Wide-field microscopy involves illumination of the whole sample, and relies on the optical characteristics of the imaging system to capture a single focal plane. The limitation of these kinds of imaging systems is that often, out of focus light (from differing sample planes) are captured in your images, resulting in image-blur. This image blur will lower your signal to noise, which is the contrast of your images. One way to overcome this is by using specialised optics with very narrow depths of field (High NA objectives) coupled with computational algorithms that can correct for out of focus light, this process is called deconvolution.

The light coming from your sample scatters (diffraction) in all directions when illuminated, and this can be affected by coverslip thickness, sample mounting medium and the many components of the microscope between your sample and the camera. The way that the light diffracts can be characterised by the point spread function (PSF) whereby a single point of light of known size (fluorescent bead) is captured in xyz and shows the scattering behaviour of light in all dimensions for that particular optical system. The PSF is then used to reverse the effects of out of focus light by correcting for these known distortions in all dimensions.

The PSF can be generated for your particular sample using a fluorescent bead that is smaller than the smallest resolvable structure by the particular imaging system, or it can be theoretically generated using known algorithms. Two main deconvolution algorithms exist, the Richardson-Lucy and Wiener deconvolution algorithms. Deconvolution is an iterative process which is cycled on your images. Deconvolution is a post processing method, and is available either on the microscope (Deltavision, Nikon Deconvolution Microscope) or via software (Analysis Computers, Microvolution, AutoQuant).

Deltavision

Resource Links:


Room 6.025b - Olympus IX80 Inverted Microscope Stand with Photometrics Coolsnap HQ2 camera

Inverted widefield microscope with Lumencor 7 line LED lightsource, high speed filter wheels and CCD camera. Suited for imaging fixed samples on slides, samples with low fluorescence and live imaging.

Features Include:

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

Transmitted Light100W LED Lamp

Reflected Light:  Lumencor Spectra LED Lightsource

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 U-Apochromat 40x 1.35 Oil - DIC   M27
2 Plan Apochromat 60x 1.35 Oil - DIC   M27
3 UPLS-Apochromat 100x 1.4 Oil - DIC   M27
4 - - - - - -    
5 - - - - - -    
6 - - - - - -    

Fluorescent Filter Sets:

Position Name Excitation Dichroic Emission Suitable Dyes
1 DAPI 360/40 Quad Band 457/40 DAPI, Hoechst
2 FITC 490/20 Quad Band 526/38 EGFP, Alexa Fluor 488
3 TRITC 555/28 Quad Band 617/63 mCherry, Alexa Fluor 555
4 Cy5 640/20 Quad Band 685/40 Cy5, Alexa Fluor 647

LEDs:

Laser

Wavelength

Laser Power

BFP Power*

Suitable Dyes

390

390nm

20mW

4.5mW

DAPI / Hoechst

440

440nm

5mW

1mW

CFP

485

485nm

20mW

8mW

GFP / A488

514

514nm

3mW

1mW

YFP

563

563nm

10mW

5mW

mCherry / Cy3

594

594nm

5mW

1mW

A594

640

640nm

5mW

2mW

Cy5 / A647

*BFP = Back Focal Plane of Objective

Computer:

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

1TB Raid Array

30" LCD Monitor

Software: 

Softworx

Accessories:

Incubation chamber with CO2 and Temperature control

Nikon Deconvolution

Resource Links:


Room 6.025c - Nikon Ti-E Inverted Microscope Stand with Hamamatsu Flash 4.0 sCMOS camera

Inverted widefield microscope with Lumencor 7 line LED lightsource, high speed filter wheels and CMOS camera. 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
  • CO2 and temperature incubation for live cells and tissue
  • Tiled imaging
  • Multi-position imaging
  • Transmitted Light:  Sutter 100W LED Lamp

Reflected Light:  Lumencor Spectra LED Lightsource

Condensor:

Name N.A. W.D. mm Position 1 Position 2 Position 3 Position 4 Position 5 Position 6
Universal LWD 0.45 54 - - - - - -

Objectives:

Position Objective Magnification N.A. Immersion W.D. (mm) Type Type Thread
1 Plan Apochromat 4x 0.2 Dry - -   M27
2 Plan Apochromat 10x 0.45 Dry 4 DIC   M27
3 Plan Apochromat 20x 0.75 Dry 1 DIC   M27
4 Plan Apochromat 40x 0.95 Dry 0.25 DIC   M27
5 S Fluor 40x 1.30 Oil 0.22 DIC   M27
6 Plan Apochromat 60x 1.4 Oil 0.13 DIC   M27

 

Fluorescent Filters:

Excitation Dichroic Emission
390/18 410/504/582/669 440/521/607/700
438/24 462/523 440/40
485/20 505/606 479/40
513/17 436/514/604 525/30
560/25 488LP 542/27
585/29   631/36
650/13   607/36
    692/40
    488LP

 

LEDs:

Designation

Wavelength

Power

BFP Power*

Suitable Dyes

390

390nm

-

-

DAPI / Hoechst

440

440nm

-

-

CFP

485

485nm

-

-

GFP / A488

514

514nm

-

-

YFP

563

563nm

-

-

mCherry / Cy3

594

594nm

-

-

A594

640

640nm

-

-

Cy5 / A647

*BFP = Back Focal Plane of Objective

Computer:

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

3TB Raid Array

30" LCD Monitor

Software: 

NIS Elements 4.3

Accessories:

Incubation chamber with CO2 and Temperature control

OBS

Room 6.018 - Olympus IX81 Inverted Microscope Stand with Hamamatsu Orca CCD camera

Inverted widefield microscope with Xenon lightsource, high speed filter wheels and CCD camera. Suited for imaging fixed samples on slides, samples with low fluorescence and live imaging.

Features Include:

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

Transmitted Light:  Olympus 100W Halogen Lamp

Reflected Light:  Olympus MT20 Xenon White Lightsource

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 10x 0.4 Dry - DIC   M27
2 Plan Apochromat 20x 0.75 Dry - DIC   M27
3 Plan Apochromat 40x 1.0 Oil - DIC   M27
4 Plan Apochromat 60x 1.35 Oil - DIC   M27

 

Fluorescent Filter Sets:

Position Name Excitation Dichroic Emission Suitable Dyes
1 GFP/RFP S500/20 dual band dual band GFP, RFP, mCherry
2 CFP/YFP S430/25

dual band

dual band

CFP, YFP
3   S565/55      
4   S470/30      

 

Computer:

Dell PC - 3GHz Xeon Processor - 8GB RAM - 256GB nVidia Quadro GPU

1TB Raid Array

30" LCD Monitor

 

Software: 

Cell^R

 

Accessories:

Incubation chamber with CO2 and Temperature control

 

Lattice Lighsheet Microscope

Room 6.019B

The Lattice Lightsheet microscope is an advanced live cell microscope developed by Nobel Prize winner Eric Betzig in 2014. This microscope is extremely gentle on cells allowing for very long acquisitions, as well as being super-fast, (capturing full 3D volumes in under a second).

Highlights

  • Very High spatial resolution (230 nm, 230 nm, 300 nm, xyz)
  • Very High Temporal resolution (100 slices / second)
  • Very low photo-toxicity
  • Opportunity to generate very rare data (very few working systems world wide)
  • 4 Individual lasers (440 nm (CFP), 488 nm (GFP), 560 nm (RFP), 640 nm (Far Red))

Challenges

  • Massive data sets are generated by this system (terabytes)
  • Visualisation and analysis software solutions are not equipped for large volumes of LLSM data, instead MIPS are generated for indexing of files, and finding sections of movies for further analysis. Large collaborations are underway to develop tools to fix this.
  • This system is not a ‘turnkey solution’ like our Zeiss systems, therefore the software is not as refined, and often many alignments are required to optimise the imaging output.

Sample Preparation

  • This microscope takes 5 mm coverslips (thickness is not important) mounted on the holder shown below.
  • The stage can be moved within a 3 x 3 mm region from the centre of the coverslip
  • Samples must be adhered to the coverslip (for non-single cell imaging please consult with facility staff)
  • Samples are imaged with water dipping lens’ with your sample in a 8 mL water bath (take this into account if your require stimulants/drugs in your imaging media)
  • The stage/water-bath is heated, however CO2 is not available while imaging.
  • The LLSM room has an incubator set to 37 oC 5% CO2.