Imaging Facility

The LSM 780 scanner has high sensitivity and is equipped with a 32 element galanium arsenide phosphide (GaAsP) Quasar detector, plus 2 standard PMTs and 1 transmitted light T-PMT. Equipped with a tuneable AOTF and capable of imaging up to 6 channels in multitrack configuration. The AxioObserver.Z1 inverted stand, is equipped with an environmental chamber with CO₂ for live cell imaging. Focus stabilisation is through software and hardware based mechanisms. Interchangeable stage inserts for slides, chamber slides, 35mm dishes and multiwall plates. Image capture in 3D and 4D, plus Lambda scanning, and spectral un-mixing capability.

Optics

Objective lens	Magnification / NA / Immersion	Contrast Method
C-ACHROMAT	10x / 0.45 / Water
LD LCI Plan-NEOFLUAR	25x / 0.8 /  Water or Oil	DIC II
Plan-ACHROMAT	40x / 1.2 / Water	DIC III
Plan-APOCHROMAT	63x / 1.2 / Water	DIC III
Plan-APOCHROMAT	63x / 1.4 / Oil	DICIII
Plan-APOCHROMAT	100x / 1.4 / Oil	DIC III

Epifluorescence filter sets

Filter set	Ex BP nm	Em BP nm
Fs49 wf	365	445/50
Fs38 wf	470/40	525/50
Fs43 wf	545/25	635/70

Laser Lines

• Lasos 30mW Diode 405nm
• Lasos 25mW LGN3001 multiline Argon, 458, 488, 514nm
• Lasos 20mW (DPSS 561)   561nm
• Lasos 2mW   HeNe 594nm
• Lasos 5mW   HeNe 633nm

Experiment Modes

• Z stack
• Multi-positions
• Time lapse
• Tile scanning / image stitching of large samples
• Bleaching

Software and Network Drives

• Zen 2.1 allows for multidimensional experiment design in 2D, 3D, and 4D
• PC, HPZ840 4TB 3 RAID Hard drives and 64GB of memory.
• Desk top script connections to personal offline storage folders 

Location

Imaging Suite, Room WXG86

Accounts/access/training

For internal users, all requests via the Stratocore MISBU page; https://ppms.eu/bham/login/?MISBU

Contacts

Dr Robert K. Shaw

Imaging Officer

Technology Hub Imaging Facility

The Imaging Suite – Ground floor IBR West Extension

College of Medical and Dental Sciences

University of Birmingham B15 2TT

T: +44(121)414(4)9072

E: r.k.shaw@bham.ac.uk

 The LSM 880 scanner has high sensitivity and is equipped with a 32 element galanium arsenide phosphide (GaAsP) Quasar detector, plus 2 standard PMTs and 1 transmitted light T-PMT. Equipped with a tuneable AOTF and capable of imaging up to 6 channels in multitrack configuration. The AxioObserver.Z1 inverted stand, is equipped with an environmental chamber with CO2 for live cell imaging. Focus stabilisation is through software and hardware based mechanisms (Definite focus II). Interchangeable stage inserts for slides, chamber slides, 35mm dishes and multiwall plates. Image capture in 3D and 4D, plus Lambda scanning, and spectral un-mixing capability.

https://www.zeiss.com/microscopy/int/products/confocal-microscopes/lsm-880-with-airyscan-.html

 

Airyscanning

An additional scan detector providing 1.7x isotropic increase in resolution from widefield ~120nm. The Airy Disk or Point spread function from every light emitting point is projected through zoom optics and scanned by a hexagonal array for 32 GaAsP detectors which then undergoes a process of pixel reassignment and subsequent processing.  All dyes and fluorophores that are scanned by the conventional detectors can be Airy-scanned using specific emission filter sets in simultaneous or sequential acquisition modes. 

Airyscanning in Fast mode

In fast mode the beam is shaped to scan not one but 4 lines of pixels in each scanning sweep, acquiring 4x faster than normal Airyscan. Only the central 16 elements of the array are utilised providing a 1.5 x increase in resolution. Excellent for fast imaging of live cell dynamics with increased resolution   

 

https://www.zeiss.com/microscopy/int/products/confocal-microscopes/lsm-880-with-airyscan-.html 

 

https://www.youtube.com/watch?v=sGTNBXq8omg 

 

https://www.youtube.com/watch?v=QjKrLTHGnc8

 

Optics

Objective lens	Magnification / NA / Immersion	Contrast Method
C-ACHROMAT	10x / 0.45 / Water
LD LCI Plan-NEOFLUAR	25x / 0.8 /  Water or Oil	DIC II
Plan-ACHROMAT	40x / 1.2 / Water	DIC III
Plan-APOCHROMAT	63x / 1.2 / Water	DIC III
Plan-APOCHROMAT	63x / 1.4 / Oil	DICIII
Plan-APOCHROMAT	100x / 1.4 / Oil	DIC III

Epifluorescence Filter Sets

Airy Scan Emission Filter Sets

Laser Lines

Unlike the Confocal microscope the Light Sheet microscope illuminates a single, thin plane at one time thus producing an optical section without the need for a confocal pinhole, but increasing SNR significantly. The detection lens is set at 90 degrees to the plane of illumination, and this perpendicular light path and focal plane illumination significantly reduces photo toxicity of delicate samples.

Application and advantages 

Light sheet fluorescence microscopy is the way to observe small animal models, such as Zebrafish and Drosophila in a more gentle and physiological manner, but is also applicable to Arabidopsis, marine organisms, excised tissue and biopsies.

The sample is usually embedded in agarose and mounted vertically in an imaging chamber via glass a capillary. This allows the sample to be rotated in 360^O for optimisation of the light sheet angle, but also to obtain a “Multiview” of a sample for whole organism reconstruction.

The system does not image pixel by pixel but will grab full frame using two sCMOS cameras allowing high resolution time lapse recordings for efficient analysis of dynamics within the sample.

Further background reading including downloads on sample preparation and other protocols can be found here;

https://www.zeiss.com/microscopy/int/products/imaging-systems/lightsheet-z-1.html

Specification

• Twin sCMOS PCO Edge 16bit cameras for simultaneous 2 channel acquisition
• Twin illumination objectives for single or dual sided sample illumination with dual side fusion of the live image
• Pivot scanning to reduce shadowing
• Time lapse
• Multiview
• Chamber incubation with CO₂ available
• System configured for imaging cleared tissue
• Objective lenses, 10x 20x and 40x
• Laser lines, 405,445, 488,514,561 and 638nm
• 32TB 3 RAID drive processing workstation networked to RDS

Software and Network Drives

• Zen SP1 Lightsheet,  allows for multidimensional experiment design in 3D, and 4D
• PC 1, HPZ840 4TB acquisition workstation
• PC 2, 32TB 3 RAID drive image processing workstation memory.

Location

Imaging Suite, Room WXG89

Accounts / access / training

For internal users, all requests via the Stratocore MISBU page; https://ppms.eu/bham/login/?MISBU

Contacts

Dr Robert K. Shaw

Imaging Officer

Technology Hub Imaging Section

The Imaging Suite – Ground floor IBR West Extension

College of Medical and Dental Sciences

University of Birmingham B15 2TT

T: +44(121)414(4)9072

E: r.k.shaw@bham.ac.uk

Axio Scan .Z1

The fully automated Zeiss Axio Scan .Z1 slide scanner creates high resolution virtual slides of whole tissue sections. Full tissue scanning with automatic or interactive detection of the selected area. The large capacity 100 slide magazine allows for scanning of large batches. Easy slide loading with dedicated imaging frames. Fully adaptable and programmable with synchronised external high speed excitation and internal emission filter wheels. Auto file saving to personalised storage in a secure server location.

https://www.youtube.com/watch?v=WBfXicOtIok

Sample illumination and cameras 

Light Source	Camera	Pixel size and resolution	Function
Bright field LED	3CCD colour 2MP Hitachi 1200x1600 HV-F202SCL	4.4 x 4.4 µm 0.22 µm per pixel*	Immunohistochemistry e.g. H&E, HRP, IHC
HXP120 fluorescent white  light	Hamamatsu Orca Flash 4 -4MP 2048x204816bit sCMOS	6.45 x 6.45 µm 0.32 µm / pixel*	Multichannel      fluorescence
		*20x magnification

Optics

Objective lens	Magnification / NA
Fluar	5x / 0.25 M27 (course focusing)
Plan-Apochromatic	10x / 0.45 M27
Plan-Apochromatic	20x / 0.8 M27
Plan-Apochromatic	40x/ 0.95 M27

Fluorescence filter sets

Filter set	Excitation nm	Beam Splitter	Emission nm	Examples of fluorophore combinations
Zeiss 49 HE	365	FT385	445/50	Dapi / Hoescht / BV421/ Pacific Blue/ eFluor450 / Alexa 405 / Alexa 350
Zeiss 38 HE	470/40	FT495	525/40	eGFP / YFP / Alexa 488 / FITC / pacific Green / Qdot525 (with ET380nm exciter) / Horizon BV500 / BV510
Zeiss 43 HE	545/25	FT570HE	635/70	Cy3 / Alexa 555/ Rhodamine / pacific Orange / Qdot605 (with ET380nm exciter) / tdTomato / Texas Red / eFLuor605nv / BV605 / Propidium Iodide / PE
Zeiss 64 HE	587/25	FT605HE	647/70	mPlum / Alexa 594 / Alexa 610 / Texas Red / Qdot655 (with ET 380nm exciter) / BV650 /
Chroma ET49006	620/60	ET700/75	700/75	Cy5 / Alexa 633 / 647 / 700 / Qdot705 (with ET380nm exciter)

Slide acceptability and capacity

Single width slide; (max 4 per mounting frame) x 25 width 24-26mm, length 74-76mm, depth 0.8-1.3mm.

Double width slide; (max 2 per mounting frame) x 2 width 52mm, length 74-76mm, depth 0.8-1.3mm.

Quad width slide; (max 1 per frame) width 104mm, length 74-76mm, depth 0.8-1.3mm (optional extra).

Software

ZEN blue (2012) slide scan software has capability to drive whole tissue or multi-region tiling with Z-stacking plus specific profiles for TMA’s and cell spreads etc.

Location

Imaging Suite: Room WXG87

Accounts / access / training

For internal users, all requests via the Stratocore MISBU page; https://ppms.eu/bham/login/?pf=4

Contacts 

Dr Robert K. Shaw

Imaging Officer

Technology Hub Imaging Section

The Imaging Suite – Ground floor IBR West Extension

College of Medical and Dental Sciences

University of Birmingham B15 2TT

T: +44(121)414(4)9072

E: r.k.shaw@bham.ac.uk

The Leica DM6000B is multimodal and equipped with twin Leica cameras for capture in 4 fluorescence channels and colour bright field for histological examination. Based on an upright DM6000B stand with fully motorised stage and filter turret. Applicable for slide analysis only.

http://www.leica-microsystems.com/home/

Optics

Objective lens	Magnification / NA / Immersion	Contrast Method
HC Plan Apo	10x / 0.45 / Dry	Phase 1
HC Plan Apo	20x / 0.7 /  Dry	Phase 3
Plan Apo	40x / 1.25 / Oil
Plan Apo	100x / 1.4 / Oil	Phase 3

Epifluorescence filter sets

Filter cube	Excitation nm	Emission nm	Fluorophores
A4	360	Blue 470	Dapi / Hoescht / Pacific Blue / eFluor450 / Alexa Fluor 405
L5	480	Green 527	eGFP / YFP / Alexa 488 / FITC / pacific Green
N3	546	Red 600	Cy3 / Alexa 555/ Rhodamine / pacific Orange / tdTomato / Texas Red / Propidium Iodide / PE
Y5	620	Far Red 700	Cy5 / Alexa 633 / 647 / APC

Software and Network Drives

• Leica LASX core
• Desk top script connections to personal offline storage folders

Location

Confocal Microscopy Room, Centre for Translational Inflammation Research

Institute of Inflammation and Aging, QEH/B

Accounts/access/training

For internal users, all requests via the Stratocore MISBU page; https://ppms.eu/bham/login/?MISBU

Contacts

Dr Robert K. Shaw

Imaging Officer

Technology Hub Imaging Section

The Imaging Suite – Ground floor IBR West Extension

College of Medical and Dental Sciences

University of Birmingham B15 2TT

T: +44(121)414(4)9072

E: r.k.shaw@bham.ac.uk

Based in the Centre for Translational Inflammation Research (CTIR), the Leica DMI 6000B is equipped with a DFL350 FX camera for capture in 3 fluorescence channels and bright field. Based on a fully motorised inverted DMI 6000B stand and environmental chamber with CO₂ for live cell imaging. A time-lapse module is available for migration studies. Applicable for imaging slides, cell dishes, multi-well plates and chamber slides.

http://www.leica-microsystems.com/home/

Optics

Objective lens	Magnification / NA / Immersion	Contrast Method
HCX Pl Fluotar	20x / 0.4 / Corr Dry	Phase 1
HCX Pl Fluotar	40x / 0.6 /  Corr Dry	Phase 2
HCX Pl Fluotar	63x / 0.7 / Corr Dry	Phase 2

 

Filter cube	Excitation nm	Emission nm	Fluorophores
A4	360	Blue 470	Dapi / Hoescht / Pacific Blue / eFluor450 / Alexa Fluor 405
L5	480	Green 527	eGFP / YFP / Alexa 488 / FITC / pacific Green
N3	546	Red 600	Cy3 / Alexa 555/ Rhodamine / pacific Orange / tdTomato / Texas Red / Propidium Iodide / PE

Software and Network Drives

• Leica LASX core
• Desk top script connections to personal offline storage folders

Location

Confocal Microscopy Room, Centre for Translational Inflammation Research

Institute of Inflammation and Aging, QEH/B

Accounts/access/training

For internal users, all requests via the Stratocore MISBU page; https://ppms.eu/bham/login/?MISBU

Dr Robert K. Shaw

Imaging Officer

Technology Hub Imaging Section

The Imaging Suite – Ground floor IBR West Extension

College of Medical and Dental Sciences

University of Birmingham B15 2TT

T: +44(121)414(4)9072

E: r.k.shaw@bham.ac.uk

LCM Is the contamination-free method for separating and collecting cells of interest out of tissue samples mounted on slides or live cell cultures. The isolated pure cell populations are the perfect starting point for specific genetic, proteomic and molecular analysis. PALM MicroBeam makes isolating uncontaminated source material simple, via precise detection of specified areas, and contact free laser microdissection technology, ensuring your results are exact and reproducible. The PALM laser technology can also be used for DNA damage of live cells which can be coupled with live cell time lapse fluorescence imaging of the downstream effects.

Background and introduction to Zeiss PALM LCM

https://www.youtube.com/watch?v=Qs8onaLfq8c

https://www.youtube.com/watch?v=B8AHqgOtob0

Microscope stand

• Axio Observer.Z1 inverted microscope
• Environmental camber for live cell work
• Fully motorised stage with joystick
• Twin CCD cameras for colour brightfield (AxioCamCc1) and greyscale fluorescence (AxioCamMRM)
• Sample illumination through 100W HBO Mercury epi-fluorescence burner and HAL 100W tungsten transmitted light
• Sample collection to 200µl and 500µl PCR tubes or Zeiss adhesive caps.

Optics

Objective lens	Magnification / NA / Immersion	Contrast Method
Fluar	5x / 0.25
Fluar	10x / 0.5
LD Plan Neofluer	20x / 0.4 / Korr
LD Plan Neofluer	40x / 0.6/ Korr
EC Plan Neofluer	100x / 1.3 / Oil
Plan Apochromat	150x / 1.35 / Oil	DIC

Widefield Epi-fluorescence Dichroic Filter sets

Filter set	Ex BPnm	Em BPnm	Fluorophores
FS49	365	445/50	Dapi / Hoescht / Pacific Blue / eFluor450 / Alexa Fluor 405
FS38 HE	470/40	525/50	eGFP / YFP / Alexa 488 / FITC / pacific Green
FS20	546/12	575/640	Cy3 / Alexa 555/ Rhodamine / pacific Orange / tdTomato / Texas Red / Propidium Iodide / PE

Software and Network Drives

• Zeiss PALM Robo software version 4.6
• Desk top script connections to personal offline storage folders

Location

Imaging Suite: Room WXG87

Accounts/access/training

For internal users, all requests via the Stratocore MISBU page; https://ppms.eu/bham/login/?pf=4

Contacts

Dr Robert K. Shaw

Imaging Officer

Technology Hub Imaging Section

The Imaging Suite – Ground floor IBR West Extension

College of Medical and Dental Sciences

University of Birmingham B15 2TT

T: +44(121)414(4)9072

E: r.k.shaw@bham.ac.uk

The IVIS Spectrum uses Xenogen’s novel optical imaging technology to facilitate non-invasive longitudinal monitoring of disease progression, cell trafficking and gene expression patterns in living animals. The IVIS Spectrum is a versatile and advanced in-vivo imaging system equipped with an optimized set of high efficiency filters and spectral un-mixing algorithms to take full advantage of bioluminescent and fluorescent reporters across the blue to near infrared wavelength region. It also offers single-view 3D tomography for both fluorescent and bioluminescent reporters that can be analysed in an anatomical context using a Digital Mouse Atlas.

For advanced fluorescence imaging, the IVIS Spectrum has the capability to use either trans-illumination (from the below) or epi-illumination (from above) to illuminate in vivo fluorescent sources. 3D diffuse fluorescence tomography can be performed to determine source localization and concentration using the combination of structured light and trans illumination fluorescent images. The instrument is equipped with 10 narrow band excitation filters (30nm bandwidth) and 18 narrow band emission filters (20nm bandwidth) that assist in significantly reducing autofluorescence by the spectral scanning of filters and the use of spectral unmixing algorithms. In addition, the spectral unmixing tools allow the researcher to separate signals from multiple fluorescent reporters within the same animal.

Main Features

• Different stage heights allow adjustable field of view, from well plates (3.9 cm field of view = highest resolution at 20µm) to high-throughput animal imaging (5 mice)
• Integrated isofluorane anaesthesia system
• Heated stage with for animal optimisation
• Safety electromagnetic door latch
• Brightfield LED photographic overlay image
• Motorised stage, filter wheels, lens positions, and f-stop controls
• Laser illuminated stage layout for mouse positioning and alignment
• Multifunctional filter wheel for bioluminescence, fluorescence and trans-illumination imaging

CCD Camera and Optics

• Back-illuminated 16 bit Andor Ikon CCD (2024x 2024 pixel) is thermoelectrically cooled to -90^OC for reduced dark noise / increased sensitivity down to single cell
• High quantum efficiency over the entire visible to near-infrared spectrum
• f-stop settings from  f/1– f/8, 150mm imaging lens
• CCD Size 26 x 26 mm
• Minimum Detectable Luminance <70 photons/s/sr/cm2

Accounts/access/training

For internal users, all requests via the Stratocore MISBU page; https://ppms.eu/bham/login/?MISBU

Contacts

Dr Robert K. Shaw
Imaging Officer
Technology Hub Imaging Section
The Imaging Suite – Ground floor IBR West Extension
College of Medical and Dental Sciences
University of Birmingham B15 2TT

T: +44(121)414(4)9072
E: r.k.shaw@bham.ac.uk 

Perkin Elmer home: http://www.perkinelmer.co.uk/default.xhtml 

IVIS Spectrum: http://www.perkinelmer.com/product/ivis-instrument-spectrum-120v-andor-c-124262