  • It is possible to make a Single, Stack or Time Sequence. Select the corresponding radio button and set the parameters.
  • By default the captured image will be scaled between the minimum and maximum pixel-value per color. For absolute (quantitative) values unselect the AutoScale checkbox.

Taking a picture

  • Press the “Acquire” button in the left menu and wait until Image Pro Plus shows the captured image.

Optimizing settings

  • The CCD camera uses 14bits to digitize the image. This means 16384 greyvalues.

 

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