IGC

WARNING

This is to remind you the basic rules of fluorescence lamps and gas lasers:

  • Never turn OFF the fluorescent lamp if it's been on for less than 30 min.
  • Never turn OFF the lasers if they've been on for less than an hour (1 Hour).
  • Never turn on the lamp if it's been off for less than 30 min (check to see if it's still warm).
  • Never turn on the lasers if they've been off for less than hour (1 hour).
  • Never turn off the lamp or lasers if someone else is using the microscope within the next 4 hours.
  • The Mercury lamp should always be first-on and last-off.
  • Not respecting these instructions may lead to instrument malfunction and complete loss of function! Both lasers and lamps are extremely expensive.

Leica DMRA2 | Reservations

  • DMRA2
  • The Leica DMRA2 is an automated upright microscope coupled to a CoolSNAPHQ CCD camera, capable of imaging fluorescently labelled samples, or unstained samples with Differential Interference Contrast (DIC).

    Location : Bartolomeu Dias wing. UIC Room 1

    Microscope : Leica DMRA2

    Year Installed: 2001

    Camera : CoolSNAP HQ CCD (1.3MPx monochrome)

    Optics : Fluorescence and DIC

    Operating temp : RT

Suggestion for description in "Materials and Methods":

Images were acquired on a Leica DMRA2 upright microscope, equiped with a CoolSNAP HQ CCD camera, using the a 63x 1.4NA Oil immersion objetive, DAPI + CY5 fluorescence filtersets and DIC optics, controlled with the MetaMorph V7.5.1 software.

Suggestion for "Acknowledgments":

We would like to thank the technical support of IGC's Advanced Imaging Facility (AIF-UIC), which is supported by the national Portuguese funding ref# PPBI-POCI-01-0145-FEDER-022122, co-financed by Lisboa Regional Operational Programme (Lisboa 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) and Fundação para a Ciência e a Tecnologia (FCT; Portugal).

Maintenance log:

  • 01-01-2015 - 20x objective replaced by a slightly better and newer one (0,5NA -> 0,7NA). The image quality should have little change in terms of detail.

Available objectives

Move mouse over me!
Magnification
Objective type
NA1
Immersion
Working distance (mm)
Resolution2 @525nm in XY (µm)
Pixel Size (µm)
DIC prisms-up
DIC prisms-low
Nyquist Sampling @525nm in Z (µm)
Link
10x
N PLAN
0.25
-
17.6
1.281
1.02
D1
10/20 (K2)
1.922
20x
HC PL APO CS
0.70
-
0.59
0.458
0.51
D
10/20
0.686
40x
HCX PL FLUOTAR
0.75
-
0.40
0.427
0.25
-
-
0.641
63x
HCX PL APO CS
1.40-0.603
OIL
0.10
0.229
0.16
D1
100 (K7)
0.343
100x
HC PL APO
1.40-0.703
OIL
0.09
0.229
0.10
D
100 (K4)
0.343

(1) NA - Numerical Aperture. For more information, follow this link.

(2) These values are the theoretical optical resolution limits depending on the NA of the objective and were calculated using the Resolution formula adapted to fluorescence as seen in the Olympus micro website: R = 0.61*λ/NA

(3)Attention: These objectives have an iris which can be accidentally closed by turning a dial in the objective. ALWAYS keep this iris open for maximum light and resolution! If you are experiencing low light check this first.

Installed filtersets

Filter Cube
Excitation filter
Dichroic mirror
Emission filter
DAPI
BP 360/40
400
BP 470/40
GFP/FITC
BP 480/40
505
BP 527/30
CY3/TRITC
HQ 546/12
565
BP 600/40
CY5
BP 620/60
660
BP 700/75
YFP
BP 500/20
515
BP 535/30
CFP
BP 436/20
455
BP 480/40
FM 4-64
BP 535/50
590 LP
LP 590

Spectra, lines & filters:


Setup

Turning system ON

  1. Turn on the mercury lamp power source.
  2. Turn on the computer.
  3. Turn on the microscope control box.
  4. Turn on the camera.
  5. Log-in to Windows using your Agendo credentials.
  6. Start the MetaMorph software.
  7. Set up your experiments on the microscope.

Turning OFF

  1. Log-off from Windows. If you are NOT the last user of the day, you don't need to do the following steps.
  2. Turn off the microscope.
  3. Turn off the camera.
  4. If you used the mercury light source and there is no one using the microscope for the next 4 hours or you’re sure that the next user won’t use this light source, then turn it off.
  5. If you used any immersion lenses be sure to clean them.
  6. TAKE OUT EVERYTHING YOU BROUGHT!, eg, pipettes, slides, etc...

Widefield Fluorescence experiments

  1. Set the upper filter wheel to the “BF” position.
  2. Push the Nomarski prism slid outward.
  3. Open the shutter (”shutter closed” button) and select your fluorescence filter.
  4. Focus the image with the coarse drive. The “step” button gives you three different sensitivities to the coarse drive, there is also two elevator buttons for approaching the sample to the objective.

Note: it’s easy to bleach a sample due to over exposure to light, don’t forget to close the shutter when you’re not viewing or acquiring images.


Differential Interference Contrast (DIC) experiments

  1. Turn the brightfield illumination on.

    DMRA2

  2. Set up your samples in the microscope and focus them.
  3. Close the field aperture ("A") and adjust for Koehler illumination. Adjust the condenser iris ("F") to approximately half (or remove of eyepiece, look down tube and adjust iris to cover no more than ~80% of the field-of-view area).

    DMRA2

  4. If it isn’t already on the default position push the polarizer to under the condenser.

    DMRA2

  5. Push the analiser slid inward into the microscope (upper left side of the microscope), confirm that there is total extinction of light (dark field), and choose on the dotating wheels the correct upper and lower Wallaston prisms (for the objetive in use).

    DMRA2

  6. Turn the small screw on the upper Nomarski prism slid to adjust contrast

Acquisition

  1. Make sure the microscope view is set to the CCD camera, pull outward the little rod on the left of the eyepieces.
  2. If you didn’t already do it start the MetaMorph Online software available both as a Desktop shortcut and the Windows start menu.
  3. On MetaMorph go to the menu Acquire and press Acquire.
  4. On the Acquire dialog:
    • Press “Show Live” to preview your image.
    • Adjust the “Exposure Time” for optimal contrast.
    • Adjust the dynamic range of your image in the “Image Scaling” dialog, it modifies the contrast without changing the exposure time.
    • When you’re happy with your image press “Acquire”.
  5. Save the image to your folder on D:\users.
  6. DONT FORGET TO COPY YOUR IMAGES IMMEDIATLY AND MAKE A SECOND BACKUP ASAP. IMAGES ARE DELETED FROM THE MICROSCOPE COMPUTERS AUTOMATICALLY AFTER 1 MONTH!

Extra Info

Widefield Fluorescence

Differential Interference Contrast (DIC)