pl en

Laboratory of Confocal and Fluorescence Microscopy

Head: Dr. Anna Anielska-Mazur

A. Instruments available for viewing and grabbing microscopic images:
1.    Nikon Upright Fluorescence Microscope Eclipse E800 equipped with:

  • Dia-illuminator for bright field - halogen lamp 12V 100W Philips;
  • Epi-illuminator for fluorescence: high pressure mercury lamp HBO 103W/2 OSRAM;
  • Wide range of objectives (see bellow Attachment 1);
  • C-CU universal system condenser N.A. 0.90;
  • Wide range of filters for Epi-fluorescence (see bellow Attachment 2);
  • Controller CO 102-D (Linkam) with two warm stages wit temperature range between 10 and 60˚C ± 0.1˚C;
  • Motorized Z axis Drive Märzäuser ECO-STEP (Märzäuser Wetzlar);
  • Detector for Epi-fluorescence and transmitted light (Dia):
  • Monochrome cooled digital camera ORCA ER camera (Hamamatsu) - Peltier cooling system, resolution 1344x1024 pixels, 12 bit, cooling down 20˚C below ambient temperature, pixel size 6.45 μm x 6.45 μm;
       o  System for Image Processing and Analysis Lucia General 4.82 and 5.0 – softweare for for image grabbing by
           CCD cameras and simple measurements;
  • Huygens Essential 2.9 (Scientific Volume Imaging b.v.)- image processing software for deconvolution of microscopic images and for co-localization analysis, 3D visualization and 3D measurements.

2.    Nikon C1 Laser Scanning Confocal System:

  • Inverted microscope Eclipse TE2000-E mounted on antivibration table;
  • 3 H107 Motorized Microscope stage Pro Scan II (PRIOR Scientific);
  • T-RCP Remote Control Pad with Joystick, XY axis;
  • Dia-illuminator for bright field - halogen lamp 12V 100W Philips;
  • Epi-illuminator for fluorescence: high pressure mercury lamp HBO 103W/2 OSRAM;
  • Wide range of objectives (see bellow Attachment 3);
  • Universal system condenser N.A. 0.52 W.D. 30 mm;
  • Wide range of filters for Epi-fluorescence (see bellow Attachment 4);
  • Detector for fluorescence and transmitted light:
      o    Monochrome cooled digital camera DS.-1QM (Nikon) - with built-in Peltier cooling mechanism, maximal resolution  
           1000x1000 pixels, 12 bit, cooling down to -30˚C (in 5 min) or -10˚C (without cooling fan), pixel size 8 μm x 8 μm;
  • System for Image Processing and Analysis Lucia General 5.0 – software for image grabbing by CCD cameras and simple measurements;
  • Confocal Scanning Head C1 (Nikon);
  • Four-channel confocal system C1 (Nikon) with 3 independent PMT (Photomultiplier Tube) detectors for three fluorescence channels and detector for transmitted light 
  • Laser lines available in confocal system C1:
      o    Violet Diode Laser MOD (44.8 mW) 404 nm;
      o    Argon-Ion Laser (40 mW) 457/476/488/514 nm
      o    Green He-Ne Laser (1.0 mW) 543 nm;
      o    Red Diode Laser MOD (10.5 mW) 638 nm;
      o    Yellow He-Ne Laser (1 mW) 594 nm (currently not included in confocal settings);
  • TE2000 Control Software v. 2.33 - control of TE2000 microscope settings (only for advanced users);EZ-C1 for Nikon C1 Confocal v. 3.0 - image acquisition and analysis; 1D, 2D, 3D reconstruction and animation; FRAP and FRET analysis; morphological and densitometric quantitative measurements;
    Detector for Epi-fluorescence and transmitted light (Dia):
      o    Color camera DS-5Mc (Nikon) - with built-in Peltier cooling mechanism, maximal resolution 2560x1920 pixels, 12 bit,
            cooling down 20˚C below room temperature;
  • Huygens Essential 2.9 (Scientific Volume Imaging b.v.) - image deconvolution; co-localization analysis; 3D visualization; 3D measurements;
  • High Speed Lifetime Imaging Module LIMO - Enable FLIM analysis by Time-domain method; Optimal detection of lifetimes between 0.5-15 ns;
  • Laser for FLIM analysis - Picosecond pulsed diode laser 440 nm with an active thermoelectric cooling mechanism; master frequency 40 MHz (PicoQuant GmbH);
  • EZ-LIMO for Nikon C1 Confocal v. 2.30 - image acquisition and FLIM analysis;

B. Available techniques:

  • The generation of a 3D image: Applying Z-stack allows the collection of several optical sections in the specimen to create a three dimensional image.  The confocal system is more suited for thick specimens as it removes the out-of-focus light in the specimen.
  • The effective removal of haze from obtained images by using the deconvolution method: The deconvoluted data has improved in resolution and in contrast. Huygens Essential is able to deconvolve time series of 3D and 2D images. Through automatically correcting the bleaching and varying background, some structures and details become visible which would have otherwise remained hidden. The deconvolution method is better suited for thin, small specimens that require  maximum sensitivity and resolution. 
  • Simultaneous detection of two or three fluorescently-tagged proteins and their co-localization in examined specimen.
  • Observation and collection of  images during  physiological process using time lapse recording.
  • Our system is suitable for analysis of protein dynamics in live cells using the techniques such as:
      o    Fluorescent Resonant Energy Transfer (FRET) through a channel comparison;
      o    Fluorescent Recovery after Photo-bleaching (FRAP);
      o    Fluorescent Loss in Photo-bleaching (FLIP);
  • Fluorescence Lifetime Imaging (FLIM) is a great system to experiment with multiple fluorophores in living cells, especially when they’re spectra overlaps. Imaging the fluorescence lifetime (decay rate) yields extra information about the fluorescent molecules and their chemical environment. The fluorescent lifetime can be measured by exciting the specimen with a short pulse and recording the exponential decay of the fluorescent intensity. The fluorescence decay is measured for each pixel in the image. FLIM is the best tool to measure FRET data because the lifetime of excited state decreases strongly when FRET provides an additional means of decay.

Attachment 1. Objectives for Nikon Fluorescence Microscope Eclipse E800.

Plan UW 2X Dry

N.A. 0.06

W.D. 7.50 mm

∞/-

BF

CFI Plan Fluor 10X Dry

N.A. 0.30

W.D. 16.0 mm

∞/0.17

BF, DIC L*

CFI Plan Fluor 20X Dry

N.A. 0.50

W.D. 2.1 mm

∞/0.17

BF, DIC M*

CFI Plan Fluor 40X Dry

N.A. 0.75

W.D. 0.72 mm

∞/0.17

BF, DIC M

CFI Plan Apochromat 60X Oil

N.A. 1.40

W.D. 0.21 mm

∞/0.17

BF, DIC H

CFI Plan Apochromat 100X Oil

N.A. 1.40

W.D. 0.13 mm

∞/0.17

BF, DIC H

* non-available due to a lack of an objective or condenser DIC prism
N.A = Numerical Aperture
W.D. = Working Distance
BF = Brightfield
DIC = Differential Interference Contrast = Nomarski contrast
∞/- = use without cover slip
∞/0.17 = use with cover glass, thickness 0.17 mm

 

Attachment 2. Filters for for Nikon Fluorescence Microscope Eclipse E800.

UV-2A (AMCA)

EX 330-380

DM 400

BA 420

UV-2EC (DAPI, Hoechst)

EX 340-380

DM 400

BA 435-485

CFP

EX 436/20

DM 455

BA 480/30

B-2A (Oregon Green)

EX 450-490

DM 505

BA 520

B2-EC (FITC, GFP)

EX 465-495

DM 505

BA 515-555

YFP

EX 500/20

DM 515

BA 535/30

G-2A (Cy3)

EX 510-560

DM 575

BA 590

G2-EC (TRITC, Rodamine, PI, EB)

EX 540/25

DM 565

BA 605/5

EX  = Excitation Filter
DM = Dichroic Mirror
BA  = Barrier Filter

 

Attachment 3. Objectives for Nikon Inverted Microscope Eclipse TE2000-E.

CFI Plan Apochromat 10X  Dry

N.A. 0.45

W.D. 4.0 mm

∞/0.17 

BF, DIC N1 

CFI Plan Fluor ELWD 20X C Dry (correction ring)

N.A. 0.45

W.D. 7.4 mm

∞/0-2 

BF, DIC L/N1 

CFI Plan Fluor 20X MImm (Multi-Immersion: Oil, Glycerin and Water)

N.A. 0.75

Oil W.D. 0.35 mm

Glycerin W.D. 0.34 mm

Water W.D. 0.33 mm

∞/0.17 

BF, DIC N2 

CFI Plan Fluor 40X Oil

N.A. 1.30

W.D. 0.2 mm

∞/0.17

BF, DIC H/N2

CFI Plan Apochromat 60X Oil

N.A. 1.40

W.D. 0.21 mm

∞/0.17

BF, DIC H/N2

CFI Plan Apochromat VC 100X Oil

N.A. 1.40

W.D. 0.13 mm

∞/0.17 

BF, DIC N2 

N.A = Numerical Aperture
W.D. = Working Distance
BF = Brightfield
DIC = Differential Interference Contrast = Nomarski contrast
∞/0-2 = use with cover glass, thickness 0.17 mm or to work in culture dishes, thickness to 2 mm
∞/0.17 = use with cover slip, thickness 0.17 mm
 
CFI Plan Fluor ELWD 20X Dry objective allows working in culture dishes.

 

Attachment 4. Filters for Epi-fluorescence in Nikon Inverted Microscope Eclipse TE2000-E.

UV-2EC (DAPI, Hoechst)

EX 340-380

DM 400

BA 435-485

B2-EC (FITC, GFP)

EX 465-495

DM 505

BA515-555

YFP-B (YFP)

EX 500/20

DM 515 

BA535/30 

G2-EC (TRITC, Rhodamine, PI, EB) 

EX 510-560 

DM 575 

BA605/55 

Y2-EC (TEXAS RED)

EX 540-580 

DM 595 

BA600-660 

EX  = Excitation Filter
DM = Dichroic Mirror
BA  = Barrier Filter