Confocal microscopy is an advanced light microscopy method which utilises a ‘pinhole’ to eliminate out of focus light and is suitable for both live and fixed cells and tissues. The advantage of a confocal microscope over conventional wide-field microscopes is that discrete optical sections can be collected whereby out of focus light can be eliminated. Because of this, high powered lasers are used to illuminate the sample in order to collect enough light only from the desired focal plane.

Focused beams of laser light are scanned across the sample (Laser Scanning Microscopy, LSM) and light only from the desired focal plane is allowed to enter the detector. Depending on the fluorophores in your sample, and the number of detectors available, multiple fluorophores can be excited and detected simultaneously. Traditional detectors are photo-multiplier tubes, however more recent Gallium Arsenide Phosphide (GaAsP) detectors have been developed to increase sensitivity (Confocal 4, Airyscan; Confocal 5, BiG detectors).

These microscopes provide excellent axial resolution, and very high signal to noise sampling, however this often comes with a sacrifice in temporal resolution due to the slow nature of scanning pixel-by-pixel across the image during capture. Some systems overcome this speed issue with either faster scanners (Leica SP8, resonant scanners) or with multiple pinholes (Spinning Disc Confocal, Yokogawa CSU-X1) microscopes overcome this speed limitation in unique ways.

Confocal 3 - Upright LSM 710 Meta

Resource Links:


Room 6.019b - Zeiss Axiovert 200 Inverted Microscope Stand with LSM 710 Meta Confocal Scanner

Upright point-scanning laser confocal microscope with spectral detection  Suited for imaging fixed samples on slides, samples with low fluorescence and live imaging.

Features Include:

  • Fully motorised X-Y-Z stage
  • Tiled imaging
  • Multi-position imaging
  • Spectral imaging

 

Transmitted Light:  Zeiss 100W Halogen Lamp

Reflected Light:  Exfo Xcite 120W White Light Lamp

Condensor:

Name N.A. W.D. mm Position 1 Position 2 Position 3 Position 4 Position 5 Position 6
- - - - - - - - -

Objectives:

Position Objective Magnification N.A. Immersion W.D. (mm) Type Type Thread
1 Plan Apochromat 10x 0.45 Dry - -   M27
2 Plan Apochromat 20x 0.80 Dry - - - M27
3 Plan Apochromat 40x 1.3 Oil - -   M27
4 Plan Apochromat 63x 1.4 Oil - -   M27

 

Fluorescent Filter Sets:

Position Name Excitation Dichroic Emission Suitable Dyes
1 Zeiss #63 HE 572/25 FT 590 629/62 mCherry, Texas Red
2 Zeiss #38 HE 470/40 FT 495 525/50 EGFP, Alexa Fluor 488
3 Zeiss #47 HE 436/25 FT 455 480/40 DAPI, Hoechst
4 - - - - -
5 - - - - -

 

Lasers:

Laser

Wavelength

Laser Power

BFP Power*

Suitable Dyes

405

405nm

-

-

DAPI / Hoechst

Argon Ion

458nm

- -

CFP

Argon Ion

488nm

- -

GFP / Alexa Fluor488

Argon Ion

514nm

- -

YFP

561

561nm

- -

mCherry / Cy3

633

633nm

- -

Cy5 / A647

*BFP = Back Focal Plane of Objective

 

Computer:

Fujitsu PC - 3GHz Xeon Processor - 32GB RAM - 1GB nVidia Quadro GPU

3TB Raid Array

30" LCD Monitor

 

Software: 

Zeiss Zen 2012 Black

 

Accessories:

Incubation chamber with CO2 and Temperature control

Confocal 4 - Inverted LSM 710 Meta NLO AiryScan

Resource Links:


Room 6.034a - Zeiss Axiovert 200 Inverted Microscope Stand with LSM 710 Meta Confocal Scanner

Inverted point-scanning laser confocal microscope with spectral detection and Airyscan super resolution detector. 2-photon imaging with Mai Tai eHP 760-1040nm laser with dedicated BiG(GaAsP) detectors. Suited for imaging fixed samples on slides, samples with low fluorescence and live imaging.

Features Include:

  • Fully motorised X-Y-Z stage
  • Piezo Z-drive for rapid Z-stacks
  • High-sensitivity BiG detectors (GFP/mCherry) for 2-photon imaging
  • AiryScan detector for super resolution imaging to 130nm (XY)
  • CO2 and temperature incubation for live cells and tissue
  • Tiled imaging
  • Multi-position imaging
  • Spectral imaging

Transmitted Light:  Zeiss 100W Halogen Lamp

Reflected Light:  Exfo Xcite 120W White Light Lamp

Condensor:

Name N.A. W.D. mm Position 1 Position 2 Position 3 Position 4 Position 5 Position 6
Universal LWD 0.55 26 - - - - - -

Objectives:

Position Objective Magnification N.A. Immersion W.D. (mm) Type Type Thread
1 Plan Apochromat 20x 0.8 Dry - -   M27
2 Plan Apochromat 40x 1.3 Oil - -   M27
3 Plan Apochromat 63x 1.40 Oil - -   M27
4 Plan Apochromat 100x 1.46 Oil - -   M27
5 - - - - - -   M27
6 Plan Apochromat 10x 0.45 Dry - -   M27

 

Fluorescent Filter Sets:

Position Name Excitation Dichroic Emission Suitable Dyes
1

Zeiss #63 HE

572/25

FT 590

629/62

mCherry, Texas Red

2

Zeiss #38 HE

470/40

FT 495

525/50

EGFP, Alexa Fluor 488

3

Zeiss #47 HE

436/25

FT 455

480/40

DAPI, Hoechst

4 - - - - -
5 - - - - -

 

Lasers:

Laser

Wavelength

Laser Power

BFP Power*

Suitable Dyes

405

405nm

20mW

4.5mW

DAPI / Hoechst

Argon Ion

458nm

1mW

0.4mW

CFP

Argon Ion

488nm

20mW

8mW

GFP / A488

Argon Ion

514nm

3mW

1mW

YFP

561

561nm

10mW

5mW

mCherry / Cy3

633

633nm

5mW

1mW

Cy5 / A647

Mai Tai

780 - 1040 nm

2000mW

10mW - 50mW

2 Photon Dyes

*BFP = Back Focal Plane of Objective

 

Computer:

Fujitsu PC - 3GHz Xeon Processor - 32GB RAM - 1GB nVidia Quadro GPU

3TB Raid Array

30" LCD Monitor

 

Software:

Zeiss Zen 2012 Black

 

Accessories:

Incubation chamber with CO2 and Temperature control

Confocal 5 - Inverted LSM 710 Meta BIG

Resource Links:


Room 6.028 - Zeiss Axiovert 200 Inverted Microscope Stand with LSM 710 Meta Confocal Scanner

Inverted point-scanning laser confocal microscope with spectral detection and Airyscan super resolution detector. 2-photon imaging with Mai Tai eHP 760-1040nm laser with dedicated BiG(GaAsP) detectors. Suited for imaging fixed samples on slides, samples with low fluorescence and live imaging.

Features Include:

  • Fully motorised X-Y-Z stage
  • High-sensitivity BiG detectors (GFP/mCherry and CFP/YFP) 
  • CO2 and temperature incubation for live cells and tissue
  • Tiled imaging
  • Multi-position imaging
  • Spectral imaging

 

Transmitted Light:  Zeiss 100W Halogen Lamp

Reflected Light:  Exfo Xcite 120W White Light Lamp

Condensor:

Name N.A. W.D. mm Position 1 Position 2 Position 3 Position 4 Position 5 Position 6
Universal LWD 0.55 26 - - - - - -

Objectives:

Position Objective Magnification N.A. Immersion W.D. (mm) Type Type Thread
1 Plan Apochromat 10x 0.45 Dry - -   M27
2 Plan Epiplan-Neofluar 20x 0.5 Dry - DIC   M27
3 EC Plan-Neofluar 40x 0.75 Dry - -   M27
4 LD C-Apochromat 40x 1.1 Water - -   M27
5

LD C-Apochromat

63x 1.15 Water - -   M27

 

Fluorescent Filter Sets:

Position Name Excitation Dichroic Emission Suitable Dyes
1

Zeiss #63 HE

572/25

FT 590

629/62

mCherry, Texas Red

2

Zeiss #38 HE

470/40

FT 495

525/50

EGFP, Alexa Fluor 488

3

Zeiss #47 HE

436/25

FT 455

480/40

DAPI, Hoechst

4 mCherry LP 560/20 580LP 585LP mCherry, Cy3
5

-

- - -

-

6

-

- - -

-

 

Lasers:

Laser

Wavelength

Laser Power

BFP Power*

Suitable Dyes

405

405nm

-

-

DAPI / Hoechst

Argon Ion

458nm

-

-

CFP

Argon Ion

488nm

-

-

GFP / A488

Argon Ion

514nm

-

-

YFP

561

561nm

-

-

mCherry / Cy3

594

594nm

-

-

A594

633

633nm

-

-

Cy5 / A647

*BFP = Back Focal Plane of Objective

 

Computer:

Fujitsu PC - 3GHz Xeon Processor - 32GB RAM - 1GB nVidia Quadro GPU

3TB Raid Array

30" LCD Monitor

 

Software: 

Zeiss Zen 2012 Black

 

Accessories:

Incubation chamber with CO2 and Temperature control

 

Leica STED Confocal - Inverted SP8 STED 3X

Current Status:  Fully Operational


Resource Links:


Room 6.029a - Leica DMi8 Inverted Microscope Stand with SP8 Galvo and Resonant Confocal Scanners, White Light Laser (WLL), STED 3X super-resolution, and FLIM/FRET

Inverted point-scanning laser confocal microscope with spectral, super resolution and FLIM/FRET detection. Tuneable 470nm - 670nm WLL, 2 x PMT and 2 x HyD detectors. Suited for imaging fixed samples on slides, samples with low fluorescence and live imaging.

Features Include:

  • Fully motorised X-Y-Z stage
  • High-sensitivity HyD detectors 
  • CO2 and temperature incubation for live cells and tissue
  • Tiled imaging
  • Multi-position imaging
  • Spectral imaging
  • Super-Resolution Imaging with 592nm, 660nm and 775nm STED lasers
  • Fast Resonant Imaging
  • FLIM/FRET Imaging with WLL and 440nm pulsed lasers

 

Transmitted Light:  Leica LED Lamp

Reflected Light:  Leica EL6000 120W White Light Lamp

Condensor:

Name N.A. W.D. mm Position 1 Position 2 Position 3 Position 4 Position 5 Position 6
Universal LWD 0.55 28 - K3 K6 K7 - -

Objectives:

Position Objective Magnification N.A. Immersion W.D. (mm) Type Type Thread
1 HC Plan Apochromat 10x 0.4 Dry 2.2 DIC    
2 HC Plan Apochromat 20x 0.75 Multi 0.67 DIC    
3 HC Plan Apochromat 40x 1.1 Water 0.65 DIC    
                 
5

HC Plan Apochromat

93x 1.30 Glyc 0.3 DIC    
6 HC Plan Apochromat 100x 1.40 Oil 0.13 DIC    

 

Fluorescent Filter Sets:

Position Name Excitation Dichroic Emission Suitable Dyes
1

DAPI

350/50

 

460/50

DAPI, Hoechst

2

GFP

470/40

 

525/50

EGFP, A488

3

YFP

500/20

 

535/30

YFP

4 Texas Red 560/40   630/75 Texas Red, mCherry
           

 

Lasers:

Laser

Wavelength

Laser Power

BFP Power*

Suitable Dyes

405

405nm

50mW

-

DAPI / Hoechst

440 pulsed

440nm

-

-

CFP FLIM/FRET

442

442nm 40mW - CFP
WLL

470nm-670nm

1.5mW

-

Visible Dyes

92 STED

592nm

1.5W

-

Star 488, Oregeon Green

660 STED

660nm

1.5W

-

Star 470sx, A546

775 STED

775nm

1.5W

-

SiR

*BFP = Back Focal Plane of Objective

 

Computer:

HP Z640 PC - 3GHz Xeon Processor - 64GB RAM - 24GB nVidia Quadro M6000 GPU

3TB Raid Array

2 x 30" LCD Monitor

 

Software: 

Leica LASX Software

Co-localisation, 3D visualisation, FRAP, FRET, SymphoTime FLIM

 

Accessories:

Incubation chamber with CO2 and Temperature control

 

Spinning Disc Confocal - Inverted Cell Observer SD

Resource Links:


Room 6.029 - Zeiss Axiovert 200 Inverted Microscope Stand with CSU-X1 scanhead

Inverted spinning disc laser confocal microscope  Suited for live imaging.

Features Include:

  • Fully motorised X-Y-Z stage
  • Piezo Z-drive for rapid Z-stacks
  • CO2 and temperature incubation for live cells and tissue
  • Tiled imaging
  • Multi-position imaging

Transmitted Light:  Zeiss 100W Halogen Lamp

Reflected Light:  Exfo Xcite 120W White Light Lamp

Condensor:

Name N.A. W.D. mm Position 1 Position 2 Position 3 Position 4 Position 5 Position 6
Universal LWD 0.45 54 Open PhL Ph1 Ph2 DIC I DIC II

Objectives:

Position Objective Magnification N.A. Immersion W.D. (mm) Type Type Thread
1 Plan Apochromat 40x 0.95 Dry - DIC III - M27
2 C Apo 40x 1.2 Water - DIC III   M27
3 LCI PlanN 63x 1.3 Water - DIC III   M27
4 Plan Apochromat 10x 0.45 Dry - DIC II   M27
5 Plan Apochromat 20x 0.8 Dry - DIC II   M27
6

-

-

-

-

-

-

 

-

Eyepiece Fluorescent Filter Sets:

Position Name Excitation Dichroic Emission Suitable Dyes
1 CFP (47HE) 436/25 455 480/40 ECFP, Cerulean
2 GFP (38HE) 470/40 495 525/50 EGFP, Alexa Fluor 488
3 YFP (46HE) 500/25 515 535/30 EYFP, Citrine
4 RFP (63HE) 572/25 590 629/62 mCherry, Cy3
5 - - - - -
6

-

- - - -

Confocal Fluorescence Filter Sets:

First Dichroic

Second Dichroic Camera1 Camera2

405/488/561

Empty 485/30 562/45
457/514/647 510LP 535/30 630/98
405/488/561/647 560LP - -

Computer:

Fujitsu PC - 3GHz Xeon Processor - 32GB RAM - 1GB nVidia Quadro GPU

3TB Raid Array

30" LCD Monitor

Software: 

Zeiss Zen 2012 Blue

Accessories:

Incubation chamber with CO2 and Temperature control