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Biomedical Imaging core facility

TissueFAXSiPlus image cytometry system

The TissueFAXSiPlus inverted imaging systems from Tissue Gnostics is available in our Department (since 2012) for quantitative analysis of histological samples in brightfield (HistoQuest), and of fluorescent live or fixed cell samples (TissueQuest). The automatic control of microscope stage by TissueFAXS Plates/Slides modules allows scanning and reconstitution of entire sample areas for both image analysis and archiving purposes. Samples can be investigated in bright field light (histology staining) using the PixelLink camera or fluorescence using the PCOPixelFly camera. Our services include quantitative cytometry statistical analysis of cell subpopulations based upon specific marker quantitation. The advanced analysis software StrataQuest allows the automatic stratification of analysed tissues sections (i.e. in tumor and stroma regions) based on various cell or nuclei parameters. Dedicated APPs were configured and made available by the TissueGnostics team of software developers in Iasi.

Contact persons: Dr. Livia Sima, Dr. Gabriela Negroiu

 

The system has benefited of several upgrades that were supported by national grants:

 

Technical parameters

HARDWARE

Fully motorized inverted microscopy system for the automated scan and analysis of up to 12 slides or alternatively two cell culture multiwell plates in epifluorescence and brightfield 

Optical Components based on Zeiss AxioObserver.Z1

Inverted optical system – dedicated to cell cultures

Objectives

5×, 20×, 40×, 63× Oil

The system is calibrated for each objective, providing fort the calculation of sample area and cell count per mm2. 

Can be equipped with a total of 7 objectives

Filters - 5 filter sets for fluorescence applications:

  • DAPI (Ex. 360nm/Em. 462nm)
  • Alexa 488/Cy2 (Ex. 488nm/ Em. 520nm)
  • Alexa 568/Cy3 (Ex. 568nm/ Em. 603nm)
  • Alexa 633/Cy5/Allophycocyanin (APC) (Ex. 633nm/ Em. 651nm)
  • Cy5.5 (Ex. 650/45nm; Em. 720/60nm)

Can be equipped with a total of 6 filter sets (simultaneously inserted)

All TissueGnostics fluorescence filters are pixelshiftfree

Ultra-precise motorized stage for 12 slides 

Motorised stage specially adapted to the system, for the automated acquisition and creation of overview images and ROI details. Specific fields of view on the sample can be reacquired.

Equipped for the analysis of: 

  • multiwell plates
  • paraffin sections
  • cryocut sections
  • biopsies
  • tissue microarrays (TMA)
  • cell cultures
  • cell smears
  • cytospin preparations

Digital high-speed camera for brightfield microscopy (PixelLink), suitable for the acquisition of H&E, IHC, ISH

High-sensitivity digital monochrome (PCO Pixelfly) camera for fluorescence microscopy, suitable for the acquisition of direct and indirect IF, FISH, FRET, FRAP

High performance PC/Workstation for microscope control and analysis - MS Windows 7 

SOFTWARE 

Acquisition module TissueFAXS

  • Control software for AxioObserver.Z1
  • Microscope stage
  • Pixelink color camera
  • PCO monochrome camera
  • Preview scan (1x to 10x) “Fluorescence”
  • Preview scan (1x to 10x) „Brightfield“
  • Autofocus for freely selectable magnification and performance in fluorescence mode & brightfield mode
  • Generation of stitched overview images with selectable resolution 
  • Automatic acquisition of brightfield images and fluorescence images 

Fluorescence analysis module TissueQuest 

Tissue cytometry in immunofluorescence 

  • Innovative software for single-cell identification in tissues
  • Dynamic data connection “from cells to dotplot” (forward connection) and “from dotplot to cells“ (backward connection)
  • Up to 9 analysis parameters available
  • Logarithmic and linear data display
  • Copy & Paste functionality using Windows clipboard for image- and data export into other applications (e.g. MS PowerPoint)
  • Single-site licence 

Brightfield analysis module HistoQuest

Tissue cytometry in immunohistochemistry 

Innovative solutions for single-cell identification in tissues using brightfield microscopy Measurement of characteristic chromogen-mediated cellular staining patterns 

  • Results presented in editable scattergrams
  • Dynamic data connection “from cells to dotplot” (forward connection) and “from dotplot to cells“ (backward connection)
  • Up to 13 analysis parameters available
  • Logarithmic and linear data display
  • Data evaluation and data processing in dotblots and tables
  • Copy & Paste functionality using Windows clipboard for image- and data export into other applications (e.g. MS PowerPoint)
  • Single-site licence

 

Publications

Publications citing our TissueFAXSiPlus system:

Spinning Disk Inverted Microscope with VivaTome

The AxioObserver Z.1 Zeiss microscope with VivaTome spinning disk and two alternative illumination systems (Colibri LED and HAL 100 halogen lamp) is available in our Institute (since 2012) for semi-confocal and widefield analysis of live and fixed cells. The Colibri LED illumination prevents phototoxicity during live cells imaging as compared to classic laser scanning systems. Live cell imaging techniques enable visualization and recording image data of cellular processes. The dual camera systems together with the Physiology Software Module allows real-time recording of fast intracellular events, such as calcium signaling.

Contact persons: Dr. Livia Sima, Dr. Ionut Popescu

 

The system has benefited of several upgrades that were supported by national grants:

 

Technical parameters

HARDWARE

Microscope: AxioObserver Z.1 Zeiss microscope

Objectives:

  • Objective EC "Plan-Neofluar" 10x/0.3 M27 (WD=5.2mm)
  • Objective "Plan-Apochromat" 20x/0.8 M27 (WD=0.55mm)
  • Objective "Plan-Apochomat" 40x/1.3 Oil DIC M27 (WD=0.2mm)
  • Objective "Plan-Apochromat" 63x/1.40 Oil DIC M27 (WD=0.19mm)
  • Objective "Plan-Apochromat" 100x/1.40 Oil DIC M27 (WD=0.17mm)
  • DIC slider EC PN 10x/0.30 I, 20x/0.50 II, PA 20x/0.80 II
  • DIC slider P A 40x/1.3 III
  • DIC slider P A 63x/1.40 III
  • DIC slider P A 100x/1.40 III

Scanning stage compatible with Definite Focus

Alternative illumination systems:

HAL 100 illuminator with quartz collector lamp mount and heat-reflecting filter 6-position reflector turret mot. ACR for P&C modules reflector modules exchangeable without removal of reflector turret

Illumination system "Colibri.2" continously variable, quickly switchable and stable on the long term fluorescence light source incl. control box and control panel for use with up to 4 high brightness LED modules changeable by customer.

  • LED module 385 nm for Colibri
  • LED module 470 nm for Colibri
  • LED module 505 nm for Colibri
  • LED module 540 - 580 nm for Colibri
  • Beam combiner 425 nm for illumination system Colibri
  • Beam combiner 490 nm for illumination system Colibri
  • Beam combiner 565 nm for illumination system Colibri

Filters:

  • Filter set 49 DAPI shift free
  • EX G 365, BS FT 395, EM BP 445/50
  • Filter set 38 HE eGFP shift free 
  • EX BP 470/40, BS FT 495, EM BP 525/50
  • Filter set 43 HE Cy 3 shift free 
  • EX BP 550/25, BS FT 570, EM BP 605/70

VivaTome System with Axiovision SW and PC

Spinning Disk Unit "VivaTome"

Filter module DAPI/FITC/Rhod for "VivaTome"

Filter module CFP/YFP for "VivaTome"

Filters

Filter set 78 HE ms CFP+YFP dual camera for excitation with multi spectral light sources. Consisting of

EX BP 445/25 for CFP

EX BP 510/15 for YFP

main beam splitter DFT 460+520 secondary beam splitter FT 510

EM BP 480/22 and EM LP 530

Filter set 79 HE ms GFP+mRFP dual camera for excitation with multi spectral light sources. Consisting of

EX BP 470/27 for GFP

EX BP 566/25 for mRFP

main beam splitter DFT 490+575

secondary beam splitter FT 565

EM BP 512/30 and EM BP 630/98

Filter set 62 HE BFP + GFP + HcRed shift free optimized for use with the illumination system Colibri EX BP 475/30

  1. BP 585/35
  2. BP 370/40

BS TFT 395+495+605

EM DBP 425/46+527/54+LP 615

 

Live cell imaging components:

OKOLAB Stage top incubator (Frame+Lid+Temperature controller)

Zeiss humidity and CO2 controllers

OKOLAB specimen holders for:

  • 12 well plates
  • 96 well plates
  • 2 POC chambers
  • 2 LabTek-II chamber slides

 

SOFTWARE

AxioVision Rel. 4.8.2 Software

Image Acquisition and Management Software for Light Microscopy under Windows XP and Windows 7 (32 bit) incl. Manual on DVD

AxioVision 4 Module Dual Camera

Dual Camera is a module which enables the simultaneous use of two identical cameras. Both cameras are synchronized using TTL signals with a patented method and can produce images without any time lag. This makes acquisition of very fast processes in two channels possible. This module is a prerequisite for emission rationing or FRET

AxioVision 4 Module Physiology

AxioVision module for measurement of fast ion fluctuation (such as intracellular calcium) or for doing FRET measurements

AxioVision Mozaix module for automatic stage control during time lapse experiments

Main vendors of microscopy hardware and software:

Main vendors of microscopy chamber slides, reagents and kits:

Microscopy web learning portals:

Free analysis software: